Detection of time-dependent and oxidatively induced antigens of bovine liver rhodanese with monoclonal antibodies

Rhodanese has been extensively utilized as a model protein for the study of enzyme structure-function relationships. An immunological study of conformational changes occurring in rhodanese as a result of oxidation or thermal inactivation was conducted. Three monoclonal antibodies (MABs) to rhodanese were produced. Each MAB recognized a unique epitope as demonstrated by binding of the MABs to different proteolytic fragments of rhodanese. While none of the MABs significantly bound native, soluble, sulfur-substituted bovine rhodanese, as indicated in indirect enzyme-linked immunosorbent assay experiments, each MAB was immunoadsorbed from solution by soluble rhodanese as a function of the time rhodanese was incubated at 37 degrees C. Thus, as rhodanese was thermally inactivated, conformational changes resulted in the expression of three new epitopes. Catalytic conformers demonstrated different rates of thermally induced antigen expression. Each MAB also recognized epitopes expressed when rhodanese was immobilized on microtiter plates at 37 degrees C. Two conformers resulting from oxidation of rhodanese by hydrogen peroxide were identified immunologically. All MABs recognized rhodanese that was oxidized with peroxide and allowed to undergo a secondary cyanide-dependent reaction which also resulted in a fluorescence shift and increased proteolytic susceptibility. Only one MAB was capable of recognizing an epitope expressed when rhodanese was oxidized with peroxide and then separated from the reactants to prevent induction of the secondary conformational changes.     

Potentiation of diacylglycerol-activated protein kinase C by acyl-coenzyme A thioesters of hypolipidaemic drugs

Acyl-Coenzyme A thioesters of the hypolipidaemic and cancerinogenic peroxisome proliferators clofibric acid, nafenopin, ciprofibrate, bezafibrate and tibric acid were found to greatly increase the activity of rat brain protein kinase C. Maximal activation required the simultaneous presence of Ca+2, phosphatidylserine and diolein, thus differentiating their action from that of other tumor promoters such as phorbol esters. Under similar conditions the unesterified drugs were comparatively ineffective. Similar results were obtained using the rat liver enzyme. The data suggest that acylcoenzyme A thioesters of hypolipidaemic drugs, may play a role in the induction of liver tumors by these compounds, through the potentiation of protein kinase C.     

C5-blocking antibody reduces fluid requirements and improves responsiveness to fluid infusion in hemorrhagic shock managed with hypotensive resuscitation.

Hypotensive resuscitation strategies and inhibition of complement may both be of benefit in hemorrhagic shock. We asked if C5-blocking antibody (anti-C5) could diminish the amount of fluid required and improve responsiveness to resuscitation from hemorrhage. Awake, male Sprague-Dawley rats underwent controlled hemorrhage followed by prolonged (3 h) hypotensive resuscitation with lactated Ringer's or Hextend, with or without anti-C5. Anti-C5 treatment led to an estimated 62.3 and 58.5% reduction in the volume of Hextend and lactated Ringer's, respectively. In the subgroup of animals with a positive mean arterial pressure (MAP) response to fluid infusion following prolonged hypotension, anti-C5 treatment led to an estimated 4.7- and 4.1-fold increase in mean arterial pressure response per unit Hextend and lactated Ringer's infused, respectively. We observed no significant postresuscitation metabolic differences between the anti-C5 groups and controls. Whether anti-C5 could serve as a volume-sparing adjunct that improves responsiveness to fluid administration in humans deserves further study.     

Autonomous induction of recombinant proteins by minimally rewiring native quorum sensing regulon of E. coli

Quorum sensing (QS) enables an individual bacterium's metabolic state to be communicated to and ultimately control the phenotype of an emerging population. Harnessing the hierarchical nature of this signal transduction process may enable the exploitation of individual cell characteristics to direct or “program” entire populations of cells. We re-engineered the native QS regulon so that individual cell signals (autoinducers) are used to guide high level expression of recombinant proteins in E. coli populations. Specifically, the autoinducer-2 (AI-2) QS signal initiates and guides the overexpression of green fluorescent protein (GFP), chloramphenicol acetyl transferase (CAT) and β-galactosidase (LacZ). The new process requires no supervision or input (e.g., sampling for optical density measurement, inducer addition, or medium exchange) and represents a low-cost, high-yield platform for recombinant protein production. Moreover, rewiring a native signal transduction circuit exemplifies an emerging class of metabolic engineering approaches that target regulatory functions.     

Comparison of chloroplast DNAs by specific fragmentation with EcoRI endonuclease

Summary Mutants ofEscherichia coli K12, deficient in up to three major outer membrane proteinsb,c andd have been constructed. Mutants that lack the lipopolysaccharide sugar heptose are deficient in proteinb. All heptose-deficient strains are supersensitive to lysozyme, various antibiotics and detergents. They excrete the periplasmic enzyme ribonuclease I. Mutants deficient in proteinsc and/ord have the same sensitivity towards these compounds as the parent strain. Cells of single, double and triple mutants are all rod-shaped. Electrophoretic analysis of cell evelope proteins indicates that in some mutants the protein deficiency is partially compensated for by increased amounts of one or two of the other major outer membrane proteins. Heptose-deficient strains have an increased amount of 2-keto-3-deoxyoctonate.     

Electroporation of cultured adult rat hepatocytes with the c-myc gene potentiates DNA synthesis in response to epidermal growth factor

The human c-myc gene was introduced and transiently expressed in adult rat hepatocyte cultures by the technique of electroporation and its effect on DNA synthesis was examined. Epidermal growth factor (EGF) has been found to stimulate a wave of DNA synthesis in electroporated rat hepatocytes. Hepatocyte cultures electroporated with the c-myc gene showed a potentiation of this EGF effect exhibiting rates of DNA synthesis up to 50% greater than those of control electroporated cultures, as determined by [3H]thymidine labeling of cell nuclei. This potentiation was dependent on the amount of c-myc DNA transfected. The potentiation was due neither to an alteration in the dose-response of the stimulatory effect of EGF nor to a change in the time course of the DNA synthesis wave.