Effects of methylmalonyl-CoA mutase gene knockouts on erythromycin production in carbohydrate-based and oil-based fermentations of Saccharopolyspora erythraea

In carbohydrate-based fermentations of Saccharopolyspora erythraea, a polar knockout of the methylmalonyl-CoA mutase (MCM) gene, mutB, improved erythromycin production an average of 126% (within the range of 102–153% for a 0.95 confidence interval). In oil-based fermentations, where erythromycin production by the wild-type strain averages 184% higher (141–236%, 0.95 CI) than in carbohydrate-based fermentations, the same polar knockout in mutB surprisingly reduced erythromycin production by 66% (53–76%, 0.95 CI). A metabolic model is proposed where in carbohydrate-based fermentations MCM acts as a drain on the methylmalonyl-CoA metabolite pool, and in oil-based fermentations, MCM acts in the reverse direction to fill the methylmalonyl-CoA pool. Therefore, the model explains, in part, how the well-known oil-based process improvement for erythromycin production operates at the biochemical level; furthermore, it illustrates how the mutB erythromycin strain improvement mutation operates at the genetic level in carbohydrate-based fermentations.     

From Transient to Steady-state Response of Ecosystems to Atmospheric CO2-enrichment and Global Climate Change: Conceptual Challenges and Need for an Integrated Approach

Evidence continues to accumulate that humans are significantly increasing atmospheric CO₂ concentrations, resulting in unprecedented changes in the global climate system. Experimental manipulations of terrestrial ecosystems and their components have greatly increased our understanding of short-term responses to these global perturbations and have provided valuable input to ecosystem, dynamic vegetation, and global scale models. However, concerns exist that these initial experimental responses may be transitory, thereby limiting our ability to extrapolate short-term experimental responses to infer longer-term effects. To do these extrapolations, it will be necessary to understand changes in response patterns over time, including alterations in the magnitude, direction, and rate of change of the responses. These issues represent one of our largest challenges in accurately predicting longer-term changes in ecosystems and associated feedbacks to the climate system. Key issues that need to be considered when designing future experiments or refining models include: linear vs. non-linear responses, direct vs. indirect effects, lags in response, acclimation, resource limitation, homeostasis, buffers, thresholds, ecosystem stoichiometry, turnover rates and times, and alterations in species composition. Although experimental and landscape evidence for these response patterns exist, extrapolating longer-term response patterns from short-term experiments will ultimately require a unified multidisciplinary approach, including better communication and collaboration between theoreticists, experimentalists and modelers.     

The cardiovascular responses to mu opioid agonist and antagonist in conscious normal and obese rats

Beta-endorphin decreases blood pressure in normal rats but increases blood pressure in obese rats. Since beta-endorphins can bind both mu opioid and kappa-opioid receptors we investigated the effect of a mu specific receptor agonist, D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and a mu specific antagonist, D-Phe-Cys-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) on cardiovascular responses in conscious control and obese rats. Rats were also implanted with telemetry transmitters and intracerebroventricular (ICV) cannulas for recording and peptide administration. The mu agonist, DAMGO, increased blood pressure (BP) in control rats. DAMGO also increased BP in obese rats but only at high concentrations. The heart rate responses paralleled the MAP responses. CTAP, the mu antagonist, paradoxically increased the MAP in both control and obese rats. The responsiveness to the mu agonist and antagonist was greater in controls. In other animals the brains were excised and the ventral medial hypothalamic area removed and mu receptor expression determined using PCR. The expression of mu opioid receptors was increased in obese rats. We conclude that the mu opioids can stimulate cardiovascular responses, but the excitatory responsiveness was not increased in conscious obese rats.     

Triumphalone, a diketone from the volatile oil of the leaves of Melaleuca triumphalis, and its spontaneous conversion into isotriumphalone

The major component (35-65%) of the volatile oil obtained by steam distillation of the leaves of Melaleuca triumphalis has been identified as (rel)-1beta-pentyl-1alpha,6alpha-dihydroxy-3,3,5,5-tetramethylcyclohexa-2,4-dione (trivial name triumphalone). Relative stereochemistry was established by nuclear Overhauser experiments and X-ray studies on the 2-(3,5-dinitrobenzoic acid) derivative. The remainder of the oil was composed of mono- and sesquiterpene hydrocarbons and alcohols. On prolonged standing the presence of a rearrangement product of triumphalone was observed which was characterized as (rel)-1beta-pentyl-1alpha,3alpha-dihydroxy-4,4,6,6-tetramethylcyclohexa-2,5-dione (trivial name isotriumphalone), presumably arising from an acid catalyzed shift of the pentyl group from C-1 to C-2.     

Response of the leaf cell wall to desiccation in the resurrection plant Myrothamnus flabellifolius

The Myrothamnus flabellifolius leaf cell wall and its response to desiccation were investigated using electron microscopic, biochemical, and immunocytochemical techniques. Electron microscopy revealed desiccation-induced cell wall folding in the majority of mesophyll and epidermal cells. Thick-walled vascular tissue and sclerenchymous ribs did not fold and supported the surrounding tissue, thereby limiting the extent of leaf shrinkage and allowing leaf morphology to be rapidly regained upon rehydration. Isolated cell walls from hydrated and desiccated M. flabellifolius leaves were fractionated into their constituent polymers and the resulting fractions were analyzed for monosaccharide content. Significant differences between hydrated and desiccated states were observed in the water-soluble buffer extract, pectin fractions, and the arabinogalactan protein-rich extract. A marked increase in galacturonic acid was found in the alkali-insoluble pectic fraction. Xyloglucan structure was analyzed and shown to be of the standard dicotyledonous pattern. Immunocytochemical analysis determined the cellular location of the various epitopes associated with cell wall components, including pectin, xyloglucan, and arabinogalactan proteins, in hydrated and desiccated leaf tissue. The most striking observation was a constitutively present high concentration of arabinose, which was associated with pectin, presumably in the form of arabinan polymers. We propose that the arabinan-rich leaf cell wall of M. flabellifolius possesses the necessary structural properties to be able to undergo repeated periods of desiccation and rehydration.     

spe-10 encodes a DHHC-CRD zinc-finger membrane protein required for endoplasmic reticulum/Golgi membrane morphogenesis during Caenorhabditis elegans spermatogenesis

C. elegans spermatogenesis employs lysosome-related fibrous body-membranous organelles (FB-MOs) for transport of many cellular components. Previous work showed that spe-10 mutants contain FB-MOs that prematurely disassemble, resulting in defective transport of FB components into developing spermatids. Consequently, spe-10 spermatids are smaller than wild type and contain defective FB-MO derivatives. In this article, we show that spe-10 encodes a four-pass integral membrane protein that has a DHHC-CRD zinc-finger motif. The DHHC-CRD motif is found in a large, diverse family of proteins that have been implicated in palmitoyl transfer during protein lipidation. Seven spe-10 mutants were analyzed, including missense, nonsense, and deletion mutants. An antiserum to SPE-10 showed significant colocalization with a known marker for the FB-MOs during wild-type spermatogenesis. In contrast, the spe-10(ok1149) deletion mutant lacked detectable SPE-10 staining; this mutant lacks a spe-10 promoter and most coding sequence. The spe-10(eb64) missense mutation, which changes a conserved residue within the DHHC-CRD domain in all homologues, behaves as a null mutant. These results suggest that wild-type SPE-10 is required for the MO to properly deliver the FB to the C. elegans spermatid and the DHHC-CRD domain is essential for this function.     

Temporal kinetics and concentration-response relationships for induction of CYP1A, CYP2B, and CYP3A in primary cultures of beagle dog hepatocytes

Compared to other species, little information is available on the xenobiotic-induced regulation of cytochrome P450 enzymes in the beagle dog. Dogs are widely used in the pharmaceutical industry for many study types, including those that will impact decisions on compound progression. The purpose of this study was (1) to determine the temporal kinetics of drug-induced changes in canine CYP1A, CYP2B, and CYP3A mRNA and enzymatic activity, and (2) to characterize concentration-response relationships for CYP1A2, CYP2B11, and CYP3A12 using primary cultures of canine hepatocytes treated with beta-naphthoflavone (BNF), phenobarbital (PB), and rifampin (RIF), respectively. CYP1A1 and CYP1A2 mRNA exhibited maximal expression (12,700-fold and 206-fold, respectively) after 36 h of treatment with BNF. PB treatment, but not RIF treatment, caused maximal induction of CYP2B11 mRNA (149-fold) after 48 h of treatment. CYP3A12 and CYP3A26 mRNA levels were increased maximally after 72 h of treatment with PB and RIF (CYP3A12, 35-fold and 18-fold, and CYP3A26, 72-fold and 22-fold with PB and RIF treatment, respectively). Concentration-response relationships for BNF induced 7-ethoxyresorufin O-dealkylation (EROD) (EC(50) = 7.8 +/- 4.2 microM), PB induced 7-benzyloxyresorufin O-dealkylation (BROD) (EC(50) = 123 +/- 30 microM), and PB and RIF induced testosterone 6beta-hydroxylation (EC(50) = 132 +/- 28 microM and 0.98 +/- 0.16 microM) resembled the relationship for human CYP induction compared to that of rodent. Interestingly, RIF had no effect on CYP2B11 expression, which represents a species difference overlooked in previous investigations. Overall, the induction of dog CYP1A, CYP2B, and CYP3A exhibits characteristics that are intermediate to those of rodent and human.     

2-(Alkylthio)pyrimidin-4-ones as novel, reversible inhibitors of lipoprotein-associated phospholipase A2

Starting from two weakly active hits from high throughput screening, a novel series of 2-(alkylthio)-pyrimidin-4-ones with high potency and selectivity for lipoprotein-associated phospholipase A2 has been designed. In contrast to previously known inhibitors, these have been shown to act by a non-covalent and substrate competitive mechanism.     

NAD(P)H:quinone oxidoreductase 1 protects lungs from oxidant-induced emphysema in mice

Emphysema is currently a leading cause of mortality with no known effective therapy to attenuate progressive loss of lung function. Previous work supports that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is protective to the lung through induction of hundreds of antioxidant genes. In models of lung injury, the expression of NAD(P)H:quinone oxidoreductase 1 (NQO1) is upregulated in a manner dependent on Nrf2 and human emphysema is associated with reduced levels of NQO1. However, the functional role of NQO1 in emphysema remains unknown. In this study, we demonstrate the protective role of NQO1 in the development of emphysema using mouse models. NQO1-deficient animals demonstrated premature age-related emphysema and were more susceptible to both elastase and inhaled lipopolysaccharide models of emphysema. The absence of NQO1 was associated with enhanced markers of oxidant stress. Treatment of NQO1-deficient animals with the antioxidant N-acetylcysteine reversed the NQO1-dependent emphysematous changes. In vitro studies utilizing either inhibition or induction of NQO1 demonstrated a potent antioxidant role of NQO1 in macrophages, suggesting a role for macrophage-derived oxidants in the pathogenesis of emphysema. These novel findings support a functional role for NQO1 in protecting the lung from development of emphysema.