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.     

Use of the inferior epigastric artery as a free graft for myocardial revascularization

From March 1990 through January 1991, 47 patients undergoing myocardial revascularization had one (37) or both (10) inferior epigastric arteries (IEA) used as a conduit for bypass with 62 distal anastomoses. The internal thoracic artery (ITA) was used bilaterally in 41 patients and unilaterally in 6 with 100 distal anastomoses. Five patients had a single saphenous vein graft. In total, 167 anastomoses (3.55 per patient) were performed. Single IEA grafts were harvested through a paramedian incision and bilateral grafts, a midline incision. Harvest time was 36.5 minutes for IEA grafts and 29.6 minutes for ITA grafts (p less than 0.0001). Graft length was 11.9 cm for IEA grafts and 16.5 cm for ITA grafts (p less than 0.0001). Distal graft diameter was 2.0 mm for IEA grafts and 2.1 mm for ITA grafts (p less than 0.01). Graft flow was 49.7 mL/min for IEA grafts and 48.7 mL/min for ITA grafts. Microscopic assessment of segments of both the IEA and ITA from 14 patients revealed similar internal elastic laminae and an equal number of fenestrations. Combined intimal and medial thickness was comparable in both conduits. Medial elastic tissue was more prominent in ITA grafts and lacking in eight of the 14 IEA grafts. Gross plaque formation was noted in the proximal 1 to 3 cm of 50% of IEA grafts, but the lumen was not compromised and microscopic thickening was minimal. An unexpected finding was medial calcifications (M?nckeberg's disease) in two of the 14 IEAs without associated atherosclerosis. There was one hospital death, one abdominal wound infection, and one instance of fat necrosis superficial to the sternum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Non-target visual event-related potentials in evaluation of children with minor head trauma

In 50 children, 4 months to 12 years of age, with minor head trauma non-target visual event-related potentials were performed and compared to a second registration of the potentials some months later. On following-up there was a clear tendency for a relative improvement of the latencies of the endogenous potentials. In this way non-target visual event-related potentials proved to be of value in the investigation of mental impairment in early childhood.     

The N-methyl-D-aspartate antagonist, MK-801, fails to protect against neuronal damage caused by transient, severe forebrain ischemia in adult rats

The neuroprotective effects of dizocilipine maleate (MK-801), a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptor/channel, were tested in the 4-vessel occlusion rat model of forebrain ischemia. Adult Wistar rats, treated intraperitoneally with MK-801 or saline using several different treatment paradigms were subjected to 5 (n = 208) or 15 (n = 62) min of severe, transient forebrain ischemia. In saline-treated animals, 15 min of ischemia (n = 13) produced extensive and consistent loss of pyramidal neurons in the CA1 zone of hippocampus. The degree and distribution of cell loss were not reduced by single dose preischemic administration of MK-801 at 1 (n = 7), 2.5 (n = 4), or 5 mg/kg (n = 8). In other animals subjected to 15 min of forebrain ischemia, multiple doses of MK-801 (5, 2.5, and 2.5 mg/kg) given immediately and at approximately 8 and 20 hr after cerebral reperfusion (n = 5) did not alter CA1 injury compared to saline-treated controls (n = 5). Five minutes of forebrain ischemia in saline-treated animals, (n = 82) resulted in significantly fewer (p less than 0.001) dead CA1 pyramidal cells and a greater variance compared to animals subjected to 15 min of ischemia. Power analysis of the preliminary saline-treated animals subjected to 5 min of ischemia (n = 22) indicated that 60 animals per group were necessary to detect a 15% difference between MK-801 and vehicle-treated groups. Multidose treatment with MK-801 (1 mg/kg) given 1 hr prior to 5 min of ischemia (n = 60) and again at approximately 8 and 16 hr after recirculation failed to attenuate hippocampal injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induced tolerance to ischemia in gerbil hippocampal neurons

Brief ischemia induced tolerance to subsequent ischemia in the hippocampal neurons. Male Mongolian gerbils were subjected to 2 min of ischemia in an awake condition. This ischemic insult only rarely produced neuronal damage in the gerbil brain. One day (n = 9), 2 days (n = 9), or 4 days (n = 10) following the first brief ischemia, the animals (double-ischemia group) were subjected to the second ischemia for 5 min. The single-ischemia group received a sham procedure instead of the first ischemia and was identically subjected to the second ischemia 1 day (n = 9), 2 days (n = 10), and 4 days (n = 13) following the sham procedure. One week following the second ischemia, all gerbils were perfusion fixed and the neuronal density in the hippocampal CA1 sector was measured. In double-ischemia groups, the neuronal density per 1-mm length of the pyramidal cell layer was 103.4 +/- 93.1 (SD) in the 1-day subgroup, 125.6 +/- 64.2 in the 2-day subgroup, and 176.2 +/- 93.7 in the 4-day subgroup, while the density in normal gerbils was 254.7 +/- 18.6. The average neuronal density in the single-ischemia group was much lower than that in the double-ischemia group (whole control group: 10.9 +/- 27.4). Immunostaining using monoclonal antibody raised against 70-kDa heat-shock protein revealed an increase in 70-kDa heat-shock protein in the CA1 area following 2 min of ischemia. Very brief ischemia induces heat-shock proteins and, presumably, thereby renders neurons more tolerant to subsequent metabolic stress.     

Pituitary abnormalities in eating disorders: further evidence from MRI studies

1. The frequent occurrence of hypothalamo-pituitary dysfunction in patients with eating disorders as well as prior reports that nutritional and endocrine status influence pituitary morphology, led us to hypothesize that pituitary size and shape may be altered in patients with eating disorders. 2. Magnetic resonance imaging (MRI) does not use ionizing radiation and is currently one of the most feasible modalities available to study the pituitary gland in vivo. Using MRI, we have previously reported in a preliminary study that female patients with eating disorders had significantly smaller pituitary glands than controls. In addition MRI excluded any pituitary mass lesions. 3. In this report, we confirm our previous MRI findings and provide further evidence of pituitary abnormalities in an expanded sample of eating disorder patients. Preliminary data on pituitary volume estimates from MRI scans are provided for a subset of patients and controls.