Optimization of polyphosphate degradation and phosphate secretion using hybrid metabolic pathways and engineered host strains

Polyphosphate degradation and phosphate secretion were optimized in Escherichia coli strains overexpressing the E. coli polyphosphate kinase gene (ppk) and either the E. coli polyphosphatase gene (ppx) or the Saccharomyces cerevisiae polyphosphatase gene (scPPX1) from different inducible promoters on medium- and high-copy plasmids. The use of a host strain without functional ppk or ppx genes on the chromosome yielded the highest levels of polyphosphate, as well as the fastest degradation of polyphosphate when the gene for polyphosphatase was induced. The introduction of a hybrid metabolic pathway consisting of the E. coli ppk gene and the S. cerevisiae polyphosphatase gene resulted in lower polyphosphate concentrations than when using both the ppk and ppx genes from E. coli, and did not significantly improve the degradation rate. It was also found that the rate of polyphosphate degradation was highest when ppx was induced late in growth, most likely due to the high intracellular polyphosphate concentration. The phosphate released from polyphosphate allowed the growth of phosphate-starved cells; excess phosphate was secreted into the medium, leading to a down-regulation of the phosphate-starvation (Pho) response. The production of alkaline phosphatase, an indicator of the Pho response, can be precisely controlled by manipulating the degree of ppx induction. Copyright 1998 John Wiley & Sons, Inc.     

Regulation of Glial Cell Line-Derived Neurotrophic Factor Release from Rat C6 Glioblastoma Cells

Abstract: We have monitored glial cell line-derived neurotrophic factor (GDNF) secretion from rat C6 glioblastoma cells by ELISA. Representative cytokines, neurotrophins, growth factors, neuropeptides, and pharmacological agents were tested for their ability to modulate GDNF release. Whereas most factors tested had minimal effect, a 24-h treatment with fibroblast growth factor-1, −2, or −9 elevated secreted GDNF protein levels five- to 10-fold. The proinflammatory cytokines interleukin-1β, interleukin-6, tumor necrosis factor-α, and lipopolysaccharide elevated GDNF release 1.5- to twofold. Parallel studies aimed at elucidating intracellular events that may regulate GDNF synthesis/release demonstrated the involvement of multiple signaling pathways. GDNF levels were increased by phorbol 12,13-didecanoate (10 n M ) activation of protein kinase C, the Ca²⁺ ionophore A23187 (1 µ M ), okadaic acid (10 n M ) inhibition of type-2A protein phosphatases, nitric oxide donors (1 m M ), and H₂O₂ (1 m M )-induced oxidative stress. Elevation of cyclic AMP levels by either forskolin (10 µ M ) or dibutyryl cyclic AMP (1 m M ) repressed GDNF secretion, as did treatment with the glucocorticoid dexamethasone (1 µ M ). Our results demonstrate that diverse biological factors are capable of modulating GDNF protein levels and that multiple signal transduction systems can regulate GDNF synthesis and/or release.     

The self-incompatibility phenotype in brassica is altered by the transformation of a mutant S locus receptor kinase

The self-incompatible (SI) Brassica napus line W1, which carries the 910 S allele, was transformed with an inactive copy of the 910 S locus receptor kinase (SRK) gene. Two transformed lines were analyzed based on their heritable ability to set self-seed. The first line was virtually completely self-compatible (SC), and reciprocal pollinations with the original W1 line demonstrated that only the stigma side of the SI phenotype was altered. An analysis of the expression of endogenous SRK-910 demonstrated that the mechanism of transgene action is via gene suppression. Furthermore, the expression of the S locus glycoprotein gene present in the 910 allele (SLG-910), SLG-A10, which is derived from a nonfunctional S allele, and an S locus-related gene were also suppressed. When the transgene was crossed into another SI line carrying the A14 S allele, it was also capable of suppressing the expression of the endogenous genes and of making this line SC. The second transgenic line studied was only partly SC. In this case as well, only the stigma phenotype was affected, although no gene suppression was detected for endogenous SRK-910 or SLG-910. In this line, the expression of the transgene most likely was causing the change in phenotype, and no effect was observed when this transgene was crossed into the other SI line. Therefore, this work reinforces the hypothesis that the SRK gene is required, but only for the stigma side of the SI phenotype, and that a single transgene can alter the SI phenotype of more than one S allele.     

Combined Utilisation of Rapid Assessment Procedures for Loiasis (RAPLOA) and Onchocerciasis (REA) in Rain forest Villages of Cameroon

BACKGROUND: Individuals with high microfilarial loads of Loa loa are at increased risk of neurologic serious adverse (SAE) events following ivermectin treatment against onchocerciasis. RAPLOA (Rapid Assessment Procedure for loiasis), a newly developed rapid assessment procedure for loiasis that relates the prevalence of key clinical manifestation of loiasis (history of eye worm) to the level of endemicity of the infection (prevalence of high intensity), is a very useful tool to identify areas at potential risk of L. loa post ivermectin treatment encephalopathy. In a perspective of treatment decision making in areas of co-endemicity of loiasis/onchocerciasis, it would be advantageous (both in time and cost savings) for national onchocerciasis control programmes to use RAPLOA and the Rapid epidemiologic assessment for onchocerciasis (REA), in combination in given surveys. Since each of the two rapid assessment tools have their own specificities, the workability of combining the two methods needed to be tested. METHODS: We worked in 10 communities of a forest area presumed co-endemic for loiasis and onchocerciasis in the North-West Province of Cameroon where the mass-treatment with ivermectin had not been carried out. A four-step approach was used and comprised: (i) generating data on the prevalence and intensity of loiasis and onchocerciasis in an area where such information is scarce; (ii) testing the relationship between the L. loa microfilaraemia prevalence and the RAPLOA prevalence, (iii) testing the relationship between the O. volvulus microfiladermia prevalence and the REA prevalence, (iv) testing the workability of combining RAPLOA/REA by study teams in which a single individual can perform the interview for RAPLOA and the nodule palpation for REA. RESULTS: The microfilaraemia prevalence of loiasis in communities ranged from 3.6% to 14.3%. 6 (0.61%) individuals had L. loa microfilarial loads above 8000 mf/ml but none of them attained 30,000 mf/ml, the threshold value above which the risk of developing neurologic SAE after ivermectin treatment is very high. None of the communities surveyed had RAPLOA prevalence above 40%. All the communities had microfiladermia prevalence above 60%. The microfiladermia results could be confirmed by the rapid epidemiologic method (nodule palpation), with all the 10 communities having REA prevalence above 20%. For the first time, this study has demonstrated that the two rapid assessment procedures for loiasis and onchocerciasis can be carried out simultaneously by a survey team, in which a single individual can administer the questionnaire for RAPLOA and perform the nodule palpation for REA. CONCLUSION: This study has: (i) Revealed that the Momo valley of the North West province of Cameroon is hyperendemic for onchocerciasis, but is of lower level of endemicity for L. loa. (ii) Confirmed the previous relationships established between RAPLOA and the L. loa microfilaraemia prevalence in one hand and between the REA and the O. volvulus microfiladermia prevalence in another hand (iii) Shown that RAPLOA and REA could be used simultaneously for the evaluation of loiasis and onchocerciasis endemicity in areas targeted by the African Programme for onchocerciasis Control for community-directed treatment with ivermectin (CDTI).     

Mapping by waves. Patterned spontaneous activity regulates retinotopic map refinement

A role for spontaneous spiking activity in shaping neuronal circuits has frequently been debated. Analyses of retinotopy in mutant mice with reduced correlated firing among neighboring retinal cells by Grubb et al. and McLaughlin et al. in this issue of Neuron indicate the importance of patterned spontaneous activity for retinotopic map refinement in subcortical visual targets.     

Harmane and harmalan are bioactive components of classical clonidine-displacing substance

Elucidation of the structure of the endogenous ligand(s) for imidazoline binding sites, clonidine-displacing substance (CDS), has been a major goal for many years. Crude CDS from bovine lung was purified by reverse-phase high-pressure liquid chromatography. Electrospray mass spectrometry (ESMS) and nuclear magnetic resonance ((1)H NMR) analysis revealed the presence of L-tryptophan and 1-carboxy-1-methyltetrahydro-beta-carboline in the active CDS extract. Competition radioligand binding studies, however, failed to show displacement of specific [(3)H]clonidine binding to rat brain membranes for either compound. Further purification of the bovine lung extract allowed the isolation of the beta-carbolines harmane and harmalan as confirmed by ESMS, (1)H NMR, and comparison with synthetic standards. Both compounds exhibited a high (nanomolar) affinity for both type 1 and type 2 imidazoline binding sites, and the synthetic standards were shown to coelute with the active classical CDS extracts. We therefore propose that the beta-carbolines harmane and harmalan represent active components of classical CDS. The identification of these compounds will allow us to establish clear physiological roles for CDS.     

Muscarinic receptors and gastrointestinal tract smooth muscle function

Over the last decade, several lines of evidence have shown that both muscarinic M2 and M3 receptors are postjunctionally expressed in many smooth muscles, including the gastrointestinal tract. Although in vitro data suggests that both receptors are functional in that they inhibit adenylate cyclase activity and activate non-selective cation channels, few studies support a role in vivo. Thus, data from procedures that ablate the signaling pathway of the muscarinic M2 receptor, including receptor antagonism, pertussis toxin pretreatment reveal little effect on gastrointestinal smooth muscle responsiveness to muscarinic agonists. Recently, information from knockout mice, lacking either M2 or M3 receptor, indicate reveal a role for both subtypes. However, the contribution of the M2 receptor appears greater in the ileum than in the urinary bladder. Therapeutically, non-selective, as well as selective M3 receptor antagonists are being clinically studied, although it remains to be shown which is the optimal approach to disorders of smooth muscle motility.