Glycolytic breakdown of sulfoquinovose in bacteria: a missing link in the sulfur cycle

Sulfoquinovose (6-deoxy-6-sulfo-D-glucopyranose), formed by the hydrolysis of the plant sulfolipid, is a major component of the biological sulfur cycle. However, pathways for its catabolism are poorly delineated. We examined the hypothesis that mineralization of sulfoquinovose to inorganic sulfate is initiated by reactions of the glycolytic and/or Entner-Doudoroff pathways in bacteria. Metabolites of [U-(13)C]sulfoquinovose were identified by (13)C-nuclear magnetic resonance (NMR) in strains of Klebsiella and Agrobacterium previously isolated for their ability to utilize sulfoquinovose as a sole source of carbon and energy for growth, and cell extracts were analyzed for enzymes diagnostic for the respective pathways. Klebsiella sp. strain ABR11 grew rapidly on sulfoquinovose, with major accumulations of sulfopropandiol (2,3-dihydroxypropanesulfonate) but no detectable release of sulfate. Later, when sulfoquinovose was exhausted and growth was very slow, sulfopropandiol disappeared and inorganic sulfate and small amounts of sulfolactate (2-hydroxy-3-sulfopropionate) were formed. In Agrobacterium sp. strain ABR2, growth and sulfoquinovose disappearance were again coincident, though slower than that in Klebsiella sp. Release of sulfate was still late but was faster than that in Klebsiella sp., and no metabolites were detected by (13)C-NMR. Extracts of both strains grown on sulfoquinovose contained phosphofructokinase activities that remained unchanged when fructose 6-phosphate was replaced in the assay mixture with either glucose 6-phosphate or sulfoquinovose. The results were consistent with the operation of the Embden-Meyerhoff-Parnas (glycolysis) pathway for catabolism of sulfoquinovose. Extracts of Klebsiella but not Agrobacterium also contained an NAD(+)-dependent sulfoquinovose dehydrogenase activity, indicating that the Entner-Doudoroff pathway might also contribute to catabolism of sulfoquinovose.     

Hand and body position during locomotor behavior in the aye-aye (Daubentonia madagascariensis)

Aye-ayes (Daubentonia madagascariensis) have unique hands among primates, with extraordinarily long fingers in relation to body size. These long digits may be vulnerable to damage from forces during locomotion, particularly during head-first descent-a locomotor mode that the aye-aye utilizes frequently. Previous behavioral studies of aye-aye locomotion reported that Daubentonia must curl its fingers during horizontal quadrupedalism and/or descent to reduce potential stresses on its long fingers. To test this hypothesis, we examined hand and body position in three captive adult aye-ayes while they walked quadrupedally on horizontal and oblique branches. Substantial variation in hand position was observed among individuals for each substrate orientation. While hand postures with curled fingers were preferred by one individual during descent, they were not preferred by the other two individuals, contrary to our expectations. Differences in body position were more consistent among all three individuals. The angle of the body relative to the substrate was significantly reduced during descent (8.4 degrees ) compared to horizontal locomotion (16.9 degrees ). These results suggest that changes in body position, rather than hand position, may help reduce stresses on the digits. A biomechanical model is proposed that demonstrates how a reduction in the body angle in relation to substrate may act to move the center of mass more caudally. This mechanism of moderating loads by altering body position, rather than hand position, may represent an important functional aspect of arboreal locomotion in aye-ayes and other primates.     

Marine bacteria cause false-positive results in the Colilert-18 rapid identification test for Escherichia coli in Florida waters

The Colilert-18 system for enumeration of total coliforms and Escherichia coli is approved by the U.S. Environmental Protection Agency for use in drinking water analysis and is also used by various agencies and research studies for enumeration of indicator organisms in fresh and saline waters. During monitoring of Pinellas County, Fla., marine waters, estimates of E. coli numbers (by Colilert-18) frequently exceeded fecal coliform counts (by membrane filtration) by 1 to 3 orders of magnitude. Samples from freshwater sites did not display similar discrepancies. Fecal coliforms, including E. coli, could be cultured from 100% of yellow fluorescent wells (denoting E. coli-positive results) inoculated with freshwater samples but could be cultured from only 17.1% of the "positive" wells inoculated with marine samples. Ortho-nitrophenyl-beta-D-galactopyranoside (ONPG)-positive or 4-methylumbelliferyl-beta-D-glucuronide (MUG)-positive noncoliform bacteria were readily cultured from Colilert-18 test wells inoculated with marine samples. Filtered cell-free seawater did not cause false positives. Coculture preparations of as few as 5 CFU of Vibrio cholerae (ONPG positive) and Providencia sp. (MUG positive) ml(-1) inoculated into Colilert-18 caused false-positive E. coli results. Salinity conditions influenced coculture results, as the concentration of coculture inoculum required to cause false positives in most wells increased from about 5 CFU ml(-1) in seawater diluted 1:10 with freshwater to approximately equal to 5,000 CFU ml(-1) in seawater diluted 1:20 with freshwater. Estimated E. coli numbers in various marine water samples processed at the 1:10 dilution ranged from 10 to 7,270 CFU.100 ml(-1), while E. coli numbers in the same samples processed at the 1:20 dilution did not exceed 40 CFU.100 ml(-1). The lower estimates of E. coli numbers corresponded well with fecal coliform counts by membrane filtration. This study indicates that assessment of E. coli in subtropical marine waters by Colilert-18 is not accurate when the recommended 1:10 sample dilution is used. The results suggest that greater dilution may diminish the false-positive problem, but further study of this possibility is recommended.     

Receptor-dependent and tyrosine phosphatase-mediated inhibition of GSK3 regulates cell fate choice

Asymmetric body axis formation is central to metazoan development. Dictyostelium establishes an anterior/posterior axis utilizing seven-transmembrane cAMP morphogen receptors (CARs) and GSK3-mediated signal transductions that has a parallel with metazoan Wnt/Frizzled-GSK3 pathways. In Dictyostelium, GSK3 promotes posterior cell patterning but inhibits anterior cell differentiation. Tyrosine kinase ZAK1 mediates GSK3 activation. We now show that CAR4 regulates a tyrosine phosphatase that inhibits GSK3 activity. We have also identified essential phosphotyrosines in GSK3, confirmed their role in activated/deactivated regulation and cell fate decisions, and relate them to the predicted 3D structure of GSK3beta. CARs differentially regulate GSK3 activity by selectively activating a tyrosine phosphatase or kinase for pattern formation. The findings may provide a comparative understanding of CAR-GSK3 and Wnt/Frizzled-GSK3 pathways.     

Regulation of osteoclast differentiation by statins

HMG-CoA reductase inhibitor (statin) treatment is frontline therapy for lowering plasma cholesterol levels in patients with hyperlipidemia. In a few case studies, analysis of clinical data has revealed a decreased risk of fracture in patients on statin therapy. However, this reduction in the incidence of fracture is not always observed nor is it supported by an increase in bone density, which further complicates our understanding of the role of statins in bone metabolism. Thus, the precise role of statins in bone metabolism remains poorly understood. In this study, we examined the effect of statin treatment on osteoclastogenesis. Treatment with lovastatin resulted in a significant, dose-dependent decrease in the numbers of differentiated osteoclasts and decreased cholesterol biosynthesis activity with an EC(50) similar to that observed in freshly isolated rat or cultured human liver cells. Studies assessing the role of mevalonate metabolites in the development of the osteoclasts demonstrated that geranylgeraniol, but not squalene or farnesol was important for the development and differentiation of osteoclasts, implicating protein geranylgeranylation rather than protein farnesylation as a key factor in the osteoclast differentiation process. In conclusion, our data indicate that lovastatin inhibits osteoclast development through inhibition of geranylgeranylation of key prenylated proteins and that the bone effects of statins are at least partially due to their effects on osteoclast numbers.     

Glycogen synthase kinase-3 inhibition by lithium and beryllium suggests the presence of two magnesium binding sites.

Lithium inhibits (Li(+)) glycogen synthase kinase-3 (GSK-3) by competition for magnesium (Mg(2+)), but not ATP or substrate. Here, we show that the group II metal ion beryllium (Be(2+)) is a potent inhibitor of GSK-3 and competes for both Mg(2+) and ATP. Be(2+) also inhibits the related protein kinase cdc2 at similar potency, but not MAP kinase 2. To compare the actions of Li(+) and Be(2+) on GSK-3, we have devised a novel dual inhibition analysis. When Be(2+) and ADP are present together each interferes with the action of the other, indicating that both agents inhibit GSK-3 at the ATP binding site. In contrast, Li(+) exerts no interference with ADP inhibition or vice versa. We find, however, that Li(+) and Be(2+) do interfere with each other. These results suggest that Be(2+) competes for two distinct Mg(2+) binding sites: one is Li(+)-sensitive and the other, which is Li(+)-insensitive, binds the Mg:ATP complex.     

Charged amino acids conserved in the aromatic acid/H+ symporter family of permeases are required for 4-hydroxybenzoate transport by PcaK from Pseudomonas putida

Charged amino acids in the predicted transmembrane portion of PcaK, a permease from Pseudomonas putida that transports 4-hydroxybenzoate (4-HBA), were required for 4-HBA transport, and they were also required for P. putida to have a chemotactic response to 4-HBA. An essential amino acid motif (DGXD) containing aspartate residues is located in the first transmembrane segment of PcaK and is conserved in the aromatic acid/H+ symporter family of the major facilitator superfamily of transporters.     

Genetic structure of Anopheles gambiae (Diptera: Culicidae) in São Tomé and Príncipe (West Africa): implications for malaria control

The impact of a vector eradication programme, conducted in the 1980s, on Anopheles gambiae populations from the islands of São Tomé and Príncipe, was evaluated by microsatellite DNA analysis. Significant genetic differentiation was observed within and between the two islands and between the islands and a population from Gabon, suggesting a degree of isolation between them. Large estimates of long‐term N_e suggested that the control programme did not affect the effective population size of the vector. Heterozygosity tests were also not consistent with a recent bottleneck.     

Dd-Alix, a conserved endosome-associated protein, controls Dictyostelium development

We have characterized the Dictyostelium homolog of the mammalian protein Alix. Dd-Alix is encoded by a single gene and is expressed during vegetative growth and multicellular development. We showed that the alx null strain fails to complete its developmental program. Past the tight aggregate stage, morphogenesis is impaired, leading to markedly aberrant structures containing vacuolated and undifferentiated cells but no mature spores. The developmental defect is cell-autonomous as most cells remain of the PstB type even when mixed with wild-type cells. Complementation analysis with different Alix constructs allowed the identification of a 101-residue stretch containing a coiled-coil domain essential for Alix function. In addition, we showed that the protein associates in part with vesicular structures and that its distribution on a Percoll gradient overlaps that of the endocytic marker Vamp7. Dd-Alix also co-localizes with Dd-Vps32. In view of our data, and given the role of Vps32 proteins in membrane protein sorting and multivesicular body formation in yeast and mammals, we hypothesize that the developmental defects of the alx null strain result from abnormal trafficking of cell-surface receptors.     

Inhibition of mammalian target of rapamycin activates apoptosis signal-regulating kinase 1 signaling by suppressing protein phosphatase 5 activity

Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces a cellular stress response characterized by rapid and sustained activation of the apoptosis signal-regulating kinase 1 (ASK1) signaling pathway and selective apoptosis of cells lacking functional p53. Here we have investigated how mTOR regulates ASK1 signaling using p53-mutant rhabdomyosarcoma cells. In Rh30 cells, ASK1 was found to physically interact with protein phosphatase 5 (PP5), previously identified as a negative regulator of ASK1. Rapamycin did not affect either protein level of PP5 or association of PP5 with ASK1. Instead, rapamycin caused rapid dissociation of the PP2A-B" regulatory subunit (PR72) from the PP5-ASK1 complex, which was associated with reduced phosphatase activity of PP5. This effect was dependent on expression of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Down-regulation of PP5 activity by rapamycin coordinately activated ASK1, leading to elevated phosphorylation of c-Jun. Amino acid deprivation, which like rapamycin inhibits mTOR signaling, also inhibited PP5 activity, caused rapid dissociation of PR72, and activated ASK1 signaling. Overexpression of PP5, but not the PP2A catalytic subunit, blocked rapamycin-induced phosphorylation of c-Jun, and protected cells from rapamycin-induced apoptosis. The results suggest that PP5 is downstream of mTOR, and positively regulated by the mTOR pathway. The findings suggest that in the absence of serum factors, mTOR signaling suppresses apoptosis through positive regulation of PP5 activity and suppression of cellular stress.