bluuebunny/crossref_metadata_2025 huggingface dataset:
Covalent structure of mutacin 1140 and a novel method for the rapid identification of lantibiotics
Authors: Smith Leif , Novák Jan , Rocca Jim , McClung Scott , Hillman J. D. , Edison Arthur S.
Published: August 2003

The primary structure of the Streptococcus mutans lantibiotic mutacin 1140 was elucidated by NMR spectroscopy, mass spectrometry, and chemical sequencing. The structure is in agreement with other closely related lantibiotics, such as epidermin. A novel method was developed in which mutacin 1140 was chemically modified with sodium borohydride followed by ethanethiol, allowing the differentiation of the thioether‐containing residues from the dehydrated residues. This double‐labeling strategy provides a simple method to reliably identify all modified lantibiotic residues with a minimal amount of material. While NMR spectroscopy is still required to obtain thioether bridging patterns and thus the complete covalent structure, the double‐labeling technique, along with mass spectrometry, provides most of the information in a fraction of the time required for a complete NMR analysis. Thus, with these new techniques lantibiotics can be rapidly characterized.
Critical role of C/EBPδ and C/EBPβ factors in the stimulation of the cyclooxygenase‐2 gene transcription by interleukin‐1β in articular chondrocytes
Authors: Thomas Béatrice , Berenbaum Francis , Humbert Lydie , Bian Huimin , Béréziat Gilbert , Crofford Leslie , Olivier Jean Luc
Published: August 2003

The activity of the [−831; +103] promoter of the human cyclooxygenase‐2 gene in cultured rabbit chondrocytes is stimulated 2.9 ± 0.3‐fold by interleukin‐1β and this stimulation depends on [−132; −124] C/EBP binding‐and [−223; −214] NF‐κB binding‐sites. The C/EBPβ and C/EBPδ factors bind to the [−132; −124] sequence. The [−61; −53] sequence is also recognized by C/EBPβ and C/EBPδ as well as USF. Mutation of the whole [−61; −53] sequence abolished the stimulation of transcription but single mutations of the C/EBP or USF site did not alter the activity of the promoter, suggesting that the factors bound to the proximal [−61; −53] sequence interact with different members of the general transcription machinery. The [−223; −214] site binds only the p50/p50 homodimer and a non‐rel‐related protein, but not the transcriptionally active heterodimer p50/p65. The p50/p50 homodimer could interact with the C/EBP family members bound to the [−132; −124] sequence for full stimulation of the COX‐2 transcription by interleukin‐1β in chondrocytes. By contrast, the [−448; −449] sequence binds with a low affinity both the p50/p50 homodimeric and p50/p65 heterodimeric forms of NF‐κB but has no role in the regulation of the human COX‐2 promoter in chondrocytes.
Regulation of pyc1 encoding pyruvate carboxylase isozyme I by nitrogen sources in Saccharomyces cerevisiae
Authors: Huet Carine , Menendez Javier , Gancedo Carlos , François Jean M.
Published: August 2003

In Saccharomyces cerevisiae, the existence of PYC1 and PYC2 encoding cytosolic pyruvate carboxylase isoform I and II is rather puzzling, owing to the lack of potent differential gene regulation by the carbon sources. We report several findings indicating that these two genes are differentially regulated by the nature of the nitrogen source. In wild‐type cells, the activity of pyruvate carboxylase, which is the sum of pyruvate carboxylase isoform I and II, was two‐ to fivefold lower in carbon medium containing aspartate, asparagine, glutamate or glutamine instead of ammonium as the nitrogen source, whereas it was 1.5‐ to threefold higher when the ammonium source was substituted by arginine, methionine, threonine or leucine. These enzymatic changes were independent of the nature of the carbon source and closely correlated to the changes in β‐galactosidase from PYC1‐lacZ gene fusion and in PYC1 transcripts. Transfer of exponentially growing cells of the pyc2 mutant from an aspartate or a glutamate medium to an ammonium medium caused a fivefold increase in PYC1 mRNA in less than 30 min, whereas in the inverse experiment, PYC1 transcripts returned within 30 min to the low levels found in aspartate/glutamate medium. By contrast, these conditions affected neither the pyruvate carboxylase activity encoded by PYC2 nor PYC2 mRNA. Considering that changes in PYC1 expression inversely correlated with changes in α‐ketoglutarate concentration or in α‐ketoglutarate/glutamate ratio following the nitrogen shift experiments, and taking into account the pivotal role of this metabolite in ammonium assimilation, it is suggested that changes in α‐ketoglutarate or in the α‐ketoglutarate/glutamate ratio might be implicated in triggering the nitrogen effects on PYC1 expression. The physiological significance of the differential sensitivity of PYC1 and PYC2 genes with respect to the nitrogen source in the growth medium is also discussed.
Purification, cloning and autoproteolytic processing of an aspartic proteinase from Centaurea calcitrapa
Authors: Domingos Ana , Cardoso Paula C. , Xue Zhong‐tian , Clemente Alda , Brodelius Peter E. , Pais Maria S.
Published: August 2003

Plant aspartic proteinases (APs) have been isolated from several seed and leaf sources but the only well characterized enzymes from flowers are cardosins and cyprosins from cardoon, Cynara cardunculus L. Here we report a full‐length cDNA clone encoding an AP named cenprosin from the flowers of Centaurea calcitrapa L., a thistle related to cardoon. As found for all eukaryotic APs, the deduced primary sequence consists of a signal sequence, a propart and a mature enzyme. In addition, an internal sequence region of 104 residues typical only of plant APs (a plant‐specific insert) is present in the primary structure. Northern analysis revealed that the strongest expression is in fresh flowers. The enzyme is also expressed in fairly high amounts in seeds and in leaves, a feature not detected for cardoon APs. The corresponding enzyme was purified in its precursor form from fresh flowers using ammonium‐sulfate precipitation followed by ion‐exchange and hydrophobic‐interaction chromatography. The processing of the precursor into its mature form was studied in vitro. The enzyme underwent autocatalytic processing at pH 3.0 resulting in two chains of 16 and 30 kDa. When dried flowers were used as a starting material for purification, only 16‐ and 30‐kDa chains were obtained, suggesting that autoproteolytic activation of procenprosin in vivo occurs mainly during drying of the flowers. This may indicate a specific degradative role for the enzyme during senescence of the flowers.
Cloning, expression and characterization of the pig liver GDP‐mannose pyrophosphorylase
Authors: Ning Baitang , Elbein Alan D.
Published: August 2003

GDP‐Man, the mannosyl donor for most Man‐containing polymers is formed by the transfer of Man‐1‐P to GTP to form GDP‐Man and PPi. This reaction is catalyzed by the widespread and essential enzyme, GDP‐Man pyrophosphorylase (GMPP). The pig liver GMPP consists of an α subunit (43 kDa) and a β subunit (37 kDa). Purified pig GMPP catalyzes the synthesis of GDP‐Glc (from Glc‐1‐P and GTP) and GDP‐Man (from Man‐1‐P and GTP), but has higher activity for the formation of GDP‐Glc than for synthesis of GDP‐Man. In the present study, we report the cloning of the cDNA for the β subunit of GMPP, and its expression in a bacterial system resulting in the formation of active enzyme. The full length cDNA encoding the β subunit was isolated from a porcine cDNA library, and its predicted gene product showed high amino‐acid sequence homology to GMPPs from other species. The gene was expressed in Escherichia coli cells, and a 37‐kDa protein was over‐produced in these cells. This gene product reacted strongly with antibody reactive to the native β subunit of pig GMPP. Most interestingly, this recombinant protein had high activity for synthesizing GDP‐Man (from Man‐1‐P and GTP), but very low activity for the formation of GDP‐Glc (from Glc‐1‐P and GTP). Other properties of the recombinant protein were also analyzed. This study suggests that the β subunit is the GMPP, whereas the α subunit, or a combination of both subunits, may have the GDP‐Glc pyrophosphorylase activity.
Biochemical and molecular characterization of taurine:pyruvate aminotransferase from the anaerobeBilophila wadsworthia
Authors: Laue Heike , Cook Alasdair M.
Published: August 2003

Bilophila wadsworthiaRZATAU is a Gram‐negative bacterium which converts the sulfonate taurine (2‐aminoethanesulfonate) to ammonia, acetate and sulfide in an anaerobic respiration. Taurine:pyruvate aminotransferase (Tpa) catalyses the initial metabolic reaction yielding alanine and sulfoacetaldehyde. We purified Tpa 72‐fold to apparent homogeneity with an overall yield of 89%. The purified enzyme did not require addition of pyridoxal 5′‐phosphate, but highly active enzyme was only obtained by addition of pyridoxal 5′‐phosphate to all buffers during purification. SDS/PAGE revealed a single protein band with a molecular mass of 51 kDa. The apparent molecular mass of the native enzyme was 197 kDa as determined by gel filtration, which indicates a homotetrameric structure. The kinetic constants for taurine were:Km = 7.1 mm,Vmax = 1.20 nmol·s−1, and for pyruvate:Km = 0.82 mm,Vmax = 0.17 nmol·s−1. The purified enzyme was able to transaminate hypotaurine (2‐aminosulfinate), taurine, β‐alanine and with low activity cysteine and 3‐aminopropanesulfonate. In addition to pyruvate, 2‐ketobutyrate and oxaloacetate were utilized as amino group acceptors. We have sequenced the encoding gene (tpa). It encoded a 50‐kDa peptide, which revealed 33% identity to diaminopelargonate aminotransferase fromBacillus subtilis.
Purification and properties of hydroquinone hydroxylase, a FAD‐dependent monooxygenase involved in the catabolism of 4‐hydroxybenzoate in Candida parapsilosis CBS604
Authors: Eppink Michel H. M. , Cammaart Erwin , van Wassenaar Dick , Middelhoven Wouter J. , van Berkel Willem J. H.
Published: August 2003

The ascomycetous yeast Candida parapsilosis CBS604 catabolizes 4‐hydroxybenzoate through the initial formation of hydroquinone (1,4‐dihydroxybenzene). High levels of hydroquinone hydroxylase activity are induced when the yeast is grown on either 4‐hydroxybenzoate, 2,4‐dihydroxybenzoate, 1,3‐dihydroxybenzene or 1,4‐dihydroxybenzene as the sole carbon source. The monooxygenase constitutes up to 5% of the total amount of protein and is purified to apparent homogeneity in three chromatographic steps. Hydroquinone hydroxylase from C. parapsilosis is a homodimer of about 150 kDa with each 76‐kDa subunit containing a tightly noncovalently bound FAD. The flavin prosthetic group is quantitatively resolved from the protein at neutral pH in the presence of chaotropic salts. The apoenzyme is dimeric and readily reconstituted with FAD.

Hydroquinone hydroxylase from C. parapsilosis catalyzes the ortho‐hydroxylation of a wide range of monocyclic phenols with the stoichiometric consumption of NADPH and oxygen. With most aromatic substrates, no uncoupling of hydroxylation occurs. Hydroxylation of monofluorinated phenols is highly regiospecific with a preference for C6 hydroxylation. Binding of phenol highly stimulates the rate of flavin reduction by NADPH. At pH 7.6, 25 °C, this step does not limit the rate of overall catalysis.

During purification, hydroquinone hydroxylase is susceptible towards limited proteolysis. Proteolytic cleavage does not influence the enzyme dimeric nature but results in relatively stable protein fragments of 55, 43, 35 and 22 kDa. N‐Terminal peptide sequence analysis revealed the presence of two nick sites and showed that hydroquinone hydroxylase from C. parapsilosis is structurally related to phenol hydroxylase from Trichosporon cutaneum. The implications of these findings for the catalytic mechanism of hydroquinone hydroxylase are discussed.
Molecular targets for autoimmune and genetic disorders of neuromuscular transmission
Authors: Vincent Angela , Beeson David , Lang Bethan
Published: August 2003

The neuromuscular junction is the target of a variety of autoimmune, neurotoxic and genetic disorders, most of which result in muscle weakness. Most of the diseases, and many neurotoxins, target the ion channels that are essential for neuromuscular transmission. Myasthenia gravis is an acquired autoimmune disease caused in the majority of patients by antibodies to the acetylcholine receptor, a ligand‐gated ion channel. The antibodies lead to loss of acetylcholine receptor, reduced efficiency of neuromuscular transmission and muscle weakness and fatigue. Placental transfer of these antibodies in women with myasthenia can cause fetal or neonatal weakness and occasionally severe deformities. Lambert Eaton myasthenic syndrome and acquired neuromyotonia are caused by antibodies to voltage‐gated calcium or potassium channels, respectively. In the rare acquired neuromyotonia, reduced repolarization of the nerve terminal leads to spontaneous and repetitive muscle activity. In each of these disorders, the antibodies are detected by immunoprecipitation of the relevant ion channel labelled with radioactive neurotoxins. Genetic disorders of neuromuscular transmission are due mainly to mutations in the genes for the acetylcholine receptor. These conditions show recessive or dominant inheritance and result in either loss of receptors or altered kinetics of acetylcholine receptor channel properties. Study of these conditions has greatly increased our understanding of synaptic function and of disease aetiology.
Cloning and characterization of mouse Klk27, a novel tissue kallikrein expressed in testicular Leydig cells and exhibiting chymotrypsin‐like specificity
Authors: Matsui Hitoshi , Moriyama Akihiko , Takahashi Takayuki
Published: August 2003

A cDNA clone of a new mouse tissue kallikrein, designated mKlk27, was isolated from an adult mouse testis cDNA library. mKlk27 was expressed in the submaxillary glands and testis of the mouse. In testis, mKlk27 gene was expressed exclusively in the Leydig cells of the adult mouse. Active recombinant mKlk27 exhibited chymotrypsin‐like cleavage specificity. A single amino‐acid substitution of Gly for Asp at position 209 in mKlk27 resulted in complete loss of its chymotryptic activity but acquisition of tryptic activity. mKlk27 effectively hydrolyzed casein, gelatin and fibronectin. Insulin‐like growth factor binding protein‐3 was also hydrolyzed by recombinant mKlk27. These results suggest that mKlk27 plays an important role in association with the function of the adult mouse testis.
Cloning and bacterial expression of monomeric short‐chain dehydrogenase/reductase (carbonyl reductase) from CHO‐K1 cells
Authors: Terada Tomoyuki , Sugihara Yukiko , Nakamura Kayo , Sato Ryuichiro , Inazu Norihisa , Maeda Masatomo
Published: August 2003

Mammalian carbonyl reductase (EC 1.1.1.184) is an enzyme that can catalyze the reduction of many carbonyl compounds, using NAD(P)H. We isolated a cDNA of carbonyl reductase (CHO‐CR) from CHO‐K1 cells which was 1208 bp long, including a poly(A) tail, and contained an 831‐bp ORF. The deduced amino‐acid sequence of 277 residues contained a typical motif for NADP+‐binding (TGxxxGxG) and an SDR active site motif (S‐Y‐K). CHO‐CR closely resembles mammalian carbonyl reductases with 71–73% identity. CHO‐CR cDNA had the highest similarity to human CBR3 with 86% identity. Using the pET‐28a expression vector, recombinant CHO‐CR (rCHO‐CR) was expressed in Escherichia coli BL21 (DE3) cells and purified with a Ni2+‐affinity resin to homogeneity with a 35% yield. rCHO‐CR had broad substrate specificity towards xenobiotic carbonyl compounds. RT‐PCR of Chinese hamster tissues suggest that CHO‐CR is highly expressed in kidney, testis, brain, heart, liver, uterus and ovary. Southern blotting analysis indicated the complexity of the Chinese hamster carbonyl reductase gene.
Conversion between two cytochalasin B‐binding states of the human GLUT1 glucose transporter
Authors: Gottschalk Ingo , Lundqvist Andreas , Zeng Cheng‐Ming , Hägglund Christine Lagerquist , Zuo Shu‐Sheng , Brekkan Eggert , Eaker David , Lundahl Per
Published: August 2003

Two cytochalasin B‐binding states of the human red blood cell facilitative glucose transporter GLUT1 were studied, one exhibiting one cytochalasin B‐binding site on every second GLUT1 monomer (state 1) and the other showing one site per monomer (state 2). Quantitative affinity chromatography of cytochalasin B was performed on (a) biotinylated red blood cells, (b) cytoskeleton‐depleted red blood cell membrane vesicles, and (c) GLUT1 proteoliposomes. The cells were adsorbed on streptavidin‐derivatized gel beads, and the vesicles and proteoliposomes entrapped in dextran‐grafted agarose gel beads. Cytochalasin B binding to free vesicles and proteoliposomes was analyzed by Hummel and Dreyer size‐exclusion chromatography and ultracentrifugation. Analysis of the biotinylated cells indicated an equilibrium between the two GLUT1 states. GLUT1 in free membrane vesicles attained state 2, but was converted into state 1 on entrapment of the vesicles. Purification of GLUT1 in the presence of non‐ionic detergent followed by reconstitution produced GLUT1 in state 1. This state was maintained after entrapment of the proteoliposomes. Finally, GLUT1 showed slightly higher affinity for cytochalasin B in state 1 than in state 2. In summary, the cytochalasin B‐binding state of GLUT1 seemed to be affected by (a) biotinylation of the cell surface, (b) removal of the cytoskeleton at high pH and low ionic strength, (c) interaction between the dextran‐grafted agarose gel matrix and the membrane vesicles, and (d) reconstitution to form proteoliposomes.
Efficient DNA base excision repair in ataxia telangiectasia cells
Authors: Cappelli Enrico , Rossi Ottavio , Chessa Luciana , Frosina Guido
Published: August 2003

Ataxia telangiectasia (A‐T) cells are sensitive to a broad range of free‐radical‐producing and alkylating agents. Damage caused by such agents is in part repaired by base excision [base excision repair (BER)]. Two BER pathways have been demonstrated in mammalian cells: a single‐nucleotide‐insertion pathway and a long‐patch pathway involving resynthesis of 2–10 nucleotides. Although early studies failed to detect DNA‐repair defects in A‐T cells exposed to ionizing radiation and radiomimetic agents, more recent experiments performed in non‐dividing A‐T cells and the demonstrated interaction of the A‐T‐mutated protein (ATM) with the BRCA1 gene product suggest that a DNA‐repair defect may underlie, at least in part, the radiation sensitivity in A‐T cells. We have analysed BER of a single abasic site or a single uracil in two A‐T families, using an in vitro BER system. In both families, the mutation involved was homozygous and completely inactivated the ATM protein. No difference was observed between affected individuals and heterozygous or homozygous wild‐type relatives in their capacity to perform DNA repair by either one‐nucleotide insertion or the long‐patch pathway. Hence, the putative DNA‐repair defect in A‐T cells, if any, does not involve BER.
X‐ray structure of acarbose bound to amylomaltase from Thermus aquaticus
Authors: Przylas Ingo , Terada Yoshinobu , Fujii Kazutoshi , Takaha Takeshi , Saenger Wolfram , Sträter Norbert
Published: August 2003

As a member of the α‐amylase superfamily of enzymes, amylomaltase catalyzes either the transglycosylation from one α‐1,4 glucan to another or an intramolecular cyclization. The latter reaction is typical for cyclodextrin glucanotransferases. In contrast to these enzymes, amylomaltase catalyzes the formation of cyclic glucans with a degree of polymerization larger than 22. To characterize the factors that determine the size of the synthesized cycloamyloses, we have analyzed the X‐ray structure of amylomaltase from Thermus aquaticus in complex with the inhibitor acarbose, a maltotetraose derivative, at 1.9 Å resolution. Two acarbose molecules are bound to the enzyme, one in the active site groove at subsite −3 to +1 and a second one ≈ 14 Å away from the nonreducing end of the acarbose bound to the catalytic site. The inhibitor bound to the catalytic site occupies subsites −3 to +1. Unlike the situation in other enzymes of the α‐amylase family, the inhibitor is not processed and the inhibitory cyclitol ring of acarbose, which mimicks the half chair conformation of the transition state, does not bind to catalytic subsite −1. The minimum ring size of cycloamyloses produced by this enzyme is proposed to be determined by the distance of the specific substrate binding sites at the active site and near Tyr54 and by the size of the 460s loop. The 250s loop might be involved in binding of the substrate at the reducing end of the scissile bond.
Structural and serological studies of the related O‐specific polysaccharides of Proteus vulgaris O21 and Proteus mirabilis O48 having oligosaccharide‐phosphate repeating units
Authors: Bartodziejska Beata , Toukach Filip V. , Vinogradov Evgeny V. , Senchenkova Sofia N. , Shashkov Alexander S. , Ziolkowski Andrzej , Czaja Jolanta , Perry Malcolm B. , Knirel Yuriy A. , Rozalski Antoni
Published: August 2003

The O‐specific polysaccharide chains (O‐antigens) of the lipopolysaccharides (LPSs) of Proteus mirabilis O48 and Proteus vulgaris O21 were found to have tetrasaccharide and pentasaccharide repeating units, respectively, interlinked by a glycosidic phosphate. Polysaccharides and an oligosaccharide were derived from the LPSs by various degradation procedures and studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, H‐detected 1H,13C and 1H,31P HMQC experiments. The following related structures of the repeating units of the O‐antigens were established (top: Proteus mirabilis O48; bottom: Proteus vulgaris O21)

The O‐specific polysaccharide of P. vulgaris O21 has the same structure as that of Hafnia allvei 744 and PCM 1194 [Petersson C., Jachymek, W., Klonowska, A., Lugowski, C., Niedziela, T. & Kenne, L. (1997) Eur. J. Biochem., 245, 668–675], except that the GlcN residue carries the N‐acetyl rather than the N‐[(R)‐3‐hydroxybutyryl] group. Serological investigations confirmed the close relatedness of the Proteus and Hafnia O‐antigens studied.
Interaction of tyrosine phenol‐lyase with phosphoroorganic analogues of substrate amino acids
Authors: Faleev Nikolai G. , Zhukov Yurii N. , Khurs Elena N. , Gogoleva Olga I. , Barbolina Maria V. , Bazhulina Natalia P. , Belikov Vassili M. , Demidkina Tatyana V. , Khomutov Radii M.
Published: August 2003

The phosphinic analogues of tyrosine and pyruvate were first demonstrated to be substrates in the reactions of elimination and synthesis catalyzed by tyrosine phenol‐lyase. Kinetic parameters of the enzymatic process were determined, and the first enzymic synthesis of an aminophosphinic acid was carried out. Replacement of the planar HOOC‐group by the tetrahedral (HO)(O)PH‐group in the substrate slightly affected its affinity for the enzyme but substantially diminished the conversion rate. For phosphonic analogues, containing (HO)2(O)P group, the affinity to the enzyme was decreased considerably while the conversion was completely prevented. Thus, the structural parameters of the acid group are important not only for the affinity for the enzyme, but also for the formation of the catalytically competent conformation of the active site.
Molecular organization, expression and chromosomal localization of the mouse pronapsin gene
Authors: Tatnell Peter J. , Cook Matthew , Peters Christoph , Kay John
Published: August 2003

Napsins have been identified only very recently as new aspartic proteinases of the pepsin family. Isolation, sequencing and functional analysis of the mouse genomic locus indicates that the organization of the pronapsin gene into nine exons is identical to that of other mammalian aspartic proteinase precursors, including pepsinogen. However, the additional C‐terminal residues, which are a distinguishing feature of napsins, are encoded within exon 9 and not within an additional exon. Quantitation of pronapsin mRNA using RT‐PCR indicates that the gene is transcribed in lung, kidney and spleen but not in heart. Regulation of gene expression was not influenced by the extent of CpG methylation but depended on the recognition of potential binding motifs in the promoter region by specific transcription factors such as YY‐1. The single copy of the mouse pronapsin gene was located on chromosome 7. In humans, there are two pronapsin genes and, based on the mouse information, preliminary structures were deduced for these from sequences in the human genome databases. They appear to be located together on chromosome 19.
Molecular cloning of NADPH‐cytochrome P450 oxidoreductase from silkworm eggs
Authors: Horike Nanao , Takemori Hiroshi , Nonaka Yasuki , Sonobe Haruyuki , Okamoto Mitsuhiro
Published: August 2003

Using RT‐PCR, a cDNA fragment of NADPH‐cytochrome P450 oxidoreductase from silkworm, Bombyx mori, was cloned from three‐day‐old nondiapause eggs. RACE was used to isolate the ends of the DNA. The full‐length cDNA obtained was composed of 3471 bp with an open reading frame encoding a protein of 687 amino‐acid residues with a relative molecular mass of 77 700. The protein, fused with glutathione S‐transferase, was expressed in Escherichia coli and purified to homogeneity. The fused protein not only had NADPH‐dependent cytochrome c‐reducing activity, but also acted as an electron carrier from NADPH to bovine adrenal 21‐hydroxylase P450 in the steroid hydroxylation reaction, confirming that the protein is the silkworm NADPH‐cytochrome P450 oxidoreductase. Ecdysone 20‐hydroxylase activity in the nondiapause egg microsomes increased until the fourth day after oviposition, and then decreased, little being detected on the ninth day. An antibody raised against the P450 reductase inhibited the ecdysone hydroxylation. Immunoblot analyses of the microsomes indicated that the P450 reductase protein appeared distinctly in the three‐day‐old nondiapause eggs and, in contrast to the developmental pattern of ecdysone hydroxylase activity, continued to increase as the embryos developed. These results suggest that ecdysone hydroxylation in the early stage of embryogenesis is dependent on the presence of both P450 reductase and ecdysone 20‐hydroxylase P450, but its gradual reduction in the later stage may be due to the decrease in the level of ecdysone 20‐hydroxylase P450.
Synchronized transcriptional gene expression of H+‐ATP synthase subunits in different tissues of Fischer 344 rats of different ages
Authors: Himeda Toshiki , Morokami Kayoko , Arakaki Naokatu , Shibata Hirofumi , Higuti Tomihiko
Published: August 2003

Little is known about the relationship between the stoichiometry of polypeptides of multisubunit enzyme complexes and the absolute amount of each transcript of the complexes in mammalian tissues. Here we showed that the absolute amounts of the transcripts of most subunits of rat H+‐ATP synthase examined greatly differed in the different tissues, showing the following hierarchy of tissue‐specificity: heart > kidney > brain ≈ liver. However, surprisingly, there was no difference in the expression pattern of these in terms of the molar ratio of each transcript, indicating a nearly similar stoichiometric expression pattern irrespective of tissue or age of the rat. Therefore, the present finding clearly indicates that most of the transcripts of the 16 subunits of rat H+‐ATP synthase were concertedly and synchronously expressed, having a constant expression pattern of the transcripts, irrespective of tissue or age of the rats. This is the first report of the absolute amounts of the transcripts of this multisubunit enzyme.
Localization of the mosaic transmembrane serine protease corin to heart myocytes
Authors: Hooper John D. , Scarman Anthony L. , Clarke Belinda E. , Normyle John F. , Antalis Toni M.
Published: August 2003

Corin cDNA encodes an unusual mosaic type II transmembrane serine protease, which possesses, in addition to a trypsin‐like serine protease domain, two frizzled domains, eight low‐density lipoprotein (LDL) receptor domains, a scavenger receptor domain, as well as an intracellular cytoplasmic domain. In in vitro experiments, recombinant human corin has recently been shown to activate pro‐atrial natriuretic peptide (ANP), a cardiac hormone essential for the regulation of blood pressure. Here we report the first characterization of corin protein expression in heart tissue. We generated antibodies to two different peptides derived from unique regions of the corin polypeptide, which detected immunoreactive corin protein of approximately 125–135 kDa in lysates from human heart tissues. Immunostaining of sections of human heart showed corin expression was specifically localized to the cross striations of cardiac myocytes, with a pattern of expression consistent with an integral membrane localization. Corin was not detected in sections of skeletal or smooth muscle. Corin has been suggested to be a candidate gene for the rare congenital heart disease, total anomalous pulmonary venous return (TAPVR) as the corin gene colocalizes to the TAPVR locus on human chromosome 4. However examination of corin protein expression in TAPVR heart tissue did not show evidence of abnormal corin expression. The demonstrated corin protein expression by heart myocytes supports its proposed role as the pro‐ANP convertase, and thus a potentially critical mediator of major cardiovascular diseases including hypertension and congestive heart failure.
Characterization of a rainbow trout matrix metalloproteinase capable of degrading type I collagen
Authors: Saito Masataka , Sato Kenji , Kunisaki Naomichi , Kimura Shigeru
Published: August 2003

Matrix metalloproteinases (MMPs) are widely distributed in vertebrate tissues and form a large family consisting of at least four distinct subfamilies. Higher vertebrate MMP‐13 is well‐known as collagenase‐3, which represents the third member of a collagenase subfamily. In this study, we cloned cDNA coding for a unique fish homologue of human MMP‐13 from a rainbow trout fibroblast cDNA library. The cDNA was 2.1 kb long and contained an open reading frame encoding a protein of 475 amino acids. The catalytic domain of the protein was 66% identical to the human counterpart with the greatest degree of identity occurring in the zinc binding site. In addition, it possessed three amino‐acid residues (Tyr122, Asp233 and Gly235) characteristic of the collagenase subfamily, together with a six residue insertion which did not occur in the collagenase subfamily. Then the isolated cDNA was expressed in Escherichia coli and the recombinant protein was found to degrade gelatin and skin type I collagen. It is worth noting that rainbow trout type I collagen was more susceptible to proteolysis with the recombinant protein when compared with the calf one. It appeared that the recombinant protein also cleaved the nonhelical regions of rainbow trout muscle type V collagen. These results have revealed that the cDNA encodes a unique MMP‐13 of rainbow trout. This is the first report of cDNA coding for fish MMP capable of degrading type I collagen.
Multiple meanings of self harm: A critical review
Authors: McAllister Margaret
Published: August 2003

ABSTRACT: The issue of self harm is a popular inclusion in various contemporary journals focusing on health and in particular women's health. This paper seeks to condense, critically analyse and more simply explain selected literature in order to raise awareness of the multiple ways of understanding self harm. Raised awareness may be a useful strategy in thinking about self harm in novel ways and thus providing alternate pathways for responding to the individual and society.
Understanding the need for personal space boundary restoration in women‐client survivors of intrafamilial childhood sexual abuse
Authors: Geanellos Rene
Published: August 2003

ABSTRACT: A large volume of literature exists on childhood sexual abuse (CSA) and nursing literature on this topic continues to develop. However, discussion of boundaries in relation to intrafamilial childhood sexual abuse (ICSA) and to its significance in terms of personal space boundary (PSB) violation, is lacking. This is important, as PSB violation creates difficulties for adult survivors of ICSA and for nurses who care for these people as clients. As all experiences of CSA cannot be addressed, discussion focuses on women who are clients of mental health services and who have experienced ICSA. Using Scott's work on boundaries and her diagrammatic conceptualization of PSBs, links are created between ICSA boundary violation, resultant PSB regulation difficulties and principles to guide nursing practice. Discussion centres on understanding the need for PSB restoration in women‐client survivors of ICSA.
The challenges of mental health care reform for contemporary mental health nursing practice: Delivery of nursing care
Authors: Cleary Michelle
Published: August 2003

ABSTRACT:
This ethnographic account of mental health nursing in a 22‐bed acute inpatient facility in New South Wales, Australia, uncovered cultural meaning and cultural realities associated with the delivery of nursing care within the context of current challenges, demands, and influences brought about by service reforms. The findings demonstrate that mental health nurses have been responsive to changes brought about by the reforms. The ability of nurses to readily identify service gaps in their everyday practice provides them with the opportunity to develop strategies to respond to workplace challenges. As such, findings of the study contribute to current discussions concerning acute inpatient mental health nursing practice.
Administration of ‘as needed’ psychotropic medication: A retrospective study
Authors: Curtis Janette , Capp Kim
Published: August 2003

ABSTRACT:
Mental health nurses are increasingly encouraged to discuss and document the outcomes of their nursing practice in order to maximize the care that they offer clients. One frequent function performed by mental health nurses working in inpatient units is the administration of ‘as required’ (PRN) psychotropic medications. However, according to the literature, this important aspect of nursing practice has been poorly explored and studies that have been undertaken demonstrate that nursing documentation of PRN medication administration is often inadequate. This study presents the results of a descriptive study examining the administration of PRN psychotropic medication at a secure acute inpatient mental health unit attached to a regional hospital in Australia. Of major concern is the findings of inadequate or no documentation regarding PRN administration. In 41% of cases the results of the PRN administration was not documented in the nursing progress notes, in 38.6% of entries it was not possible to identify who initiated the request for medication and in 9.1% of cases no reason was given for the administration of PRN medication.
Close‐observation areas in acute psychiatric units: A literature review
Authors: O'Brien Louise , Cole Rose
Published: August 2003

ABSTRACT:Close‐observation areas in Australian inpatient psychiatric units are locked areas usually within an open ward. Despite patient acuity, and the inherent difficulties in this area, little has been written that addresses either the processes or goals of containing patients, the role of nurses, or the skills involved. This paper examines the literature related to close‐observation areas and argues that they are highly demanding of expert psychiatric nursing skills. Nurses need to advocate for humane, well‐resourced areas, staffed with highly skilled nurses in order to fulfil the obligations of the national nursing and mental health service standards and to reduce the deleterious effects of hospitalization on patients.
Is informed consent necessary for paediatric computed tomography?
Authors: De Campo JF , Lau KKP , De Campo MP
Published: August 2003

Abstract:  Computed tomography (CT) is a valuable examination in children, but the lifetime cancer mortality attributed to CT radiation in children is an order of magnitude higher than in adults. Therefore, it is important that radiologists and referring clinicians ensure that parents are fully informed of the radiation risks, the appropriateness of the examination is considered and imaging protocols are tailored to reduce the radiation burden.
Soy infant formula and phytoestrogens
Authors: Tuohy PG
Published: August 2003

Abstract:  Soy infant formula contains high levels of the isoflavones, genistein and daidzein, which are commonly referred to as phytoestrogens. These are non‐steroidal chemicals with structural similarities to estrogen. Infants consuming soy formula have high levels of circulating isoflavones. These are an order of magnitude greater than the levels of isoflavones which have been shown to produce physiological effects in adult women consuming a high soy diet. There is conflicting evidence about the risks and benefits of soy phytoestrogens, with research presenting a contradictory picture. Some reviewers suggest that early exposure to soy may prevent cancer and heart disease. However, there is very little research on the effects of consumption of soy phytoestrogens by human neonates. Against this generally positive view there is an increasing number of recent reports that suggest that in experimental animals, phytoestrogens have adverse effects with respect to carcinogenesis, reproductive function, immune function, and thyroid disease. Despite the absence of adequate scientific research that quantifies the level of risk to infants, most would argue for a precautionary approach to be taken in situations where there are potential developmental effects from the consumption of pharmacologically active compounds in infancy and childhood.
Rules for clinical diagnosis in babies with ambiguous genitalia
Authors: Low Y , Hutson JM ,   Murdoch Childrens Research Institute Sex Study Group
Published: August 2003

Abstract:  Intersex disorders are rare and complex; yet, in each case of genital ambiguity, accurate and expeditious management is required of the clinician. This article reviews the embryology of sexual differentiation, from which some ‘rules’ of diagnosis are derived. A simplified approach to the interpretation of clinical signs in ambiguous genitalia is presented and discussed.
Tolerability of topiramate in children and adolescents
Authors: Reith D , Burke C , Appleton DB , Wallace G , Pelekanos J
Published: August 2003

Objective:  To examine the tolerability of topiramate (TPM) in paediatric practice.

Methodology:  A retrospective cohort study of patients aged less than 18 years commenced on TPM by paediatric neurologists. Patients were identified from the dispensing databases of two paediatric tertiary referral hospitals and from the authority prescription records of four paediatric neurologists. The clinical data were obtained from the patients’ medical records.

Results:  There were 159 patients who were identified as having been commenced on TPM and follow‐up data were available for 127 (80%) patients. The median (range) age at commencement of TPM was 8.1 (0.5−17.9) years, with 12 patients aged less than 2 years. After 4 years, 60% of patients had ceased the medication. Treatment limiting adverse effects included aggression/psychosis (n = 10), cognitive impairment/sedation (n = 6), anorexia/weight loss (n = 4) and desquamation (n = 1). Prior aggression (hazard ratio 5.91 (2.12−16.44)) and female gender (hazard ratio 2.94 (1.02−8.41)) were risk factors for ceasing TPM because of an adverse event. Thirty percent of children commenced on TPM experienced a treatment limiting adverse effect within 2 years of commencement.

Conclusions:  The frequency of treatment limiting adverse events in children receiving topiramate is higher than previously reported.
Unintentional residential child injury surveillance in Hong Kong
Authors: Chan CC , Luis Bpk , Chow Cb , Cheng JCY , Wong Tw , Chan K
Published: August 2003

Objectives:  To provide an overall pattern of morbidity in unintentional residential childhood injuries (URCI) in Hong Kong.

Methodology:  A cross‐sectional telephone survey of caregivers of children aged under 16‐years and adolescents suffering from URCI and admitted to three selected local Accident and Emergency Departments.

Results:  Falls, cuts and scalds were the most common external causes of URCI observed, while boys predominated in the sample population. Most of the observed URCI were of moderate to mild severity. Children of new immigrant mothers were more likely to receive first aid immediately after the incidents. Parents were aware of potentially injurious behaviour and intervened on occasion, but most resorted to verbal warnings only.

Conclusions:  Prevalence of falls among observed URCI offers evidence in support of the hypothesis that the high population density in Hong Kong plays an integral role in understanding mechanisms of morbidity. Parents show concern about URCI but often lack substantial action that modifies injury risk. Considering the local injury differentials, an active prevention effort such as behavioural intervention and education for parents may be useful.
Gastroenteritis and environmental health among Aboriginal infants and children in Western Australia
Authors: Gracey M , Cullinane J
Published: August 2003

Objective:  To retrospectively examine rates of hospitalization of infants and children in Western Australia for gastroenteritis from 1994 through 2000.

Methodology:  Analysis of hospital separations data from the Hospital Morbidity Data System of the Department of Health, Western Australia.

Results:  Rates of hospitalization of Aboriginal infants and children for gastroenteritis from 1994 to 2000 in Western Australia were approximately seven times higher than for their non‐Aboriginal peers. This was despite some decline in Aboriginal hospitalization rates over the study period. This may have been due to a simultaneous decline in hospital admissions of non‐Aboriginal infants and children. Rates of hospitalization of Aboriginal infants and children were much higher in non‐metropolitan rather than in metropolitan regions. There was a remarkable fall in the deaths of Aboriginal infants and children from gastroenteritis between 1970 and 2000. There were no deaths recorded in Western Australian hospitals from this disease from 1990 to 2000.
Background severity of asthma in children discharged from the emergency department
Authors: Khan Msr , O'Meara M , Henry Rl
Published: August 2003

Objective:  Attendance at an Emergency Department (ED) with an acute attack of asthma may be indicative of undertreatment of persistent disease. However, many presentations are in children with infrequent episodic asthma. The aim of this study was to characterize the pattern of asthma of children discharged from ED to determine whether there was potential to improve underlying disease control.

Methodology:  This was a cohort study. Three hundred and ten parental caretakers of 1 to 15‐year‐old children, attended and discharged from an ED with asthma, completed an asthma control questionnaire, an asthma knowledge questionnaire and a caregiver's quality of life questionnaire. Background severity of asthma was classified and medication history was assessed. Also included were those with their first attack of asthma.

Results:  One hundred and thirty‐two (43%) children had infrequent episodic asthma, 105 (34%) frequent episodic, 40 (13%) persistent asthma and 33 (11%) first attack asthma. Thirty‐nine per cent of children were not receiving preventer therapy and this seemed appropriate; 14% of children with frequent episodic and persistent asthma were not receiving appropriate preventer therapy; and a further 34% had frequent symptoms despite receiving preventer therapy.

Conclusions:  We observed deficiencies in use of preventer medications, use of written asthma management plans and lack of parental knowledge in some children with established asthma who presented to an ED. There was also a large number of children who did not have frequent background symptoms or who presented with their first episode.
Association of bullying with adolescent health‐related quality of life
Authors: Wilkins‐Shurmer A , O'Callaghan Mj , Najman Jm , Bor W , Williams Gm , Anderson Mj
Published: August 2003

Objective:  To examine the relationship between being bullied and health‐related quality of life (HR‐QOL) in adolescence.

Methodology:  Subjects were a cohort of 805 adolescents with a mean age of 13.6 years (standard deviation 0.2 years). An adolescent questionnaire elicited the frequency of being bullied. HR‐QOL was measured using the Child Health Questionnaire − Parent Report Form (CHQ‐PF50) and Dartmouth COOP Functional Health Assessment Charts for Adolescents.

Results:  Thirty‐six per cent of boys and 38 per cent of girls reported being bullied at least weekly. Adolescent psychosocial HR‐QOL was inversely related to frequency of being bullied, while physical HR‐QOL was not related.

Conclusion:  Peer bullying is an important determinant of adolescent HR‐QOL with a negative impact on psychosocial well‐being.
Dental visits by Australian preschool children
Authors: Slack‐Smith LM
Published: August 2003

Objective:  To provide population‐based data on dental visits in preschool children, to assist in the planning of such services and preventive programs.

Methodology:  Dental visits in young children are described and associated factors are investigated using data from the 1995 National Health Survey.

Results:  Thirty‐eight per cent of children aged 3−4 years had ever visited a dental professional with 31% having visited a dental professional in the previous 12 months (total n = 1596). Factors associated with having ever visited a dental professional included age in years (χ2 = 74.16, P < 0.0001), state of residence (χ2 = 25.81, P= 0.0005) and private health insurance (χ2 = 7.96, P= 0.005). Similar factors were associated with visits in last 12 months. Western Australia had the lowest proportion of preschool children aged 3−4 years having ever visited a dental professional (29%) while South Australia had the highest proportion (48%). The major reasons given for dental visits were check‐up and fillings.

Conclusion:  Data regarding children not as likely to attend dental services can be used to target services and assist in ensuring preschool children receive appropriate dental care.
Children under 5 years presenting to paediatricians with near‐drowning
Authors: Ross Fi , Elliott Ej , Lam Lt , Cass Dt
Published: August 2003

Objective:  To characterize children aged under 5 years who present to paediatricians following near‐drowning and the circumstances surrounding the event, identify high‐risk groups and document short‐term outcome.

Methodology:  Monthly notifications to the Australian Paediatric Surveillance Unit (an active, national surveillance system) between 1994 and 1996. Collection of additional case information from reporting doctors by postal questionnaire.

Results:  All 169 reported cases of near‐drowning were admitted to hospital (mean (SD) stay 6 (17) days) and 15% required intensive care (mean (SD) stay 19 (32) days). The mean (SD) age for near drowning was 26 (13) months and 22% children were aged between 12 and 18 months. Males predominated (1.6:1) and 69 (41%) of episodes occurred in summer (December − February). The majority (82%) of near‐drownings occurred in the child's home, usually in a swimming pool or bath. Children who nearly drowned at home were significantly younger than those who nearly drowned in natural waterways or public pools. Neurological damage at discharge following near‐drowning was reported in 7%.

Conclusions:  Children reported in this national case series represent the severe end of the spectrum of those who nearly drown, as indicated by their presentation to a paediatrician, universal hospitalization and adverse neurological outcome. The home is the site of most near‐drownings and males and toddlers were at particular risk. Unimpeded access to pools and lack of supervision were identified as potentially modifiable factors for prevention. The study suggests the need for additional community education regarding the risks of near‐drowning and for further research on long‐term neuro‐developmental outcomes following near‐drowning.