Rachel Baribault Kent

Inhibitors of human neutrophil elastase

Human neutrophil elastase (HNE) is a serine protease that is responsible for a number of diverse pathological conditions. Synthetic inhibitors which can regulate the in vivo activity of HNE will therefore have broad therapeutic applications. Over the past 15 years a diverse set of strategies have been pursued for the development of HNE inhibitors, the most successful of which have been electrophilic ketones and acylating agents. Many of these inhibitors have been demonstrated to prevent the deleterious effects of elastase-mediated proteolysis in a number of in vivo models. This report reviews electrophilic ketones and acylating agents that have appeared in the patent literature since 1993. Supporting primary literature which highlights the therapeutic potential of these inhibitors is also included.

A mutant rho ATPase from Escherichia coli that is temperature-sensitive in the presence of RNA

SummaryThe Escherichia coli mutant rho-115 suppresses lac operon polarity conferred by the lacZ::IS1 insertion MS319. The ATPase activity of purified rho-115 protein was maximal at 40°C, in contrast to 45°C for rho+. At higher temperatures (50°C, 55°C), the fractions of activities at maximal temperature were consistently lower for rho-115 compared to rho+. The 30-minute time course of rho-115 ATP hydrolysis was linear at 37°C but at 45°C the linear kinetics of hydrolysis reached a plateau between 10 and 15 minutes. The 30-minute time courses for rho+ were linear at both 37°C and 45°C. The rho-115 and rho+ ATPase activities were equally heat-stable during preincubation at 45°C in buffer. Inclusion of ATP during preincubation protected these rho proteins from inactivation to the same extent. The presence of polyC during preincubation protected rho- activity but produced substantial inactivation of rho-115 ATPase. The presence of polyU during preincubation gave similar results. Concentrations of polyC between 625 ng/ml and 100 μg/ml yielded the same extent of rho-115 ATPase inactivation during preincubation at 45°C. Thermal inactivation of rho-115 ATPase by polyC was halted by shifting preincubation temperature from 45°C to 35°C, indicating that polyC-induced destabilization of rho-115 was irreversible.

Immune Regulation of the c-myc Oncogene in a Murine B Lymphoma

The c-myc oncogene has been implicated in a wide spectrum of B cell neoplasias (1–4) Reports of c-myc gene amplification (5), of insertion of viral elements near the c-myc promoter (1), and of point mutations within the c-myc gene (6) have appeared. However, the most frequently observed alteration is a chromosomal translocation, and usually this is to an immunoglobulin gene element (3–5). The observed amplification of the c-myc gene in some tumors led to the proposal of activation via enhanced levels of expression of c-myc RNA; however, increased transcription does not appear to be a general mechanism of activation (5). Since the first exon of the c-myc gene is often lost during translocation, it was postulated that the product of the rearranged myc gene would be different from that of the germ-line gene. However, DNA sequencing demonstrated that initiation of protein synthesis would occur within the second exon, and thus both protein products would be the same (7). Since the large majority of these modifications affect the promoter region, subtle alterations in the regulation of c-myc expression might result. Therefore we decided to examine the regulation of expression of the normal c-myc gene within a B cell system.

Temperature-sensitive mutant rho-115 rho-RNA binary complexes, and stabilization by substrates and analogues

SummaryTo determine the molecular basis for the temperature-sensitivity of pure rho RNA-dependent ATPase from Escherichia coli mutant rho-115 cells, we investigated mutant rho binding to [3H] polyC as measured by retention on nitrocellulose filters. Complexes of wild-type rho and polyC incubated at 37°C and 45°C were similarly stable. At 37°C mutant rho-polyC binary complexes were inactivated at a slightly faster rate than complexes with wild-type rho. Upon shift to 45°C the quantity of rho-115 bound to polyC declined immediately, resulting in one-fifth of the quantity of complexes observed at 37°C. Shift back to 37°C restored the level of observed complexes by two-fold. The inclusion of ATP or the analogue β-γ methylene ATP during 45°C incubation resulted in stable mutant rho-polyC complexes. The hydrolysis product ADP was also effective in stabilizing binary complexes at 45°C but this effect was observed with an order of magnitude more ADP than ATP. Adenine, adenosine, AMP or Pi had no stabilizing effect. We conclude that the mutant rho-115 protein exhibits a structural instability as a result of binding RNA. Furthermore ATP confers a wild-type phenotype upon rho-115 protein, probably as a result of conformational change due to binding of this compound. The effect of ATP on the stability of mutant rho-polyC binary complexes supports the model of ATP modulation of rho-RNA interaction proposed by Galluppi and Richardson (1980).