Engineering of complex polyketide biosynthesis — insights from sequencing of the monensin biosynthetic gene cluster

The biosynthesis of complex reduced polyketides is catalysed in actinomycetes by large multifunctional enzymes, the modular Type I polyketide synthases (PKSs). Most of our current knowledge of such systems stems from the study of a restricted number of macrolide-synthesising enzymes. The sequencing of the genes for the biosynthesis of monensin A, a typical polyether ionophore polyketide, provided the first genetic evidence for the mechanism of oxidative cyclisation through which polyethers such as monensin are formed from the uncyclised products of the PKS. Two intriguing genes associated with the monensin PKS cluster code for proteins, which show strong homology with enzymes that trigger double bond migrations in steroid biosynthesis by generation of an extended enolate of an unsaturated ketone residue. A similar mechanism operating at the stage of an enoyl ester intermediate during chain extension on a PKS could allow isomerisation of an E double bond to the Z isomer. This process, together with epoxidations and cyclisations, form the basis of a revised proposal for monensin formation. The monensin PKS has also provided fresh insight into general features of catalysis by modular PKSs, in particular into the mechanism of chain initiation. Journal of Industrial Microbiology & Biotechnology (2001) 27, 360–367. Received 18 March 2001/ Accepted in revised form 09 July 2001     

Oviposition behavior of Edovum puttleri, reared on two hosts, Leptinotarsa decemlineata and L. texana

Female Edovum puttleri Grissell [Hymenoptera: Eulophidae], reared from eggs of Leptinotarsa decemlineata (Say) or Leptinotarsa texana Schaeffer [Coleoptera: Chrysomelidae], were videotaped as they attacked egg masses of L. decemlineata containing 20 host eggs. We identified 15 components of ovipositional behavior. Parasitoids reared on L. texana attacked and oviposited in significantly more host eggs than did females reared on L. decemlineata. Ethometric analyses of behavioral transitions and a clustering analysis of 34 behavioral parameters showed that females reared on L. texana attacked the host egg mass in a different manner than those reared from L. decemlineata. It was concluded that differences were associated with the host species upon which they were reared. Contrary to previous reports, mortality of unparasitized hosts was caused by an ovipositor probe of short duration, which was not related to host-feeding.     

Effect of endurance training on gross energy expenditure during exercise

We compared the effect of endurance exercise training on gross energy expenditure (GEE) during steady-state exercise in 20 younger men (31.2 +/- 0.6 years) and 20 middle-aged men (49.2 +/- 1.1 years). The subjects trained for eight months. The training program consisted of three 45-min walking and jogging exercise sessions per week at an intensity of approximately 60-85% of the heart rate at peak VO2. We administered bicycle ergometer tests at 0, 4, and 8 months into training. Participants exercised at a power output of 100 W for 10 min using a pedaling frequency of 50 rpm. We determined GEE (kcal/min) by measuring the oxygen consumption and respiratory exchange ratio. We found a significant reduction (p less than 0.05) in GEE (0.7-1.3 kcal/min) following 4 months of endurance training in both age groups, with a further reduction (p less than 0.05) noted in only the middle-aged group at month 8. We found no difference (p greater than 0.05) in GEE between the younger and middle-aged men. We conclude that chronic exercise may modify GEE during a submaximal exercise bout and that this adaptation is similar in magnitude in younger and middle-aged men.     

Thymic hormone-containing cells. V. Immunohistological detection of metallothionein within the cells bearing thymulin (a zinc-containing hormone) in human and mouse thymuses

Using an immunofluorescence (IF) assay, the presence of metallothionein (MT) was investigated in sections of normal and pathologic human thymuses as well as in cultures of thymic epithelial cells. This protein, known to have a high binding affinity for class II B transitional metals, such as zinc, was detected in the epithelial component of the thymus. Moreover, double labeling experiments with the anti-MT and an anti-thymulin monoclonal antibody showed that all cells containing thymulin, a thymic hormone whose active structure is known to contain zinc, also exhibited large amounts of metallothionein. These results, together with the fact that zinc and thymulin have been detected in the same type of cell organelles, lead to the conclusion that the MT present in thymic epithelial cells might be involved in the mechanism of zinc storage in these cells, thus favoring the secretion of thymulin in its biologically active, zinc-containing form.     

Interactions of astringent substances

Two-component mixtures of astringent materials were rated forperceived intensity of astringent and taste attributes overtime. Components included alum (a complex salt), gallic acid(the monomeric component of hydrolyzable tannins), catechin(the monomeric component of condensed tannins) and citric acid.Mixtures of alum and gallic acid showed mixture suppression,in that the 50/50 mixture was less intense than either componentin astringency, drying, roughing and puckery/drawing sensations.Suppression was seen at concentration levels producing moderateto strong astringency but was absent or less pronounced at lowerconcentration levels. A similar pattern held for citric acid,although the suppressive effects were less pronounced. Catechinand gallic acid mixtures were additive. Sensory interactionsbetween astringent materials appears to depend on the substancesinvolved and their concentrations (or intensity levels).     

The neuroprotective activity of the amyloid precursor protein against traumatic brain injury is mediated via the heparin binding site in residues 96‐110

We have previously shown that following traumatic brain injury (TBI) the presence of the amyloid precursor protein (APP) may be neuroprotective. APP knockout mice have increased neuronal death and worse cognitive and motor outcomes following TBI, which is rescued by treatment with exogenous sAPPα (the secreted ectodomain of APP generated by α‐secretase cleavage). Two neuroprotective regions were identified in sAPPα, the N and C‐terminal domains D1 and D6a/E2 respectively. As both D1 and D6a/E2 contain heparin binding activity it was hypothesized that this is responsible for the neuroprotective activity. In this study, we focused on the heparin binding site, encompassed by residues 96‐110 in D1, which has previously been shown to have neurotrophic properties. We found that treatment with APP96‐110 rescued motor and cognitive deficits in APP?/? mice following focal TBI. APP96‐110 also provided neuroprotection in Sprague–Dawley rats following diffuse TBI. Treatment with APP96‐110 significantly improved functional outcome as well as preserve histological cellular morphology in APP?/? mice following focal controlled cortical impact injury. Furthermore, following administration of APP96‐110 in rats after diffuse impact acceleration TBI, motor and cognitive outcomes were significantly improved and axonal injury reduced. These data define the heparin binding site in the D1 domain of sAPPα, represented by the sequence APP96‐110, as the neuroprotective site to confer neuroprotection following TBI.