William R. Trumble

Apoptosis Induced by Staphylococcus aureus in Epithelial Cells Utilizes a Mechanism Involving Caspases 8 and 3

ABSTRACT In this study, we demonstrate that the mechanism by whichStaphylococcus aureus induces apoptosis in bovine mammary epithelial (MAC-T) cells involves caspases 8 and 3, two key components of a proteolytic cascade leading to apoptosis. In addition, internalized S. aureus induces expression of the inflammatory cytokines tumor necrosis factor alpha and interleukin-1β by MAC-T cells. These data suggest that the internalization of S. aureus could induce specific cellular responses in vivo that may ultimately impact the course of infection.

Molecular Characterization of a Novel Staphylococcus aureus Serine Protease Operon

ABSTRACT The present study identified and characterized a unique operon (spl) encoding six serine protease-like proteins. In addition, native Spl proteins were isolated and characterized. Typical of most exoproteins, the spl gene products contain putative 35- or 36-amino-acid signal peptides. The Spl proteins share 44 to 95% amino acid sequence identity with each other and 33 to 36% sequence identity with V8 protease. They also contain amino acids found in catalytic triads of enzymes in the trypsin-like serine protease family, and SplB and SplC were shown to degrade casein. The sploperon is transcribed on a 5.5-kb transcript, but several nonrandom degradation products of this transcript were also identified. Similar to other S. aureus exoprotein genes, the sploperon is maximally expressed during the transition into stationary phase and is positively controlled by the Agr virulence factor regulator. The Sar regulatory system did not affect sploperon expression. PCR analysis revealed the presence of thespl operon in 64% of the S. aureus isolates tested, although one spl operon-negative isolate was shown to contain at least two of the spl genes. Finally, intraperitoneal injection of an spl operon deletion mutant revealed no major differences in virulence compared to the parental strain.

A series of meso-tris (N-methyl-pyridiniumyl)-(4-alkylamidophenyl) porphyrins: synthesis, interaction with DNA and antibacterial activity.

A series of meso-5,10,15-tris(N-methyl-4-pyridiniumyl)-20-(4-alkylamidophen yl) porphyrins were synthesized by derivatizing the amino group on the phenyl ring with the following hydrophobic groups: -C(O)C7F15, -C(O)CH=CH2, C(O)CH3, -C(O)C7H15, and -C(O)C15H31. The cationic tris-pyridiumyl porphyrin core serves as a DNA binding motif and a photosensitizer to photomodify DNA molecules. The changes of the UV-Vis absorption spectra during the titration of these porphyrins with calf thymus DNA revealed a large bathochromic shift (up to 14 nm) and a hypochromicity (up to 55%) of the porphyrins Soret bands, usually considered as proof of porphyrin intercalation into DNA. Association constants (K) calculated according to the McGhee and von Hippel model, were in the range of 10(6)-10(7) M(-1). An increase in hydrophobicity of the substituents at the 20-meso-position produced higher binding affinity. These porphyrins caused photomodification of the supercoiled plasmid DNA when a green laser beam at 532 nm was applied. Those with higher surface activity acted more efficiently as DNA photomodifiers. The porphyrin with a perfluorinated alkyl chain (-COC7F15) at the meso-20-position inhibited the growth of gram-positive bacteria (S. aureus, or S. epidermidis). Other porphyrins exhibited moderate activity against both gram-negative and gram-positive organisms.

A bioassay for cobra cardiotoxin activity using semi-isolated cockroach heart

A semi-isolated cockroach heart preparation was used to rapidly determine the activity of cobra cardiotoxin, monitored as a direct response on heart rate. This preparation produced a dose-response curve in the presence of active cardiotoxin and demonstrated that cardiotoxin retained its biological activity after boiling, although cardiotoxin activity was destroyed by heating in the presence of dithiothreitol. Experiments that cross-linked radiolabeled cardiotoxin to solubilized cockroach heart membranes suggested that cardiotoxin bound specifically to a 59,000 mol. wt membrane protein in this tissue.

Cardiotoxin from cobra venom affects the CaMg-ATPase of cardiac sarcolemmal membrane vesicles

Highly purified sarcolemmal (SL) membrane vesicles were prepared from bovine cardiac tissue and used to evaluate the effects of cardiotoxin (CTX) on Ca2+ transport systems of the SL membrane. The addition of CTX, at 1.0 microM and 10 microM, stimulated the ATP-dependent transport of 45Ca2+ by the Ca2(+)-Mg2(+)-ATPase to 138% and 193% of control levels, respectively. The increase in Ca2+ accumulation by the SL vesicles in the presence of CTX was not affected by 100 microM ouabain but was reduced to 50% of control uptake levels by the addition of 0.5 mM dicyclohexylcarbodiimide. Additionally, no effect of CTX (1.0 microM to 10 microM) was seen on 45Ca2+ transport by the Na-Ca exchange system. These effects of CTX on the SL membrane do not appear to be due to non-specific membrane disruption since SL vesicles preloaded with 45Ca2+ did not release (efflux) the accumulated Ca2+ more rapidly in the presence of CTX (1.0 microM to 250 microM). The rate of hydrolysis of ATP by the SL Ca2(+)-Mg2(+)-ATPase was observed to increase (29% to 52%) as CTX concentration increased (1.0 microM to 10 microM). The same concentrations of CTX had no effect on ATP hydrolysis by Na(+)-K(+)-ATPase. We conclude that 10 microM CTX stimulates the rate of ATP hydrolysis and Ca2+ transport by the SL Ca2(+)-Mg2(+)-ATPase.