Thermal treatment of stabilized air pollution control residues in a waste incinerator pilot plant. Part 1: Fate of elements and dioxins.

Air pollution control (APC) residues from municipal solid waste incinerator plants that are treated by means of the Ferrox process can be more safely disposed of due to reduction of soluble salts and stabilization of heavy metals in an iron oxide matrix. Further stabilization can be obtained by thermal treatment inside a combustion chamber of a municipal solid waste incinerator. The influence of the Ferrox products on the combustion process, the quality of the residues, and the partitioning of heavy metals between the various solids and the gas have been investigated in the Karlsruhe TAM-ARA pilot plant for waste incineration. During the experiments only few parameters were influenced. An increase in the SO2 concentration in the raw gas and slightly lower temperatures in the fuel bed could be observed compared with reference tests. Higher contents of Fe and volatile heavy metals such as Zn, Cd, Pb and partly Hg in the Ferrox products lead to increased concentration of these elements in the solid residues of the co-feeding tests. Neither the burnout nor the PCDD/F formation was altered by the addition of the Ferrox products. Co-feeding of treated APC residues seems to be a feasible approach for obtaining a single solid residue from waste incineration.

Thermal treatment of stabilized air pollution control residues in a waste incinerator pilot plant. Part 2: Leaching characteristics of bottom ashes.

With the perspective of generating only one solid residue from waste incineration, co-feeding of municipal solid waste and air pollution control residues stabilized by the Ferrox process was investigated in the TAMARA pilot plant incinerator as described in Bergfeldt et al. (Waste Management Research, 22, 49-57, 2004). This paper reports on leaching from the combined bottom ashes. Batch leaching test, pH-static leaching tests, availability tests and column leaching tests were used to characterize the leaching properties. The leaching properties are key information in the context of reuse in construction or in landfilling of the combined residue. In general, the combined bottom ashes had leaching characteristics similar to the reference bottom ash, which contained no APC residue. However, As and Pb showed slightly elevated leaching from the combined bottom ashes, while Cr showed less leaching. The investigated combined bottom ashes had contents of metals comparable to what is expected at steady state after continuous co-feeding of APC residues. Only Cd and Pb were partly volatilized (30-40%) during the incineration process and thus the combined bottom ashes had lower contents of Cd and Pb than expected at steady state. Furthermore, a major loss of Hg was, not surprisingly, seen and co-feeding of Ferrox-products together with municipal solid waste will require dedicated removal of Hg in the flue gas to prevent a build up of Hg in the system. In spite of this, a combined single solid residue from waste incineration seems to be a significant environmental improvement to current technology.

[Ischemia of the brain stem caused by compression of the vertebral arteries by head rotation--an etiology for SIDS?]. / Minderperfusion des Hirnstammes durch Kompression der Vertebralarterien bei Kopfrotation--Eine Ursache von SIDS?

UNLABELLED: Hypoperfusion of the brainstem during head rotation may be a risk factor for the development of SIDS. On this background we established a Doppler sonographic screening programme of the basilar cerebral arteries to evaluate the dependency of blood flow on head and body position. PATIENTS AND METHOD: We investigated 3840 newborns (1872 girls and 1968 boys) with a birth weight of 3399 +/- 497 g and a gestational age of 39.2 +/- 1.4 weeks. The investigations were performed in the neonatal period with an average age of 4.7 +/- 3 days. In all infants blood flow was measured in the basilar artery (BA) in supine position with the head in the midline. From the flow profile peak systolic flow velocity Vs and time average flow velocity TAV were measured. Additionally flow measurements were performed in supine and prone position with rotation of the head to the right and left side. A decrease of blood flow velocities below 50% of the value in neutral position was considered to be abnormal. Retrograde or biophasic flow profiles during rotation were considered to be pathologic. In infants with abnormal or pathologic flow during rotation of the head flow measurements in the vertebral arteries (VA) were additionally performed. Blood flow velocities in the VA were measured in supine and prone position with the head in the midline position and after rotation to the right and to the left. In neutral position unilateral vertebral hypoplasia, aplasia and normal VA were differentiated. The judgement after rotation was performed such as in the BA. RESULTS: In 3807 infants (99.14%) blood flow velocities during head rotation did not decrease below 50% of the value measured in neutral position. In 33 infants (0.86%) a decrease of blood flow velocities below 50% could be found during rotation. In 7 infants (0.18%) a pathologic flow could be found during head rotation. 27 of the 33 infants with abnormal and pathologic blood flow in the BA during rotation showed anatomic abnormalities of the VA. 20 of these infants (61%) had unilateral vertebral hypoplasia (11 right, 9 left side), 7 (21%) had unilateral vertebral aplasia (4 right, 3 left side). 32 of the 33 infants with abnormal flow in the BA showed a decrease of blood flow in the contralateral VA during head rotation. 9 infants had an abnormal, 19 a pathologic flow within the contralateral VA. In 4 infants the corresponding VA could not be measured during head rotation. The decrease of blood flow velocities in the BA during head rotation was caused by compression of the contralateral VA at the craniocervical junction. CONCLUSION: Blood flow within the basilar artery of healthy infants is independent of body position and rotation of the head. A decrease of the flow velocities below 50% during rotation has to be considered as an abnormality. The incidence of pathologic blood flow during head rotation with 1.8@1000 approximates the incidence of SIDS. Hypoperfusion of the brainstem during head rotation may be a risk factor of SIDS.

Reproducibility of the high-performance liquid chromatographic fingerprints obtained from two soybean cultivars and a selected progeny.

Roots from soybean cultivars Williams 82 and Hartwig along with one of their progeny 14a, were extracted with non-polar, moderately polar, and highly polar solvent systems. Extracts were compared by thin-layer chromatography and by HPLC. Methanol extractions conducted at ambient temperature coupled with analysis by reversed-phase HPLC using UV detection provided the most representative sets of reproducible fingerprints. Further optimization of the overall protocol should allow for the profiling of different soybean cultivars when their roots are exposed to various environments and insults during early growth.

Medicinal chemistry in the development of societies. Biodiversity and natural products.

This document has been elaborated by the IUPAC Medicinal Chemistry section and is backed by a large number of scientists, many of whom have had direct involvement and whose names appear at the end of the article. This work discusses the role that the discovery of new medicinal agents has in the development of societies as well as in the conservation of biodiversity in terms of work carried out on natural products. Also included are several recommendations for countries which are presently in search of their own scientific and technological development in medicinal agents. The IUPAC Medicinal Chemistry section would appreciate the collaboration of the scientific societies in every country to aid in the diffusion of this document.

B-Raf inhibits programmed cell death downstream of cytochrome c release from mitochondria by activating the MEK/Erk pathway.

Growth factor-dependent kinases, such as phosphatidylinositol 3-kinase (PI 3-kinase) and Raf kinases, have been implicated in the suppression of apoptosis. We have recently established Rat-1 fibroblast cell lines overexpressing B-Raf, leading to activation of the MEK/Erk mitogen-activated protein kinase pathway. Overexpression of B-Raf confers resistance to apoptosis induced by growth factor withdrawal or PI 3-kinase inhibition. This is accompanied by constitutive activation of Erk without effects on the PI 3-kinase/Akt pathway. The activity of MEK is essential for cell survival mediated by B-Raf overexpression, since either treatment with the specific MEK inhibitor PD98059 or expression of a dominant inhibitory MEK mutant blocks the antiapoptotic activity of B-Raf. Activation of MEK is not only necessary but also sufficient for cell survival because overexpression of constitutively activated MEK, Ras, or Raf-1, like B-Raf, prevents apoptosis after growth factor deprivation. Overexpression of B-Raf did not interfere with the release of cytochrome c from mitochondria after growth factor deprivation. However, the addition of cytochrome c to cytosols of cells overexpressing B-Raf failed to induce caspase activation. It thus appears that the B-Raf/MEK/Erk pathway confers protection against apoptosis at the level of cytosolic caspase activation, downstream of the release of cytochrome c from mitochondria.

Analysis of extemporaneous alprostadil formulaions.

Twenty-two extemporaneous alprostadil (PGE1) injection solutions samples from five different suppliers and three Caverject (Pharmacia and Upjohn, Inc., Bridgewater, NJ) samples from three different lots, all intended for the clinical treatment of erectile dysfunction, were analyzed to determine PGE1 concentration, assess formation of the PGE1 aqueous breakdown product (PGA1), define pH and assess active microbial contamination. High-pressure liquid chromatography (HPLC), pH meter and cell culture techniques were used to conduct the analyses. Of the 22 extemporaneously formulated samples, six showed PGE1 concentrations 10% greater than their listed amounts and seven showed PGA1 weight fractions corresponding to at least 1.5% of the total prostaglandidn content. It should be noted that no standard has been published in the United States Pharmacopeia/National Formulary for this preparation as of this date. All samples were within the pH range 4.5 to 6.0. Four samples tested positive for active microbial contamination. In adition, nearly all the extemporaneously formulated samples contained what appeared to be benzyl alcohol, and about one half had at least two other undefined peaks within their HPLC chromatograms. In contrast, all three Caverject samples were within +/- 7.5% of their listed PGE1 concentrations while showing PGA1 prostaglandins weight fractions of less 0.6%, all were within the pH range 4.0 to 4.5 and all tested negative for active microbial contamination. Chromatograms of the Caverject samples also diplayed peaks consistent with the presence of benzyl alcohol but did not exhibit addtional undefined peaks. The results suggest that significant variations in PGE1 concentration and in PGA1 formation, accompanied by the possibility of microbial contamination, can occur as a result of the extemporaneous formulation and subsequent transfer of this type of product as a premixed solution intended for treating erectile dysfunction.

Intact Ras function is required for sustained activation and nuclear translocation of extracellular signal-regulated kinases in nerve growth factor-stimulated PC12 cells.

PC12 pheochromocytoma cell lines expressing the dominant negative Ha-Ras Asn-17 protein at different levels were used in this study to analyze the relationship between nerve growth factor (NGF)-induced activation of members of the mitogen-activated protein kinase (MAPK) family, and neuritogenesis. In wild-type PC12 cells, NGF rapidly stimulated the extracellular signal-regulated kinases (ERKs). Kinase activation was sustained and was followed by the translocation of ERK 1 and ERK 2 into the nucleus ultimately leading to neurite outgrowth. In cells expressing relatively high levels of the inhibitory Ras protein, NGF stimulation of ERK 1 and ERK 2 as well as nuclear translocation of these protein kinases were greatly inhibited. In contrast, in PC12 subclones expressing low amounts of Ha-Ras Asn-17 the peak of ERK activation was only slightly reduced, but became transient in nature and was not followed by nuclear translocation of ERKs 1 and 2. Since all PC12 subclones expressing detectable levels of the dominant inhibitory Ras protein are resistant to NGF induction of neurite formation, our observations support the notion that sustained activation and translocation of ERKs into the nucleus are essential for NGF-induced neuronal differentiation of PC12 cells.

Stability of Magnesium Sulfate in 0.9% Sodium Chloride and Lactated Ringers solutions.

Storage stability test were peformed on two extemporaneous formulation alternatives to the commercially available magnesium sulfate injection solutions that are in 5% dextrose or in water. Preparations of the commercial water for injection formulation and two alternative formulations in lactated Ringers and in 0.9% sodium chloride were stored at room temperature in glass bottles and in polyvinyl chloride bags over a three-month period. Solutions were monitored for gross precipitation and for changes in magnesium, sulfur and calcium levels as measured by elemental analysis using atomic absorption spectroscopy and inductively coupled plasma atomic emission spectroscopy. The results demonstrate no consistent decreases in measured elemental concentrations or gross signs of precipitation for any formulation tested.

Identification of the MDM2 oncoprotein as a substrate for CPP32-like apoptotic proteases.

Programmed cell death is mediated by members of the interleukin 1-beta convertase family of proteases, which are activated in response to diverse cell death stimuli. However, the key substrates of these proteases that are responsible for apoptotic cell death have not been identified. Here we report that the MDM2 oncoprotein is cleaved by members of the CPP32 subfamily of interleukin 1-beta convertase proteases both in vitro and in vivo, resulting in the disappearance of MDM2 from apoptotic cells. Because MDM2 functions as a negative regulator of the p53 tumor suppressor and because p53 induces apoptosis in response to a variety of stimuli, this cleavage of MDM2 by CPP32-like proteases may result in deregulation of p53 and contribute directly to the process of apoptotic cell death.

p53-dependent induction of apoptosis by proteasome inhibitors.

Proteolysis by the ubiquitin/proteasome pathway controls the intracellular levels of a number of proteins that regulate cell proliferation and cell cycle progression. To determine whether this pathway of protein turnover was also linked to apoptosis, we treated Rat-1 and PC12 cells with specific proteasome inhibitors. The peptide aldehydes PSI and MG115, which specifically inhibit the chymotrypsin-like activity of the proteasome, induced apoptosis of both cell types. In contrast, apoptosis was not induced by inhibitors of lysosomal proteases or by an alcohol analog of PSI. The tumor suppressor p53 rapidly accumulated in cells treated with proteasome inhibitors, as did the p53-inducible gene products p21 and Mdm-2. In addition, apoptosis induced by proteasome inhibitors was inhibited by expression of dominant-negative p53, whereas overexpression of wild-type p53 was sufficient to induce apoptosis of Rat-1 cells in transient transfection assays. Although other molecules may also be involved, these results suggest that stabilization and accumulation of p53 plays a key role in apoptosis induced by proteasome inhibitors.

Adenovirus-mediated gene transfer of dominant negative ras(asn17) in 3T3L1 adipocytes does not alter insulin-stimulated P13-kinase activity or glucose transport.

Recent studies suggest that the ras-map kinase and PI3-kinase cascades converge. We sought to determine whether PI3-kinase is downstream of ras in insulin signaling in a classic insulin target cell. We generated a recombinant adenovirus encoding dominant negative ras by cloning the human H-ras cDNA with a ser to asn substitution at amino acid 17 (ras(asn17)) into the pACCMVpLpA vector and cotransfecting 293 cells with the pJM17 plasmid containing the adenoviral genome. Efficiency of gene transfer was assessed by infecting fully differentiated 3T3L1 adipocytes with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 70% of cells were infected. Infection of adipocytes with ras(asn17) resulted in 10-fold greater expression than endogenous ras. This high efficiency gene transfer allowed biochemical assays. Insulin stimulation of ras-GTP formation was inhibited in ras(asn17)-expressing cells. Map kinase gel mobility shift revealed that insulin (1 UM) or epidermal growth factor (100 ng/ml) resulted in the appearance of a hyperphosphorylated species of p42 map kinase in uninfected cells and those expressing beta-gal but not in cells expressing ras(asn17). In contrast, insulin increased IRS-1-associated PI3-kinase activity approximately 10-fold in control cells and high level overexpression of ras(asn17) did not impair this effect. Similarly, insulin and epidermal growth factor activation of total (no immunoprecipitation) PI3-kinase activity in both cytosol and total cellular membranes and insulin stimulation of glucose transport were not affected by expression of dominant negative ras. Thus, adenovirus-mediated gene transfer is effective for studying insulin signaling in fully differentiated insulin target cells. Inhibition of ras activation abolishes insulin-stimulated phosphorylation of map kinase but does not affect insulin stimulation of PI3-kinase activity. In normal cell physiology, PI3-kinase does not appear to be downstream of ras in mediating the actions of insulin.

Estimating xenobiotic half-lives in humans from rat data: influence of log P.

The nature of empirical allometric expressions relating dispositional and kinetic parameters for a given xenobiotic across multiple mammalian species is well known. It has also been demonstrated that a simple allometric relationship may be used to predict kinetic parameters for humans based merely on data for multiple xenobiotics from rats. We decided to explore reasons for the variance in the data arising from the latter method. We were particularly interested in learning whether any physicochemical characteristics of xenobiotics might account for outlying data points (i.e., poor prediction of human half-life from rat half-life). We have explored the influence of lipid solubility as reflected by a xenobiotic's log P value because adipose tissue comprises a significantly larger percentage of total body weight in humans than in rats. We used half-life data from the literature for 127 xenobiotics. A data subset of 102 xenobiotics for which we were able to find estimates of log P values, including several with extremely large log P values, was also analyzed. First and second order models, including and excluding log P, were compared. The simplest of these models can be recast as the familiar allometric relationship having the form Y = a(Xb). The remaining models can be seen as extensions of this relationship. Our results suggest that incorporation of log P into the prediction of xenobiotic half-life in humans from rat half-life data is important only for xenobiotics with extremely large log P values such as dioxins and polychlorinated biphenyls. Moreover, a second order model in logarithm of rat half-life accommodates all data points very well, without specifically accounting for log P values.

Activation of the CPP32 apoptotic protease by distinct signaling pathways with differential sensitivity to Bcl-xL.

In the absence of growth factors, many types of mammalian cells undergo apoptosis. We and others have shown recently that growth factors promote cell survival by activating phosphatidylinositol 3-kinase (PI 3-kinase) in several cell types. In the present study, we have compared downstream elements of the apoptotic pathways induced by PI 3-kinase inhibitors and other stimuli. In U937 cells, both PI 3-kinase inhibitors (wortmannin and LY294002) and etoposide activated the CPP32 apoptotic protease by cleavage to active p17 subunits. In contrast, treatment with tumor necrosis factor alpha (TNFalpha) resulted in the accumulation of a distinct active CPP32 subunit, p20. Furthermore, overexpression of Bcl-xL blocked DNA fragmentation, CPP32 activation and cleavage of poly(ADP-ribose) polymerase in U937 cells treated with both PI 3-kinase inhibitors and etoposide, but not in cells treated with TNFalpha. Distinct patterns of CPP32 activation and differential sensitivities to Bcl-xL thus distinguish the cell death pathways activated by PI 3-kinase inhibition and DNA damage from that activated by TNFalpha.

Differential regulation of Raf-1 and B-Raf and Ras-dependent activation of mitogen-activated protein kinase by cyclic AMP in PC12 cells.

Growth factor stimulation of the mitogen-activated protein (MAP) kinase pathway in fibroblasts is inhibited by cyclic AMP (cAMP) as a result of inhibition of Raf-1. In contrast, cAMP inhibits neither nerve growth factor-induced MAP kinase activation nor differentiation in PC12 pheochromocytoma cells. Instead, in PC12 cells cAMP activates MAP kinase. Since one of the major differences between the Ras/Raf/MAP kinase cascades of these cell types is the expression of B-Raf in PC12 cells, we compared the effects of cAMP on Raf-1 and B-Raf. In PC12 cells maintained in serum-containing medium, B-Raf was refractory to inhibition by cAMP, whereas Raf-1 was effectively inhibited. In contrast, both B-Raf and Raf-1 were inhibited by cAMP in serum-starved PC12 cells. The effect of cAMP is thus dependent upon growth conditions, with B-Raf being resistant to cAMP inhibition in the presence of serum. These results were extended by studies of Rat-1 fibroblasts into which B-Raf had been introduced by transfection. As in PC12 cells, B-Raf was resistant to inhibition by cAMP in the presence of serum, whereas Raf-1 was effectively inhibited. In addition, the expression of B-Raf rendered Rat-1 cells resistant to the inhibitory effects of cAMP on both growth factor-induced activation of MAP kinase and mitogenesis. These results indicate that Raf-1 and B-Raf are differentially sensitive to inhibition by cAMP and that B-Raf expression can contribute to cell type-specific differences in the regulation of the MAP kinase pathway. In contrast to the situation in PC12 cells, cAMP by itself did not stimulate MAP kinase in B-Raf-expressing Rat-1 cells. The activation of MAP kinase by cAMP in PC12 cells was inhibited by the expression of a dominant negative Ras mutant, indicating that cAMP acts on a target upstream of Ras. Thus, it appears that a signaling component upstream of Ras is also require for cAMP stimulation of MAP kinase in PC12 cells.

Hydroxamic acids as potent inhibitors of endothelin-converting enzyme from human bronchiolar smooth muscle.

Hydroxamic acids 6a-h, derived from malonyl amino acids, and 25a-d, derived from succinyl amino acids, were synthesized as inhibitors of human bronchiolar smooth muscle endothelin-converting enzyme (HBSM ECE). Several unexpected side reactions were discovered, particularly in the synthesis of hydroxamates derived from succinates. In vitro evaluation against human bronchiolar ECE revealed that in all cases hydroxamates derived from malonate were more potent than hydroxamates derived from succinate. Isopropyl and isobutyl P1' side chains were suitable; omission of the P1' side chain seriously diminished potency. In the P2' position, several amino acids gave potent malonate-derived hydroxamate inhibitors (6b, d-h, IC50 = 0.2-6.8 nM), and beta-Ala provided an extremely potent inhibitor (6c, IC50 = 0.01 nM). C-terminus carboxylates are much more potent ECE inhibitors than the corresponding amides. Most of the hydroxamates were also potent inhibitors of thermolysin and neutral endopeptidase (NEP); however, the P2' beta-Ala derivative 6c uniquely inhibited HBSM ECE much more potently than NEP.

Complete coding sequence, deduced primary structure, chromosomal localization, and structural analysis of murine aggrecan.

We have isolated and sequenced overlapping cDNA clones encoding the entire core protein of aggrecan (the large aggregating chondroitin sulfate/keratan sulfate proteoglycan of cartilage) from three chondrocyte cDNA libraries of BALB/c mice and localized the aggrecan gene in mouse chromosome 7. We determined 7386 bp of the cDNA sequence, including 132 and 854 nucleotides of 5' and 3' untranslated regions, respectively. The core protein precursor is 2132 amino acids long (M(r) 222,008), including a 19-residue secretory signal peptide. The overall amino acid sequence of the mouse aggrecan shows 91.6% identity to rat and 72.5% to human aggrecan. Comparison of the amino acid sequences of various domains and subdomain structures of mouse aggrecan to known sequences of other species and related proteins (versican, neurocan, link protein, and lymphocyte homing receptor CD44) revealed high levels of identity of the G1, G2, and G3 globular domains and relatively less conserved structures in the interglobular and glycosaminoglycan-attachment regions. Epidermal growth factor (EGF)-like module was detected in only a minor fraction of aggrecan clones, while the complement regulatory protein (CRP)-like domain was regularly expressed in all samples.

Hydrolysis of phosphatidylcholine couples Ras to activation of Raf protein kinase during mitogenic signal transduction.

We have investigated the relationship between hydrolysis of phosphatidylcholine (PC) and activation of the Raf-1 protein kinase in Ras-mediated transduction of mitogenic signals. As previously reported, cotransfection of a PC-specific phospholipase C (PC-PLC) expression plasmid bypassed the block to cell proliferation resulting from expression of the dominant inhibitory mutant Ras N-17. In contrast, PC-PLC failed to bypass the inhibitory effect of dominant negative Raf mutants, suggesting that PC-PLC functions downstream of Ras but upstream of Raf. Consistent with this hypothesis, treatment of quiescent cells with exogenous PC-PLC induced Raf activation, even when normal Ras function was blocked by Ras N-17 expression. Further, activation of Raf in response to mitogenic growth factors was blocked by inhibition of endogenous PC-PLC. Taken together, these results indicate that hydrolysis of PC mediates Raf activation in response to mitogenic growth factors.

ras-independent induction of rat brain type II sodium channel expression in nerve growth factor-treated PC12 cells.

Nerve growth factor (NGF) plays an important role in the development of the nervous system, and there is considerable interest in understanding the molecular mechanisms underlying its effects on neuronal differentiation. To determine if the activity of proteins of the ras gene family is necessary for the NGF-mediated induction of sodium channel expression in pheochromocytoma (PC12) cells, sodium channel expression was analyzed in PC12 sublines stably overexpressing the dominant inhibitory mutant c-Ha-ras(Asn-17). Northern blot analysis, RNase protection assays, and whole-cell patch clamp recordings indicate that the NGF-mediated increase in type II sodium channel mRNA and sodium current density can occur independent of ras activity and by doing so provide strong evidence for the importance of ras-independent mechanisms in NGF-mediated neuronal differentiation.