As procalcitonin concentrations have been shown to be elevated in patients with septicemia and gram-negative infections in particular, we proceeded to investigate the effect of endotoxin, a product of gram-negative bacteria, on procalcitonin concentrations in normal human volunteers. Endotoxin from Escherichia coli 0113:H10:k, was injected i.v. at a dose of 4 mg/kg BW into these healthy volunteers. Blood samples were obtained before and 1, 2, 4, 6, 8, and 24 h after injection of the endotoxin. Each patient's cardiovascular and overall clinical status was monitored over this period. The patients developed chills and rigors, myalgia, and fever between 1-3 h. Tumor necrosis factor-alpha levels increased sharply at 1 h and peaked at 90 min, reaching the baseline concentration thereafter by 6 h. Interleukin-6 levels increased more gradually, peaking at 3 h and reaching the baseline concentration at 8 h. The procalcitonin concentration, which was undetectable (< 10 pg/mL) at 0, 1, and 2 h, was detectable at 4 h and peaked at 6 h, maintaining a plateau through 8 and 24 h (4 ng/mL). There was no elevation of calcitonin concentrations, which remained below 10 pg/mL, the lowest sensitivity of the assay. Procalcitonin was measured by a two-antibody immunoradiometric assay specific for this peptide, with no cross-reactivity with calcitonin, katacalcin, or calcitonin gene-related peptide. We conclude that endotoxin induces the release of procalcitonin systemically, that this increase is not associated with an increase in calcitonin, and that the increase in procalcitonin associated with septicemia in patients may be mediated through the effect of endotoxin described here. Whether procalcitonin participates in the mechanisms underlying inflammation remains to be investigated.
Transcriptional feedback loops are a feature of circadian clocks in both animals and plants. We show that the plant circadian clock also incorporates the cytosolic signaling molecule cyclic adenosine diphosphate ribose (cADPR). cADPR modulates the circadian oscillator's transcriptional feedback loops and drives circadian oscillations of Ca2+ release. The effects of antagonists of cADPR signaling, manipulation of cADPR synthesis, and mathematical simulation of the interaction of cADPR with the circadian clock indicate that cADPR forms a feedback loop within the plant circadian clock.
During selenate respiration by Thauera selenatis, the reduction of selenate results in the formation of intracellular selenium (Se) deposits that are ultimately secreted as Se nanospheres of approximately 150 nm in diameter. We report that the Se nanospheres are associated with a protein of approximately 95 kDa. Subsequent experiments to investigate the expression and secretion profile of this protein have demonstrated that it is up-regulated and secreted in response to increasing selenite concentrations. The protein was purified from Se nanospheres, and peptide fragments from a tryptic digest were used to identify the gene in the draft T. selenatis genome. A matched open reading frame was located, encoding a protein with a calculated mass of 94.5 kDa. N-terminal sequence analysis of the mature protein revealed no cleavable signal peptide, suggesting that the protein is exported directly from the cytoplasm. The protein has been called Se factor A (SefA), and homologues of known function have not been reported previously. The sefA gene was cloned and expressed in Escherichia coli, and the recombinant His-tagged SefA purified. In vivo experiments demonstrate that SefA forms larger (approximately 300 nm) Se nanospheres in E. coli when treated with selenite, and these are retained within the cell. In vitro assays demonstrate that the formation of Se nanospheres upon the reduction of selenite by glutathione are stabilized by the presence of SefA. The role of SefA in selenium nanosphere assembly has potential for exploitation in bionanomaterial fabrication.nanoparticles | biomineralization | anaerobic respiration
Biofuels are the most immediate, practical solution for mitigating dependence on fossil hydrocarbons, but current biofuels (alcohols and biodiesels) require significant downstream processing and are not fully compatible with modern, mass-market internal combustion engines. Rather, the ideal biofuels are structurally and chemically identical to the fossil fuels they seek to replace (i.e., aliphatic n-and iso-alkanes and -alkenes of various chain lengths). Here we report on production of such petroleum-replica hydrocarbons in Escherichia coli. The activity of the fatty acid (FA) reductase complex from Photorhabdus luminescens was coupled with aldehyde decarbonylase from Nostoc punctiforme to use free FAs as substrates for alkane biosynthesis. This combination of genes enabled rational alterations to hydrocarbon chain length (C n ) and the production of branched alkanes through upstream genetic and exogenous manipulations of the FA pool. Genetic components for targeted manipulation of the FA pool included expression of a thioesterase from Cinnamomum camphora (camphor) to alter alkane C n and expression of the branched-chain α-keto acid dehydrogenase complex and β-keto acyl-acyl carrier protein synthase III from Bacillus subtilis to synthesize branched (iso-) alkanes. Rather than simply reconstituting existing metabolic routes to alkane production found in nature, these results demonstrate the ability to design and implement artificial molecular pathways for the production of renewable, industrially relevant fuel molecules.branched fatty acid biosynthesis | lux genes | metabolic engineering | synthetic biology
We have tested the hypothesis that circadian oscillations in the concentration of cytosolic free calcium ([Ca 21 ] cyt ) can encode information. We imaged oscillations of [Ca 21 ] cyt in the cotyledons and leaves of Arabidopsis (Arabidopsis thaliana) that have a 24-h period in light/dark cycles and also constant light. The amplitude, phase, and shape of the oscillations of [Ca 21 ] cyt and [Ca 21 ] cyt at critical daily time points were controlled by the light/dark regimes in which the plants were grown. These data provide evidence that 24-h oscillations in [Ca 21 ] cyt encode information concerning daylength and light intensity, which are two major regulators of plant growth and development.
Plants have circadian oscillations in the concentration of cytosolic free calcium ([Ca 2þ ] cyt ] cyt and CAB2 promoter activity are uncoupled in the timing of cab1 (toc1-1) mutant but not in toc1-2. We suggest that the circadian oscillations of [Ca 2þ ] cyt and CAB2 promoter activity are regulated by distinct oscillators with similar components that are used in a different manner and that these oscillators may be located in different cell types in Arabidopsis.
To investigate the endoplasmic reticulum (ER) Ca 2ϩ stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca 2ϩ -binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca 2ϩ uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent 45 Ca 2ϩ accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca 2ϩ ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of 45 Ca 2ϩ released, and a 2-to 3-fold increase in the amount of 45 Ca 2ϩ retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca 2ϩ pool. In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca 2ϩ -containing medium to Ca 2ϩ -depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca 2ϩ stores and thereby enhances the survival of plants grown in low Ca 2ϩ medium.
To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP 3 ) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP 3 . The transgenic cell lines showed a 12-to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP 3 compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP 3 in both wild-type and transgenic cells. However, even with stimulation, InsP 3 levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP 3 signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ), the lipid precursor of InsP 3 , was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP 2 metabolism showed that the activity of the PtdInsP 2 -hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of 32 P into PtdInsP 2 in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP 2 synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP 2 synthesis as a regulatory step in this system.In plants the phosphoinositide (PI) pathway has been implicated in the transduction of signals after a multitude of stimuli (Drøbak, 1992; Munnik et al., 1998a;Stevenson et al., 2000). Upon stimulation, the membrane-associated phospholipid, phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ), is hydrolyzed by phospholipase C (PLC) to produce inositol 1,4,5-trisphosphate (InsP 3 ) and diacylglycerol (Berridge, 1993). InsP 3 can act as a soluble second messenger to mediate the release of Ca 2ϩ from intracellular stores, such as the vacuole (for review, see Sanders et al., 1999). Furthermore, because InsP 3 can travel between cells through plasmodesmatal connections (Tucker and Boss, 1996), it fulfills the requirements for a rapidly diffusible signaling molecule in both intraand intercellular signal propagation.Rapid transient increases in InsP 3 have been demonstrated in various plant tissues in response to environmental stimuli and chemical effectors, including hyperosmotic stress (Srivastava et al., 1989; Heilmann et al., 1999 Heilmann et al., , 2001 DeWald et al., 2001;Takahashi et al., 2001), salinity (Drøbak and Watkins, 2000), cold shock (Smolenska-Sym and Kacperska, 1996), oligogalacturonide elicitors (Legendre et al., 1993), fusicoccin (Aducci and Marra, 1990), mastoparan (Legendre et al., 1993; Drøbak and Watkins, 1994; Cho et a...
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