Progress toward re‐engineering non‐ribosomal peptide synthetase proteins: a potential new source of pharmacological agents

Many important pharmaceutical agents, including vancomycin, bleomycin, cyclosporin, and several antibiotics, are produced by non‐ribosomal peptide synthetase (NRPS) enzymes in microorganisms. The NRPS pathway produces an extensive library of products using multienzyme complexes acting in an assembly‐line fashion. Engineering an NRPS system to produce an even greater variety of products, some of which may also have beneficial therapeutic value, would be an enormous advantage. Several approaches have been successful in generating novel NRPS products: mutational biosynthesis during which nonnatural substrates are fed to an organism; domain and module swapping between different species to generate hybrid enzymes; and rational site‐directed mutagenesis, based either on phylogeny or computational prediction, intended to switch substrate specificity and produce altered products. This review will highlight the progress in these areas and describe research in the future that will extend the capacity for re‐engineering NRPS systems. Drug Dev. Res. 66:9–18, 2006. © 2006 Wiley‐Liss, Inc.     

Clonidine in the treatment of Tourette's syndrome exacerbation due to haloperidol withdrawal

A patient with long-standing Tourette's syndrome had a dramatic exacerbation of symptoms following the rapid withdrawal of haloperidol. Clonidine administration resulted in a disappearance of tics, which recurred 6 days later. The role of clonidine in blocking withdrawal-induced symptoms in patients with Tourette's syndrome treated with long-term neuroleptics is discussed from a clinical and neurobiological perspective. The implications of this case for the treatment of supersensitivity psychosis and tricyclic withdrawal states with clonidine are also discussed.     

The effect of optical design on micromanipulator spot size using CO2 laser irradiation

Objective: The study goal was to compare the laser spot size created using reflective and refractive micromanipulators with a CO₂ laser and to determine the sensitivity of spot size to laser power. Study Design and Setting: A CO₂ laser and operating microscope (400-mm focal length) was coupled to either a reflective (Cassegrain-like) or refractive micromanipulator. Laser spot size was determined by measuring the region of ablation created by laser irradiation of wood (dry tongue depressors), exposed photographic film, and agar gel using optical micrometry. Laser power varied from 0.5 to 20 W with pulse durations of 0.1 and 0.5 second. Results: The reflective micromanipulator demonstrated overall smaller spot sizes for a given laser power and lower incremental change in spot size with increasing power. The reflective design demonstrated less sensitivity to increases in laser power. Conclusions: Micromanipulator optical design can result in significant differences in laser spot size. The reflective device used in this study demonstrated less sensitivity to increasing laser power. (Otolaryngol Head Neck Surg 2002;126:593-597.)     

The contemporary role of antioxidant therapy in attenuating liver ischemia-reperfusion injury: a review.

Oxidative stress is an important factor in many pathological conditions such as inflammation, cancer, ageing and organ response to ischemia-reperfusion. Humans have developed a complex antioxidant system to eliminate or attenuate oxidative stress. Liver ischemia-reperfusion injury occurs in a number of clinical settings, including liver surgery, transplantation, and hemorrhagic shock with subsequent fluid resuscitation, leading to significant morbidity and mortality. It is characterized by significant oxidative stress but accompanied with depletion of endogenous antioxidants. This review has 2 aims: firstly, to highlight the clinical significance of liver ischemia-reperfusion injury, the underlying mechanisms and the main pathways by which the antioxidants function, and secondly, to describe the new developments that are ongoing in antioxidant therapy and to present the experimental and clinical evidence about the role of antioxidants in modulating hepatic ischemia-reperfusion injury.     

A new transthyretin variant (Glu61Gly) associated with cardiomyopathy.

We report the identification of a new transthyretin (TTR) gene mutation and variant protein, Glu61Gly, in a 55-year-old man with progressive cardiomyopathy, mild peripheral neuropathy and bilateral carpal tunnel syndrome. A diagnosis of TTR-associated familial amyloidosis (ATTR) was considered after an endomyocardial biopsy revealed amyloid deposits in the heart of a patient who had no family history of amyloidosis and no evidence of a plasma cell dyscrasia. Serum screening for a TTR variant by isoelectric focusing (IEF) was positive and prompted further studies to identify the genetic abnormality and to characterize the amyloidogenic protein. Direct DNA sequence analysis of all four coding regions in the TTR gene demonstrated heterozygosity in exon 3. Near equal amounts of guanine (G) and adenine (A) were observed at the second base position of codon 61. The wild-type (GAG) and mutated (GGG) sequences found in codon 61 correspond to glutamic acid (Glu) and glycine (Gly) residues, amino acids which differ in mass by -72 Da. Mass spectrometric analyses of TTR immunoprecipitated from serum showed the presence of both wild-type and variant proteins. The observed mass results for the wild-type and variant proteins were consistent with the predicted values calculated from the genetic analysis data.     

Toxicity, inflammation, and anti-human immunodeficiency virus type 1 activity following exposure to chemical moieties of C31G.

C31G, which has potent activity against the human immunodeficiency virus type 1 (HIV-1) and an established record of safety in animal studies and human trials, is a microbicidal agent comprised of a buffered equimolar mixture of two amphoteric, surface-active agents: an alkyl amine oxide (C14AO) and an alkyl betaine (C16B). Studies of long-term in vitro exposure to C31G and its constituents have suggested that the components of C31G may contribute differentially to its toxicity and efficacy. In the present studies, in vitro assays of cytotoxicity and anti-HIV-1 activity demonstrated that C16B was slightly less cytotoxic compared to either C31G or C14AO, whereas the anti-HIV-1 activities of C31G and its individual constituents were similar. In the murine model of cervicovaginal microbicide toxicity, in vivo exposure to C14AO resulted in severe cervical inflammation followed by a delayed disruption of the columnar epithelium. In contrast, exposure to C16B caused severe cervical epithelial disruption and a secondary, less intense inflammatory response. These results demonstrate that (i) there are both mechanistic and temporal differences in toxicity associated with the components of C31G not necessarily predicted by in vitro assessments of cytotoxicity and (ii) contributions of each component to the anti-HIV-1 activity of C31G appear to be equal. In addition, these findings indicate that direct and indirect mechanisms of in vivo toxicity can be observed as separate but interrelated events. These results provide further insight into the activity of C31G, as well as mechanisms potentially associated with microbicide toxicity.     

Statistical clues to postoperative blood loss: Moving averages applied to medical data

With the advent of computerized databases, medical data has become easy to accumulate; however, effective use of this data continues to pose significant problems. In other circumstances, smoothing algorithms have been used to uncover non-obvious correlations, trends and relationships in noisy data. We have applied four such algorithms to a large dataset of postoperative blood replacement in cardiopulmonary bypass patients. When applied to this dataset, one of the algorithms proved surprisingly effective. It confirmed several previously observed correlations, and also provided an additional series of counterintuitive and apparently unrelated associations. These associations have been explored in an accompanying paper.