C. Mark Ensor

Pegylated arginine deiminase treatment of patients with metastatic melanoma: results from phase I and II studies.

PURPOSE Individuals with metastatic melanoma have a poor prognosis. Many human melanomas are auxotrophic for arginine, and arginine is not an essential amino acid in humans. We hypothesized that this auxotrophy may be therapeutically exploited. A novel amino acid-degrading enzyme (arginine deiminase) conjugated to polyethylene glycol (ADI-SS PEG 20,000 mw) was used to lower plasma arginine in individuals with metastatic melanoma. PATIENTS AND METHODS Two cohort dose-escalation studies were performed. A phase I study in the United States enrolled 15 patients, and a phase I to II study in Italy enrolled 24 patients. The Italian patients also received two subsequent cycles of treatment, each consisting of four once-weekly injections of 160 U/m2. The goals of these studies were to determine pharmacokinetics (PK), pharmacodynamics (PD), safety, and the antitumor activity of ADI-SS PEG 20,000 mw. RESULTS PK and PD studies indicated that a dose of 160 U/m2 lowered plasma arginine from a resting level of approximately 130 micromol/L to less than 2 micromol/L for at least 7 days; nitric oxide levels also were lowered. There were no grade 3 or 4 toxicities directly attributable to the drug. Six of 24 phase I to II patients responded to treatment (five partial responses and one complete response; 25% response rate) and also had prolonged survival. CONCLUSION Elimination of all detectable plasma arginine in patients with metastatic melanoma was well tolerated and may be effective in the treatment of this cancer. Further testing of ADI-SS PEG 20,000 mw in a larger population of individuals with metastatic melanoma is warranted.

Incidence and distribution of argininosuccinate synthetase deficiency in human cancers: a method for identifying cancers sensitive to arginine deprivation.

Argininosuccinate synthetase (ASS) was the first of two enzymes to convert citrulline to arginine. This pathway allowed cells to synthesize arginine from citrulline, making this amino acid nonessential for the growth of most mammalian cells. Previous studies demonstrated that several human tumor cell lines were auxotrophic for arginine due to an inability to express ASS. Selective elimination of arginine from the circulation of animals with these tumors is a potentially effective anticancer treatment. The purpose of these experiments was to determine the frequency of ASS deficiency and arginine auxotrophy in a variety of human malignant tumors.

Paper strip whole cell biosensors: a portable test for the semiquantitative detection of bacterial quorum signaling molecules.

Herein, we report the development of a novel, inexpensive, and portable filter-paper-based strip biosensor for the detection of bacterial quorum sensing signaling molecules, N-acylhomoserine lactones (AHLs). AHLs are generally employed by Gram-negative bacteria for their cell-cell communication to control expression of specialized genes, such as those involved in biofilm formation and production of virulence factors, in a population-density-dependent manner. First, a bacterial cell-based sensing system employing components of AHL-mediated QS regulatory system as recognition elements and beta-galactosidase as the reporter protein was designed and developed. The bacterial-sensing cells were then liquid-dried on strips of filter paper. beta-Galactosidase as the reporter allows for the visual monitoring of the analyte-induced signal when a colorimetric method of detection is applied. The paper strip biosensor was able to detect low AHL concentrations down to 1 x 10(-8) M. Furthermore, it was successfully applied to the detection of AHLs in physiological samples, such as saliva. The filter-paper-based sensing strips could provide reproducible results upon storage at 4 degrees C for at least 3 months. In conclusion, a filter-paper-based strip biosensor was developed that allows for visual, fast, and convenient detection of AHLs in a dose-dependent manner in a test sample. In addition, it does not require expensive equipment or trained personnel and allows ease of transportation and storage. Therefore, we envision that this biosensor will serve as a simple and economical portable field kit for on-site monitoring of AHL in a variety of clinical and environmental samples.

Peer Reviewed: Applications of Reporter Genes

Reporter proteins can be used in bioanalytical methods to produce signals indicating the presence of a target analyte.

Enzymic degradation of plasma arginine using arginine deiminase inhibits nitric oxide production and protects mice from the lethal effects of tumour necrosis factor α and endotoxin

Septic shock is mediated in part by nitric oxide (NO) and tumour necrosis factor alpha (TNFalpha). NO is synthesized primarily from extracellular arginine. We tested the ability of an arginine-degrading enzyme to inhibit NO production in mice and to protect mice from the hypotension and lethality that occur after the administration of TNFalpha or endotoxin. Treatment of BALB/c mice with arginine deiminase (ADI) formulated with succinimidyl succinimide polyethylene glycol of M(r) 20000 (ADI-SS PEG(20000)) eliminated all measurable plasma arginine (from normal levels of approximately 155 microM arginine to 2 microM). In addition, ADI-SS PEG(20000) also inhibited the production of NO, as quantified by plasma nitrate+nitrite. Treatment of mice with TNFalpha or endotoxin resulted in a dose-dependent increase in NO production and lethality. Pretreatment of mice with ADI-SS PEG(20000) resulted in increased resistance to the lethal effects of TNFalpha and endotoxin. These observations are consistent with NO production resulting, to some extent, from the metabolism of extracellular arginine. The toxic effects of TNFalpha and endotoxin may be partially inhibited by enzymic degradation of plasma arginine by ADI-SS PEG(20000). Interestingly, pretreatment with ADI-SS PEG(20000) did not inhibit the anti-tumour activity of TNFalpha in vitro or in vivo. This treatment may allow greater amounts of TNFalpha, as well as other cytokines, to be administered while abrogating side effects such as hypotension and death.

Pegylated arginine deiminase lowers hepatitis C viral titers and inhibits nitric oxide synthesis

Background:  The arginine‐degrading enzyme, arginine deiminase conjugated to polyethylene glycol (ADI‐SS PEG 20 000 mw), reduces extracellular arginine, has minimal toxicity, decreases tumor burden and improves liver function in patients with chronic hepatitis C virus infection (HCV) and inoperable hepatocellular carcinoma (HCC). Reduced extracellular arginine inhibits viral replication through unknown mechanisms. It is hypothesized that ADI‐SS PEG 20 000 mw reduces HCV viral titers through nitric oxide (NO)‐dependent effects.

Chapter 13:Biotechnological Improvements of Bioluminescent Systems

Genetic and biomolecular engineering are two of the leading disciplines in biotechnology that have lead to great advancements in protein engineering. Applications of analytical bioluminescence, such as genetic reporter assays, optical in vivo imaging, and cell viability assays can often be improved by enhancing wild-type bioluminescent systems. The ability to rationally or randomly modify proteins has expanded their employment in various bioanalytical applications. Specifically, a wide range of bioluminescent proteins and photoproteins have been engineered that can be utilized in many detection and diagnostic applications. Herein, we focus on the improvements of two of the most commonly studied photoproteins, aequorin and obelin, and their uses in a variety of bioanalytical applications. Techniques such as random mutagenesis, site-directed mutagenesis, bioluminescence resonance energy transfer, and the incorporation of coelenterazine analogues are discussed as ways that have expanded the palette of these designer proteins by altering their emission wavelengths and/or half-lifes. Strategic amino acid substitutions and insertions have been also used to improve luciferase stability in high temperature, extreme pH, and harsh chemical environments, and to customize their kinetic properties and bioluminescence colors. As researchers advance engineering techniques to expand the array of photoproteins, luciferases from fireflies, click beetles, marine organisms and bacteria, their use in bioanalytical applications will continue to grow and it is envisioned that photoproteins and bioluminescent proteins will become as diverse as their fluorescence counterparts.