Crystal structure of mammalian purple acid phosphatase

Background: Mammalian purple acid phosphatases are highly conserved binuclear metal-containing enzymes produced by osteoclasts, the cells that resorb bone. The enzyme is a target for drug design because there is strong evidence that it is involved in bone resorption. Results: The 1.55 Angstrom resolution structure of pig purple acid phosphatase has been solved by multiple isomorphous replacement. The enzyme comprises two sandwiched beta sheets flanked by or-helical segments. The molecule shows internal symmetry, with the metal ions bound at the interface between the two halves. Conclusions: Despite less than 15% sequence identity, the protein fold resembles that of the catalytic domain of plant purple acid phosphatase and some serine/threonine protein phosphatases. The active-site regions of the mammalian and plant purple acid phosphatases differ significantly, however. The internal symmetry suggests that the binuclear centre evolved as a result of the combination of mononuclear ancestors. The structure of the mammalian enzyme provides a basis for antiosteoporotic drug design.

D-amino acid residue in the C-type natriuretic peptide from the venom of the mammal, Ornithorhynchus anatinus, the Australian platypus

The C-type natriuretic peptide from the platypus venom (OvCNP) exists in two forms, OvCNPa and OvCNPb, whose amino acid sequences are identical. Through the use of nuclear magnetic resonance, mass spectrometry, and peptidase digestion studies, we discovered that OvCNPb incorporates a D-amino acid at position 2 in the primary structure. Peptides containing a D-amino acid have been found in lower forms of organism, but this report is the first for a D-amino acid in a biologically active peptide from a mammal. The result implies the existence of a specific isomerase in the platypus that converts an L-amino acid residue in the protein to the D-configuration. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Synthesis of alpha-aspartyl-containing cyclic peptides

alpha-Aspartyl-containing cyclic pentapeptides were synthesised in high yields using a strategy that maintained fluorenylmethyl protection on the aspartic acid side chain during chain assembly, resin cleavage and cyclisation of the linear precursors. Tetra-n-butylammonium fluoride treatment of the fluorenylmethyl-protected cyclic peptides catalysed imide formation, whereas piperidine-induced deprotection resulted in good yields of the target cyclic peptides.

Visualising the activity of the cystine-glutamate antiporter in glial cells using antibodies to aminoadipic acid, a selectively transported substrate

The cystine-glutamate antiporter is a transport system that facilitates the uptake of cystine, concomitant with the release of glutamate. The cystine accumulated by this transporter is generally considered for use in the formation of the cysteine-containing antioxidant glutathione, which is abundant in many glial cells. This study used the simple strategy of generating an antibody to aminoadipic acid, a selective substrate for the cystine-glutamate antiporter. Stereospecific accumulation of aminoadipic acid into specific cell types in rat brain slice preparations was detected immunocytochemically. Strong accumulation was detected in astroglial cells in all brain regions studied including those in white matter tracts. Strong accumulation into radial glial cells, including the retinal Muller cells and the Bergmann glial cells was also observed. Glial accumulation was observed not only in cells within the blood brain barrier, but also outside such; anterior pituitary folliculostellate cell and intermediate lobe pituitary glial cells exhibited strong accumulation of aminoadipic acid. Interestingly, some glial cells such as the posterior pituitary glial cells (pituicytes) exhibited very little if any accumulation of aminoadipic acid. Within the brain labelling was not uniform. Particularly strong labelling was noted in some regions, such as the glial cells surrounding the CA1 pyramidal cells. By contrast, neurons never exhibited uptake of aminoadipic acid. Because cystine uptake is associated with glutamate release, it is suggested that this antiporter might contribute to release of glutamate from glial cells under some pathophysiological conditions. (C) 2001 Wiley-Liss, Inc.

An ESR study of the gamma radiolysis of aromatic polyesters containing isomeric naphthalene links

Six polyesters were synthesised from 4,4 ' -oxy-bis(benzoyl chloride) and 1,4-, 1,5-, 1,6-, 2,3-, 2,6-, and 2,7-naphthalenediol isomers. The structures of the polyesters were characterised by means of IR, inherent viscosities in tetrachloroethane (TCE), solutions at 303 K and thermal analysis. The glass transition temperatures were in the range of 425-494 K by DSC thermal analysis. All of the polyesters were irradiated in an AECL Gammacell 220 unit at a dose rate of approximately 6.7 kGy/h to doses in the range of 0-15 kGy at 77 and 300 K. ESR spectroscopy was used to examine the radicals formed during radiolysis and to measure their yields. The G-values for radical formation in the polyesters were found to be in the range 0.18-1.41 at 77 K and 0.19-0.78 at 300 K. At 77 K, up to 15% of the radicals formed on radiolysis were found to be photo-bleachable anion radicals. Annealing experiments were carried out in order to identify the neutral radicals, which were assigned to naphthyl- or phenyl- and phenoxyl-type radicals. (C) 2001 Elsevier Science Ltd. All rights reserved.

A titrimetric respirometer measuring the nitrifiable nitrogen in wastewater using in-sensor-experiment

Measurement of nitrifiable nitrogen contained in wastewater by combining the existing respirometric and titrimetric principles is reported. During an in-sensor-experiment using nitrifying activated sludge. both the dissolved oxygen (DO) and pH in the mixed liquor were measured, and the FH was controlled at a set-point through titration of base or acid. A combination of the oxygen uptake rate (OUR), which was obtained from the measured DO signal, and the titration data allowed calculation of the nitrifiable nitrogen and the short-term biological oxygen demand (BOD) of the wastewater sample that was initially added to the sludge. The calculation was based solely on stoichiometric relationships. The approach was preliminarily tested with two types of wastewaters using a prototype sensor. Good correlation was obtained. (C) 2000 Elsevier Science Ltd. All rights reserved.

Effects of vitamin E and alpha-lipoic acid on skeletal muscle contractile properties

Initial experiments were conducted using an in situ rat tibialis anterior (TA) muscle preparation to assess the influence of dietary antioxidants on muscle contractile properties. Adult Sprague-Dawley rats were divided into two dietary groups: 1) control diet (Con) and 2) supplemented with vitamin E (VE) and alpha -lipoic acid (alpha -LA) (Antiox). Antiox rats were fed the Con rats' diet (AIN-93M) with an additional 10,000 IU VE/kg diet and 1.65 g/kg alpha -LA. After an 8-wk feeding period, no differences existed (P > 0.05) between the two dietary groups in maximum specific tension before or after a fatigue protocol or in force production during the fatigue protocol. However, in unfatigued muscle, maximal twitch tension and tetanic force production at stimulation frequencies less than or equal to 40 Hz were less (P < 0.05) in Antiox animals compared with Con. To investigate which antioxidant was responsible for the depressed force production, a second experiment was conducted using an in vitro rat diaphragm preparation. Varying concentrations of VE and dihydrolipoic acid, the reduced form of -LA, were added either individually or in combination to baths containing diaphragm muscle strips. The results from these experiments indicate that high levels of VE depress skeletal muscle force production at low stimulation frequencies.

SOX9 enhances aggrecan gene promoter/enhancer activity and is up-regulated by retinoic acid in a cartilage-derived cell line, TC6

SOX9 is a transcription factor that plays a key role in chondrogenesis, Aggrecan is one of the major structural components in cartilage; however, the molecular mechanism of aggrecan gene regulation has not yet been fully elucidated, TC6 is a clonal chondrocytic cell line derived from articular cartilage, The purpose of this study was to examine whether SOX9 modulates aggrecan gene expression and to further identify molecules that regulate Sox9 expression in TC6 cells. SOX9 overexpression in TC6 cells enhanced by similar to 3-fold the transcriptional activity of the AgCAT-8 construct containing S-kilobase (kb) promoter/first exon/first intron fragments of the aggrecan gene. SOX9 enhancement of aggrecan promoter activity was lost when we deleted a 4.5-kb fragment from the 3'-end of the 8-kb fragment corresponding to the region including the first intron, In TC6 cells, SOX9 enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence >10-fold. SOX9 enhancement of aggrecan gene promoter activity and SOX9 transactivation through the Sry/Sox consensus sequence were not observed in osteoblastic osteosarcoma cells (ROS17/2.8), indicating the dependence on the cellular background. Northern blot analysis indicated that TC6 cells constitutively express Sox9 mRNA at relatively low levels. To examine regulation of Sox9 gene expression, we investigated the effects of calciotropic hormones and cytokines, Among these, retinoic acid (RA) specifically enhanced Sox9 mRNA expression in TC6 cells. The basal levels of Sox9 expression and its enhancement by RA were observed similarly at both permissive (33 degrees C) and nonpermissive (39 degrees C) temperatures. Furthermore, RA treatment enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence in TC6 cells. Moreover, RA treatment also enhanced the transcriptional activity of another reporter construct containing the enhancer region of the type II procollagen gene in TC6 cells. These observations indicate that SOX9 enhances aggrecan promoter activity and that its expression is up-regulated by RA in TC6 cells.

A purple acid phosphatase from sweet potato contains an antiferromagnetically coupled binuclear Fe-Mn center

A purple acid phosphatase from sweet potato is the first reported example of a protein containing an enzymatically active binuclear Fe-Mn center. Multifield saturation magnetization data over a temperature range of 2 to 200 K indicates that this center is strongly antiferromagnetically coupled. Metal ion analysis shows an excess of iron over manganese. Low temperature EPR spectra reveal only resonances characteristic of high spin Fe(III) centers (Fe(III)-apo and Fe(III)-Zn(II)) and adventitious Cu(II) centers. There were no resonances from either Mn(II) or binuclear Fe-Mn centers. Together with a comparison of spectral properties and sequence homologies between known purple acid phosphatases, the enzymatic and spectroscopic data strongly indicate the presence of catalytic Fe(III)-Mn(II) centers in the active site of the sweet potato enzyme. Because of the strong antiferromagnetism it is likely that the metal ions in the sweet potato enzyme are linked via a mu -oxo bridge, in contrast to other known purple acid phosphatases in which a mu -hydroxo bridge is present. Differences in metal ion composition and bridging may affect substrate specificities leading to the biological function of different purple acid phosphatases.

Quantification of sulfur and sulfur-containing compounds in wastewaters by means of a combination of liquid chromatographic methods

Low-micromolar concentrations of sulfite, thiosulfate and sulfide, present in synthetic wastewater or anaerobic digester effluent, were quantified by means of derivatization with monobromobimane, followed by HPLC separation with fluorescence detection. The concentration of elemental sulfur was determined, after its extraction with chloroform from the derivatized sample, by HPLC with UV detection. Recoveries of sulfide (both matrices), and of thiosulfate and sulfite (synthetic wastewater) were between 98 and 103%. The in-run RSDs on separate derivatizations were 13 and 19% for sulfite (two tests), between 1.5 and 6.6% for thiosulfate (two tests) and between 4.1 and 7.7% for sulfide (three tests). Response factors for derivatives of sulfide and thiosulfate, but not sulfite, were steady over a 13-month period during which 730 samples were analysed. Dithionate and tetrathionate did not seem to be detectable with this method. The distinctness of the elemental sulfur and the derivatizing-agent peaks was improved considerably by detecting elution at 297 instead of 263 nm. (C) 2002 Elsevier Science B.V. All rights reserved.

Modern scientific methods and their potential in wastewater science and technology

Application of novel analytical and investigative methods such as fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM), microelectrodes and advanced numerical simulation has led to new insights into micro-and macroscopic processes in bioreactors. However, the question is still open whether or not these new findings and the subsequent gain of knowledge are of significant practical relevance and if so, where and how. To find suitable answers it is necessary for engineers to know what can be expected by applying these modern analytical tools. Similarly, scientists could benefit significantly from an intensive dialogue with engineers in order to find out about practical problems and conditions existing in wastewater treatment systems. In this paper, an attempt is made to help bridge the gap between science and engineering in biological wastewater treatment. We provide an overview of recently developed methods in microbiology and in mathematical modeling and numerical simulation. A questionnaire is presented which may help generate a platform from which further technical and scientific developments can be accomplished. Both the paper and the questionnaire are aimed at encouraging scientists and engineers to enter into an intensive, mutually beneficial dialogue. (C) 2002 Elsevier Science Ltd. All rights reserved.

Development of a novel titration and off-gas analysis (TOGA) sensor for study of biological processes in wastewater treatment systems

The development of the new TOGA (titration and off-gas analysis) sensor for the detailed study of biological processes in wastewater treatment systems is outlined. The main innovation of the sensor is the amalgamation of titrimetric and off-gas measurement techniques. The resulting measured signals are: hydrogen ion production rate (HPR), oxygen transfer rate (OTR), nitrogen transfer rate (NTR), and carbon dioxide transfer rate (CTR). While OTR and NTR are applicable to aerobic and anoxic conditions, respectively, HPR and CTR are useful signals under all of the conditions found in biological wastewater treatment systems, namely, aerobic, anoxic and anaerobic. The sensor is therefore a powerful tool for studying the key biological processes under all these conditions. A major benefit from the integration of the titrimetric and off-gas analysis methods is that the acid/base buffering systems, in particular the bicarbonate system, are properly accounted for. Experimental data resulting from the TOGA sensor in aerobic, anoxic, and anaerobic conditions demonstrates the strength of the new sensor. In the aerobic environment, carbon oxidation (using acetate as an example carbon source) and nitrification are studied. Both the carbon and ammonia removal rates measured by the sensor compare very well with those obtained from off-line chemical analysis. Further, the aerobic acetate removal process is examined at a fundamental level using the metabolic pathway and stoichiometry established in the literature, whereby the rate of formation of storage products is identified. Under anoxic conditions, the denitrification process is monitored and, again, the measured rate of nitrogen gas transfer (NTR) matches well with the removal of the oxidised nitrogen compounds (measured chemically). In the anaerobic environment, the enhanced biological phosphorus process was investigated. In this case, the measured sensor signals (HPR and CTR) resulting from acetate uptake were used to determine the ratio of the rates of carbon dioxide production by competing groups of microorganisms, which consequently is a measure of the activity of these organisms. The sensor involves the use of expensive equipment such as a mass spectrometer and requires special gases to operate, thus incurring significant capital and operational costs. This makes the sensor more an advanced laboratory tool than an on-line sensor. (C) 2003 Wiley Periodicals, Inc.

Manipulation of colonic bacteria and volatile fatty acid production by dietary high amylose maize (amylomaize) starch granules

Aims : To study the effects of amylomaize starch and modified (carboxymethylated and acetylated) amylomaize starches on the composition of colonic bacteria and the production of volatile fatty acids, in mice. Methods and Results : Balb/c mice were fed with experimental diets containing various amount of amylomaize and modified amylomaize starches. Colonic bacterial populations and short-chain fatty acids were monitored. Results showed that the increases in indigenous bifidobacteria were detected in mice fed all starches tested; however, the highest numbers were observed in the group fed with 40% unmodified amylomaize starch. The starch type influenced the populations of indigenous Lactobacillus , Bacteroides and coliforms. High Lactobacillus numbers were achieved in the colon of mice fed with high concentration of amylomaize starch. Acetylated amylomaize starch significantly reduced the population of coliforms. In addition, orally dosed amylomaize utilizing bifidobacteria reached their highest levels when fed together with amylomaize or carboxymethylated amylomaize starch and in both cases butyrate levels were markedly increased. Conclusions: These results indicate that different amylomaize starches could generate desirable variation in gut microflora and that particular starches may be used to selectively modify gut function. Significance and Impact of Study: Amylomaize starch appeared to enhance the desirable composition of colonic bacteria in mice, and suggested it possessed the potential prebiotic properties.MTherefore, resistant starch and its chemical derivatives may exert beneficial impacts to the human colon.

Lysine requirements of pre-lay broiler breeder pullets: Determination by indicator amino acid oxidation

The indicator amino acid oxidation (IAAO) method allows the determination of amino acid requirements under conditions of low growth rate as found in pre-laying broiler breeder pullets. Cobb 500 breeder pullets (20 wk old; 2290 +/- 280 g, n = 4) were adapted (6 d) to a pelleted, purified control diet containing all nutrients at greater than or equal to 110% of NRC recommendations. After recovery from surgery for implantation of a jugular catheter, each bird was fed, in random order, test diets containing one of nine levels of lysine (0.48, 0.96, 1.92, 2.88, 3.84, 4.80, 7.68, 9.60 and 14.40 g/kg of diet). Indicator oxidation was determined during 4-h primed (74 kBq/kg body), constant infusions (44 kBq (.) h(-1) (.) kg body(-1)) of L-[1-C-14]phenylalanine. Using the breakpoint of a one-slope broken-line model, the lysine requirement was determined to be 4.88 +/- 0.96 g/kg of diet or 366 +/- 72 mg (.) hen(-1) (.) d(-1) with an upper 95% Cl of 6.40 g/kg of diet or 480 mg (.) hen(-1) (.) d(-1). IAAO allows determination of individual bird amino acid requirements for specific ages and types of birds over short periods of time and enables more accurate broiler breeder pullet diet formulation.

Multiple tissue-specific promoters control expression of the murine tartrate-resistant acid phosphatase gene

Tartrate-resistant acid phosphatase (TRAP) is highly expressed in osteoclasts and in a subset of tissue macrophages and dendritic cells. It is expressed at lower levels in the parenchymal cells of the liver, glomerular mesangial cells of the kidney and pancreatic acinar cells. We have identified novel TRAP mRNAs that differ in their 5-untranslated region (5'-UTR) sequence, but align with the known murine TRAP mRNA from the first base of Exon 2. The novel 5'-UTRs represent alternative first exons located upstream of the known 5'-UTR. A similar genomic structure exists for the human TRAP gene with partial conservation of the exon and promoter sequences. Expression of the most distal 5'-UTR (Exon 1A) is restricted to adult bone and spleen tissue. Exon 1B is expressed primarily in tissues containing TRAP-positive nonhaematopoietic cells. The known TRAP 5'-UTR (Exon 1) is expressed in tissues characteristic of myeloid cell expression. In addition the Exon 1C promoter sequence is shown to comprise distinct transcription start regions, with an osteoclast-specific transcription initiation site identified downstream of a TATA-like element. Macrophages are shown to initiate transcription of the Exon 1C transcript from a purine-rich region located upstream of the osteoclast-specific transcription start point. The distinct expression patterns for each of the TRAP 5'-UTRs suggest that TRAP mRNA expression is regulated by the use of four alternative tissue- and cell-restricted promoters. (C) 2003 Elsevier Science B.V. All rights reserved.