bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of urokinase in childhood pleural empyema
Authors: Barbato A. , Panizzolo C. , Monciotti C. , Marcucci F. , Stefanutti G. , Gamba P.G.
Published: December 2002

Urokinase is an enzyme with a fibrinolytic effect that facilitates pleural empyema drainage through a chest tube. The aim of this study was to assess the risk of pneumothorax, the need for pleural debridement surgery, the persistence of fever, and the number of days in hospital in a group of children with parapneumonic pleural empyema treated with urokinase.

This was an uncontrolled retrospective study on children suffering from parapneumonic empyema. Data collected on 17 children treated with urokinase were compared with 11 children treated prior to the advent of urokinase (the “historic” group). The urokinase was instilled in the pleural cavity over a period ranging from 2–8 days, amounting to a median total dose per kilogram of body weight of 18,556 IU (range, 7,105–40,299). Surgical treatment of the empyema involved drainage tube placement and/or debridement of the pleural cavity.

Three children developed pneumothorax during their hospital stay, and one more case occurred 6 months after the child had recovered from his empyema; there were 3 cases of pneumothorax during the acute phase in the “historic” group (P = 0.54). Five children in the urokinase group were debrided and 12 were only drained, as opposed to 9 and 2, respectively, in the “historic” group (P = 0.02). The overall hospital stay was 17 days for the urokinase group, and 24 for the “historic” group (P = 0.02). No bleeding or other major complications were reported in the group treated with urokinase.

In conclusion, urokinase treatment does not carry a risk of pneumothorax, while it does reduce hospital stay and the need for pleural debridement. Pediatr Pulmonol. 2003; 35:50–55. © 2003 Wiley‐Liss, Inc.
A magnitude‐robust control chart for monitoring and estimating step changes for normal process means
Authors: Pignatiello Joseph J. , Simpson James R.
Published: December 2002

Statistical process control charts are intended to assist operators of a usually stable system in monitoring whether a change has occurred in the process. When a change does occur, the control chart should detect it quickly. If the operator can also be provided information that aids in the search for the special cause, then critical off‐line time can be saved. We investigate a process‐monitoring tool that not only provides speedy detection regardless of the magnitude of the process shift, but also supplies useful change point statistics. A likelihood ratio approach can be used to develop a control chart for permanent step change shifts of a normal process mean. The average run length performance for this chart is compared to that of several cumulative sum (CUSUM) charts. Our performance comparisons show that this chart performs better than any one CUSUM chart over an entire range of potential shift magnitudes. The likelihood ratio approach also provides point and interval estimates for the time and magnitude of the process shift. These crucial change‐point diagnostics can greatly enhance special cause investigation. Copyright © 2002 John Wiley & Sons, Ltd.
EWMA techniques for computer intrusion detection through anomalous changes in event intensity
Authors: Ye Nong , Borror Connie , Zhang Yebin
Published: December 2002

Intrusion detection is used to monitor and capture intrusions into computer and network systems, which attempt to compromise the security of computer and network systems. To protect information systems from intrusions and thus assure the reliability and quality of service of information systems, it is highly desirable to develop techniques that detect intrusions into information systems. Many intrusions manifest in dramatic changes in the intensity of events occurring in information systems. Because of the ability of exponentially weighted moving average (EWMA) control charts to monitor the rate of occurrences of events based on the their intensity, we apply three EWMA statistics to detect anomalous changes in the events intensity for intrusion detections. They include the EWMA chart for autocorrelated data, the EWMA chart for uncorrelated data and the EWMA chart for monitoring the process standard deviation. The objectives of this paper are to provide design procedures for realizing these control charts and investigate their performance using different parameter settings based on one large dataset. The early detection capability of these EWMA techniques is also examined to provide the guidance about the design capacity of information systems. Copyright © 2002 John Wiley & Sons, Ltd.
Phase I control charts for times between events
Authors: Jones L. Allison , Champ Charles W.
Published: December 2002

A count of the number of defects is often used to monitor the quality of a production process. When defects rarely occur in a process, it is often desirable to monitor the time between the occurrence of each defect rather than a count of the number of defects. An exponential distribution often provides a useful model of the time between defects. Phase I control charts for exponentially distributed processes are discussed. Methods for computing the control limits are given and the overall Type I error rates of these charts are evaluated. Copyright © 2002 John Wiley & Sons, Ltd.
Model‐based control chart for autoregressive and correlated data
Authors: Loredo Elvira N. , Jearkpaporn Duangporn , Borror Connie M.
Published: December 2002

In recent years, statistical process control for autocorrelated processes has received a great deal of attention. This is due in part to the improvements in measurement and data collection that allow processes to be sampled at higher frequency rates and, hence, data autocorrelation. A method for monitoring autocorrelated processes based on regression adjustment is presented in this paper. The performance of the residual‐based control chart in terms of the average run length is compared to observation‐based control charts via Monte Carlo simulations. In general, the observation‐based control charts perform very poorly when data are correlated over time. Under the assumption that the model is correct, the residual‐based control charts are superior for all cases considered here. This suggests using a residual‐based control chart to detect the mean shift. This is recommended particularly for chemical processes where there are often cascade processes with several inputs but only a few outputs, and where many of the variables are highly autocorrelated. Copyright © 2002 John Wiley & Sons, Ltd.
Effect of annexin I on insulin secretion through surface binding sites in rat pancreatic islets
Authors: Hong Shin-Hee , Won Jong Hak , Yoo Seung-Ah , Auh Chung-Kyoon , Park Young Min
Published: December 2002

This study investigates the effect of extracellular annexin I (Anx I) on regulating insulin secretion in isolated rat pancreatic islets. Results show that Anx I stimulates insulin release in pancreatic islets regardless of the presence or absence of extracellular Ca2+. In particular, confocal microscopy shows that Anx I binds to the surface of islet cells in the absence of extracellular Ca2+. However, insulin secretion through Anx I significantly decreases in trypsin‐treated islets. Likewise, there is minimal binding of Anx I to the surface of trypsin‐treated islets. Anti‐Anx I polyclonal antibody also inhibits the stimulating effect of Anx I on insulin secretion. These results indicate that Anx I is capable of binding to the cell surface receptor, in order to regulate the stimulation of insulin release in rat pancreatic islets.
A cluster of familial Creutzfeldt–Jakob disease mutations recapitulate conserved residues in Doppel: a case of molecular mimicry?
Authors: Mastrangelo Peter , Serpell Louise , Dafforn Tim , Lesk Arthur , Fraser Paul , Westaway David
Published: December 2002

Intrachromosomal deletions linking Dpl expression to the PrP promoter produce cerebellar degeneration that can be abrogated by the introduction of wild‐type PrP transgenes. Since Dpl‐like truncated forms of PrP are neuropathogenic in mice and likewise counterbalanced by expression of PrPC we asked whether naturally occurring mutant forms of human PrP have Dpl‐like attributes. Five PRNP missense mutations causing familial Creutzfeldt–Jakob disease (F‐CJD) map to a helical region found in both PrPC and Dpl and result in amino acids identical to conserved residues in Dpl. These F‐CJD alleles may cause mutant PrP to become a weak mimetic of Dpl structure and/or function.
Interaction of FLU, a negative regulator of tetrapyrrole biosynthesis, with the glutamyl‐tRNA reductase requires the tetratricopeptide repeat domain of FLU
Authors: Meskauskiene Rasa , Apel Klaus
Published: December 2002

Regulation of tetrapyrrole biosynthesis in plants has been attributed to feedback control of glutamyl‐tRNA reductase (GLU‐TR) by heme. Recently, another negative regulator, the FLU protein, has been discovered that operates independently of heme. A truncated form of FLU that contains two domains implicated in protein–protein interaction was co‐expressed in yeast with either GLU‐TR or glutamate‐1‐semialdehyde‐2‐1‐aminotransferase (GSA‐AT), the second enzyme involved in δ‐aminolevulinic acid (ALA) biosynthesis. FLU interacts strongly with GLU‐TR but not with GSA‐AT. Two variants of FLU that carry single amino acid exchanges within their coiled coil and tetratricopeptide repeat (TPR) domains, respectively, were also tested. Only the FLU variant with the mutated TPR motif lost the capacity to interact with GLU‐TR.
Transcriptional activation of collagenase‐3 by transforming growth factor‐β1 is via MAPK and Smad pathways in human breast cancer cells
Authors: Selvamurugan Nagarajan , Fung Ziawei , Partridge Nicola C
Published: December 2002

Transforming growth factor (TGF)‐β1, a crucial molecule in metastatic bone cancer, stimulates collagenase‐3 expression in the human breast cancer cell line, MDA‐MB231. Cycloheximide inhibited this stimulation, indicating that de novo protein synthesis was essential for this response. We examined whether mitogen‐activated protein kinase (MAPK) and/or Smad pathways are involved in TGF‐β1‐stimulated collagenase‐3 expression in MDA‐MB231 cells. Biochemical blockade of extracellular regulated kinase‐1/2 and p38 MAPK pathways partially abolished TGF‐β1‐stimulated collagenase‐3 mRNA expression; whereas overexpression of a dominant negative form of Smad3 completely blocked the TGF‐β1‐response. These data indicate that TGF‐β1‐induced MAPK and Smad pathways are involved in TGF‐β1‐stimulated collagenase‐3 expression in MDA‐MB231 cells.
Bruton's tyrosine kinase targets NF‐κB to the bcl‐x promoter via a mechanism involving phospholipase C‐γ2 following B cell antigen receptor engagement
Authors: Petro James B , Castro Iris , Lowe John , Khan Wasif N
Published: December 2002

Disruption of Bruton's tyrosine kinase (BTK) function leads to x‐linked immunodeficiency (xid) in mice. BTK‐deficient (btk
−/−) B cells are defective for survival. Prior studies show that BTK is required for the induction of Bcl‐xL following B cell antigen receptor (BCR) engagement. However, the mechanism underlying Bcl‐xL induction in response to BCR ligation remains unresolved. We now demonstrate that BTK regulates bcl‐x expression by transcriptional control in response to BCR engagement. BTK targets nuclear factor‐κB (NF‐κB) to activate the bcl‐x promoter via a phospholipase C‐γ2 (PLC‐γ2)‐dependent mechanism. Perturbation of the BTK/PLC‐γ2/NF‐κB signaling axis likely contributes to the defective expression of bcl‐x and compromised survival of xid B cells.
Insulin feedback action on pancreatic β‐cell function
Authors: Leibiger Ingo B , Leibiger Barbara , Berggren Per-Olof
Published: December 2002

Pancreatic β‐cell function is essential for the regulation of glucose homeostasis and its impairment leads to diabetes mellitus. Besides glucose, the major nutrient factor, inputs from neural and humoral components and intraislet cell–cell communication act together to guarantee an appropriate pancreatic β‐cell function. Data obtained over the last 5 years in several laboratories have revitalized a controversial concept, namely the autocrine feedback action of secreted insulin on β‐cell function. While, historically, insulin was suggested to exert a negative effect on β‐cells, recent data provide evidence for a positive role of insulin in transcription, translation, ion flux, insulin secretion and β‐cell survival.
Differential phosphorylation of SNAP‐25 in vivo by protein kinase C and protein kinase A
Authors: Hepp Régine , Cabaniols Jean-Pierre , Roche Paul A
Published: December 2002

SNAP‐25 is a key protein required for the fusion of synaptic vesicles with the plasma membrane during exocytosis. This study establishes that SNAP‐25 is differentially phosphorylated by protein kinase C and protein kinase A in neuroendocrine PC12 cells. Using phosphopeptide mapping and site‐directed mutagenesis we identified both Thr138 and Ser187 as the targets of SNAP‐25 phosphorylation by protein kinase C and Thr138 as the exclusive site of SNAP‐25 phosphorylation by protein kinase A in vivo. Finally, despite published data to the contrary, we demonstrate that stimulation of regulated exocytosis under physiological conditions is independent of a measurable increase in SNAP‐25 phosphorylation in PC12 cells.
A mitochondrial dysfunction induces the expression of nuclear‐encoded complex I genes in engineered male sterile Arabidopsis thaliana
Authors: Gómez-Casati Diego F , Busi Maria V , Gonzalez-Schain Nahuel , Mouras Armand , Zabaleta Eduardo J , Araya Alejandro
Published: December 2002

To study the effect of a mitochondrial dysfunction induced by the expression of the unedited form of the subunit 9 of ATP synthase gene (u‐atp9) in Arabidopsis, we constructed transgenic plants expressing u‐atp9 under the control of three different promoters: CaMV 35S, apetala 3 and A9. The size and shape of transgenic plants bearing the apetala3::u‐atp9 and A9::u‐atp9 genes looked normal while the 35S::u‐atp9 transformed plants showed a dwarf morphology. All u‐atp9 expressing plants, independent of the promoter used, exhibited a male sterile phenotype. Molecular analysis of male sterile plants revealed the induction of the mitochondrial nuclear complex I (nCI) genes, psst, tyky and nadh binding protein (nadhbp), associated with a mitochondrial dysfunction. These results support the hypothesis that the expression of u‐atp9 can induce male sterility and reveal that the apetala3::u‐atp9 and A9::u‐atp9 plants induced the sterile phenotype without affecting the vegetative development of Arabidopsis plants. Moreover, male sterile plants produced by this procedure are an interesting model to study the global changes generated by an engineered mitochondrial dysfunction at the transcriptome and proteome levels in Arabidopsis plants.
The HCF136 protein is essential for assembly of the photosystem II reaction center in Arabidopsis thaliana
Authors: Plücken Henning , Müller Bernd , Grohmann Dina , Westhoff Peter , Eichacker Lutz A
Published: December 2002

Hcf136 encodes a hydrophilic protein localized in the lumen of stroma thylakoids. Its mutational inactivation in Arabidopsis thaliana results in a photosystem II (PHII)‐less phenotype. Under standard illumination, PSII is not detectable and the amount of photosystem I (PSI) is reduced, which implies that HCF136p may be required for photosystem biogenesis in general. However, at low light, a comparison of mutants with defects in PSII, PSI, and the cytochrome b6f complex reveals that HCF136p regulates selectively biogenesis of PSII. We demonstrate by in vivo radiolabeling of hcf136 that biogenesis of the reaction center (RC) of PSII is blocked. Gel blot analysis and affinity chromatography of solubilized thylakoid membranes suggest that HCF136p associates with a PSII precomplex containing at least D2 and cytochrome b559. We conclude that HCF136p is essential for assembly of the RC of PSII and discuss its function as a chaperone‐like assembly factor.
The DMPC lipid phase transition influences differently the first and the second electron transfer reactions in bacterial reaction centers
Authors: Taly Antoine , Baciou Laura , Sebban Pierre
Published: December 2002

Photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides were incorporated in dimyristoylphosphatidylcholine (DMPC) liposomes. The first and second electron transfer rates (k
AB(1) and k
AB(2), respectively) between the first and the second quinone electron acceptors have been measured as a function of temperature, across the phase transition of DMPC (23°C). The Eyring plots of k
AB(1) display straight lines. In contrast, the Eyring plots for k
AB(2) in proteoliposomes show a break at about 23.5°C. This physical discrimination between the two electron transfer reactions demonstrates that the stiffness of the lipid environment of the RCs and/or the protein–protein interactions influence the parameters governing k
AB(2), but not the gating process limiting k
AB(1).
Lcb4p sphingoid base kinase localizes to the Golgi and late endosomes
Authors: Hait Nitai C , Fujita Katsuhide , Lester Robert L , Dickson Robert C
Published: December 2002

Sphingoid long chain base phosphates (LCBPs) regulate cell proliferation, survival and motility in mammals. To learn more about LCBPs in Saccharomyces cerevisiae, we determined the cellular location of Lcb4p, the major enzyme catalyzing LCBP synthesis. By indirect immunofluorescence microscopy and subcellular fractionation, Lcb4p localizes to the trans‐Golgi network and late endosomes and cycles between these compartments. Lcb4p faces the cytosol and is probably bound to membranes by protein–protein interactions. These results indicate that LCBs made in the endoplasmic reticulum must transit to the Golgi to be converted into LCBPs, which must then return to the endoplasmic reticulum to be degraded.
Pyrrolidine dithiocarbamate is a potent antioxidant against hypochlorous acid‐induced protein damage
Authors: Zhu Ben-Zhan , Carr Anitra C , Frei Balz
Published: December 2002

The antioxidant potential of the dithiol compound pyrrolidine dithiocarbamate (PDTC) against protein damage induced by hypochlorous acid (HOCl) was investigated. The effects of PDTC were compared to those of reduced glutathione (GSH) and N‐acetylcysteine (NAC). PDTC markedly and in a concentration‐dependent manner inhibited HOCl‐induced inactivation of α1‐antiproteinase, protein carbonyl formation on serum albumin and oxidation of human low‐density lipoprotein. The direct scavenging of HOCl by PDTC was demonstrated by two quantitative methods, oxidation of ferrocyanide and chlorination of monochlorodimedon. In all assay systems, PDTC was two to three times more potent than GSH and NAC, while diethyldithiocarbamate was about as effective as PDTC. These data demonstrate that PDTC is a potent antioxidant against HOCl‐induced protein oxidative damage, suggesting that PDTC might be useful in the prevention and treatment of inflammatory conditions.
Modulation of Lon protease activity and aconitase turnover during aging and oxidative stress
Authors: Bota Daniela A , Van Remmen Holly , Davies Kelvin J.A
Published: December 2002

We compared Lon protease expression in murine skeletal muscle of young and old, wild‐type and Sod2
−/+ heterozygous mice, and studied Lon involvement in the accumulation of damaged (oxidized) proteins. Lon protease protein levels were lower in old and oxidatively challenged animals, and this Lon deficiency was associated with increased levels of carbonylated proteins. We identified one of these proteins as aconitase, and another as an aconitase fragmentation product, which we can also generate in vitro by treating purified aconitase with H2O2. These results imply that aging and oxidative stress down‐regulate Lon protease expression which, in turn, may be responsible for the accumulation of damaged proteins, such as aconitase, within mitochondria.
Quantitative mRNA expression profiling of ACE 2, a novel homologue of angiotensin converting enzyme
Authors: Harmer Dan , Gilbert Maureen , Borman Richard , Clark Kenneth L
Published: December 2002

ACE 2, a novel homologue of angiotensin converting enzyme, has recently been identified. This study used QRT‐PCR to quantitatively map the transcriptional expression profile of ACE 2 (and the two isoforms of ACE) in 72 human tissues. While confirming that ACE 2 expression is high in renal and cardiovascular tissues, the novel observation has been made that ACE 2 shows comparably high levels of expression in the gastrointestinal system, in particular in ileum, duodenum, jejunum, caecum and colon. Therefore, in probing the functional significance of this novel peptidase, some consideration should be given to a role in gastrointestinal physiology and pathophysiology.
Antisense down regulation of NtBI‐1 in tobacco BY‐2 cells induces accelerated cell death upon carbon starvation
Authors: Bolduc Nathalie , Brisson Louise F
Published: December 2002

Bax inhibitor‐1 (BI‐1) protein is proposed to be a conserved programmed cell death suppressor. In this report, we investigate the anti‐apoptotic function of plant BI‐1 by antisense (AS) down regulation of NtBI‐1 in Nicotiana tabacum cv. BY‐2 cells. We observed that AS cell lines were more susceptible to autophagy, internucleosomal DNA fragmentation and death than control cells when subjected to sucrose starvation and hypo‐osmotic shock, in agreement with a role of BI‐1 as a death inhibitor.
Interaction of hagfish cathelicidin antimicrobial peptides with model lipid membranes
Authors: Basañez Gorka , Shinnar Ann E , Zimmerberg Joshua
Published: December 2002

Hagfish intestinal antimicrobial peptides (HFIAPs) are a family of polycationic peptides exhibiting potent, broad‐spectrum bactericidal activity. In an attempt to unravel the mechanism of action of HFIAPs, we have studied their interaction with model membranes. Synthetic HFIAPs selectively bound to liposomes mimicking bacterial membranes, and caused the release of vesicle‐encapsulated fluorescent markers in a size‐dependent manner. In planar lipid bilayer membranes, HFIAPs induced erratic current fluctuations and reduced membrane line tension according to a general theory for lipidic pores, suggesting that HFIAP pores contain lipid molecules. Consistent with this notion, lipid transbilayer redistribution accompanied HFIAP pore formation, and membrane monolayer curvature regulated HFIAP pore formation. Based on these studies, we propose that HFIAPs kill target cells, at least in part, by interacting with their plasma membrane to induce formation of lipid‐containing pores. Such a membrane‐permeabilizing function appears to be an evolutionarily conserved host‐defense mechanism of antimicrobial peptides.
The sea anemone Bunodosoma granulifera contains surprisingly efficacious and potent insect‐selective toxins
Authors: Bosmans Frank , Aneiros Abel , Tytgat Jan
Published: December 2002

Two sodium channel toxins, BgII and BgIII, isolated from the sea anemone Bunodosoma granulifera, have been subjected to an elaborate electrophysiological and pharmacological comparison between five different cloned sodium channels expressed in Xenopus laevis oocytes in order to determine their efficacy, potency and selectivity. Our results reveal large differences in toxin‐induced effect between the different sodium channels. These toxins possess the highest efficacy for the insect sodium channel (para). Our data also show that BgII, generally known as a neurotoxin, is especially potent on the insect sodium channel with an EC50 value of 5.5±0.5 nM. Therefore, this toxin can be used as a template for further development of new insecticides. Based on our findings, an evolutionary relationship between crustaceans and insects is also discussed.
Cellular activation of proMMP‐13 by MT1‐MMP depends on the C‐terminal domain of MMP‐13
Authors: Knäuper Vera , Bailey Louise , Worley Joanna R , Soloway Paul , Patterson Margaret L , Murphy Gillian
Published: December 2002

Procollagenase‐3 (proMMP‐13) can be activated by soluble or cell associated membrane type matrix metalloproteinase 1 (MT1‐MMP). In this study we show that the cell based activation of proMMP‐13 by MT1‐MMP was dependent on the C‐terminal domain, as Δ249–451 proMMP‐13, which lacks the haemopexin domain, and a chimaera from N‐terminal MMP‐13 and C‐terminal MMP‐19 (proMMP‐13/19) were not processed by MT1‐MMP expressing cells. Only the initial cleavage at Gly35–Ile36 was dependent on MT1‐MMP activity, as conversion to the active enzyme (Tyr85 N‐terminus) required a functional MMP‐13 active site. Unlike proMMP‐2 activation, this process was independent of tissue inhibitor of metalloproteinase‐2 (TIMP‐2) as MT1‐MMP expressing cells from the TIMP‐2−/− mouse efficiently activated proMMP‐13.
The vacuolating toxin of Helicobacter pylori mimicks the CFTR‐mediated chloride conductance1
Authors: Campello Silvia , Tombola Francesco , Cabrini Giulio , Zoratti Mario
Published: December 2002

Cystic fibrosis (CF) is caused by defects of the CF transmembrane conductance regulator (CFTR), which acts both as an anion‐selective channel and as a regulator of other proteins. The relative contribution of these two functions in CF disease is debated. The toxin VacA forms channels with properties similar to those of the CFTR, and we report here that it can insert into the membrane of various cells originating from respiratory epithelia, generating a chloride conductance comparable to that produced by activation of the CFTR. VacA may therefore become a valuable tool in the study of CF pathogenesis.
The Epstein–Barr virus nuclear antigen‐1 may act as a transforming suppressor of the HER2/neu oncogene
Authors: Chuang Tzu-Chao , Way Tzong-Der , Lin Yen-Shing , Lee Ying-Chu , Law Sai-Lung , Kao Ming-Ching
Published: December 2002

It is known that the HER2/neu proto‐oncogene is associated with a wide variety of human cancers and considered to be an attractive target for developing anti‐cancer agents. We report here for the first time that the Epstein–Barr virus nuclear antigen‐1 (EBNA1) suppresses the HER2/neu oncogene expression at the transcriptional level. Recombinant clones of EBNA1 were subcloned and stably transfected into HER2/neu‐overexpressing human ovarian cancer SKOV3.ip1 cells. These EBNA1‐containing clones down‐regulated the endogenous production of p185
HER2/neu
. In addition, the EBNA1‐expressing stable transfectants showed reduced growth rate, low soft agarose colony‐forming ability and tumorigenic potential as compared with the parental line. These data suggest that EBNA1 may act as a transforming suppressor of the HER2/neu oncogene.
Enhancement of protein expression in insect cells by a lobster tropomyosin cDNA leader sequence
Authors: Sano Ken-Ichi , Maeda Kayo , Oki Manami , Maéda Yuichiro
Published: December 2002

We describe a cis element that dramatically increases the expression levels of exogenous genes in baculovirus‐infected insect cells. This 21 bp sequence element is derived from a 5′ untranslated leader sequence of a lobster tropomyosin cDNA (L21). By using a transfer vector carrying L21, the expression levels of tropomyosin and luciferase were 20‐ and seven‐fold higher with L21 than without L21, respectively. L21 has both the Kozak sequence and the A‐rich sequence found in the polyhedrin leader sequence. We assume that both sequence elements are essential for the enhancement of protein expression in the baculovirus‐based expression system.
A novel mechanism for glucose side‐chain formation in rhamnose‐glucose polysaccharide synthesis1
Authors: Ozaki Kazuhisa , Shibata Yukie , Yamashita Yoshihisa , Nakano Yoshio , Tsuda Hiromasa , Koga Toshihiko
Published: December 2002

We have cloned two genes (rgpH and rgpI) that encode proteins for the formation of the glucose side‐chains of the Streptococcus mutans rhamnose‐glucose polysaccharide (RGP), which consists of a rhamnan backbone with glucose side‐chains. The roles of rgpH and rgpI were evaluated in a rhamnan‐synthesizing Escherichia coli. An E. coli strain that harbored rgpH reacted with antiserum directed against complete RGP, whereas the E. coli strain that carried rgpI did not react with this antiserum. Although E. coli:rgpH reacted strongly with rhamnan‐specific antiserum, co‐transformation of this strain with rgpI increased the number of glucose side‐chains and decreased immunoreactivity with the rhamnan‐specific antiserum significantly. These results suggest that two genes are involved in side‐chain formation during S. mutans RGP synthesis in E. coli: one gene encodes a glucosyltransferase, and the other gene probably controls the frequency of branching. This is the first report to identify a gene that is involved in regulation of branching frequency in polysaccharide synthesis.
Synergistic effect of retinoic acid and dehydroepiandrosterone on differentiation of human neuroblastoma cells
Authors: Silvagno Francesca , Guarnieri Vincenzo , Capizzi Anna , Piero Pescarmona Gian
Published: December 2002

Retinoic acid (RA) affects many cell types by either promoting their survival or inducing their differentiation. Dehydroepiandrosterone (DHEA), a precursor for both androgenic and estrogenic steroids and abundantly produced by brain, is known as an inhibitor of cell proliferation. Differentiation of a human neuroblastoma cell line (SK‐N‐BE) was evaluated measuring growth rate, motility, neurite extension and GAP‐43 expression. We report that DHEA enhances the differentiating effect of RA on neuroblastoma cells via a signalling that is not RA receptor‐mediated. Instead, we show a differential expression of matrix metalloproteinases: RA enhances the activity of MMP‐2, whereas MMP‐9 expression is up‐regulated by DHEA. The concerted modulation of these proteinases may support the neurite outgrowth observed after co‐treatment with the two drugs.
Integrin‐nucleated Toll‐like receptor (TLR) dimerization reveals subcellular targeting of TLRs and distinct mechanisms of TLR4 activation and signaling
Authors: Zhang Haifeng , Tay Puei Nam , Cao Weiping , Li Wei , Lu Jinhua
Published: December 2002

Toll‐like receptors (TLRs) are activated by microbial structures. To investigate the mechanisms of TLR activation, the 10 human TLRs were expressed as chimeras with the integrin αv and β5 subunits. Co‐expression of the αv‐TLR and β5‐TLR chimeras in 293T cells generated 10 TLR homodimers, but only TLR4/4 could effectively activate NF‐κB. TLR4 monomers also activated NF‐κB but it was enhanced upon homodimerization. The TLR homodimers showed differential surface/intracellular expression. In TLR heterodimers, only TLR2/1 and TLR2/6 were potent in NF‐κB activation. NF‐κB activation by TLR2/1, TLR2/6 and the TLR4 monomer, but not TLR4/4, was completely inhibited by dominant negative MyD88, suggesting that TLR4 homodimers and monomers could activate NF‐κB through different mechanisms.
Identification and characterization of the single channel function of human mucolipin‐1 implicated in mucolipidosis type IV, a disorder affecting the lysosomal pathway
Authors: LaPlante Janice M , Falardeau John , Sun Mei , Kanazirska Marie , Brown Edward M , Slaugenhaupt Susan A , Vassilev Peter M
Published: December 2002

Mucolipin‐1 (MLN1) is a membrane protein with homology to the transient receptor potential channels and other non‐selective cation channels. It is encoded by the MCOLN1 gene, which is mutated in patients with mucolipidosis type IV (MLIV), an autosomal recessive disease that is characterized by severe abnormalities in neurological development as well as by ophthalmologic defects. At the cellular level, MLIV is associated with abnormal lysosomal sorting and trafficking. Here we identify the channel function of human MLN1 and characterize its properties. MLN1 represents a novel Ca2+‐permeable channel that is transiently modulated by changes in [Ca2+]. It is also permeable to Na+ and K+. Large unitary conductances were measured in the presence of these cations. With its Ca2+ permeability and modulation by [Ca2+], MLN1 could play a major role in Ca2+ transport regulating lysosomal exocytosis and potentially other phenomena related to the trafficking of late endosomes and lysosomes.
Compromised ATP binding as a mechanism of phosphoinositide modulation of ATP‐sensitive K+ channels
Authors: Wang Congmiao , Wang Kun , Wang Wenxia , Cui Yijun , Fan Zheng
Published: December 2002

Inhibition of ATP‐sensitive K+ (KATP) channels by ATP, a process presumably initiated by binding of ATP to the pore‐forming subunit, Kir6.2, is reduced in the presence of phosphoinositides (PPIs). Previous studies led to the hypothesis that PPIs compromise ATP binding. Here, this hypothesis was tested using purified Kir6.2. We show that PPIs bind purified Kir6.2 in an isomer‐specific manner, that biotinylated ATP analogs photoaffinity label purified Kir6.2, and that this labeling is weakened in the presence of PPIs. Patch‐clamp measurements confirmed that these ATP analogs inhibited Kir6.2 channels, and that PPIs decreased the level of inhibition. These results indicate that interaction of PPIs with Kir6.2 impedes ATP‐binding activity. The PPI regulation of ATP binding revealed in this study provides a putative molecular mechanism that is potentially pivotal to the nucleotide sensitivity of KATP channels.
Maleimides stimulate oxygen reduction in illuminated thylakoids
Authors: Ivanov Boris , Ignat'ev Alexander , Khorobrykh Sergey
Published: December 2002

N‐ethylmaleimide (NEM) and N,N′‐(1,4‐phenylene)dimaleimide (PDM) were discovered to stimulate light‐induced oxygen uptake in isolated thylakoids, and PDM provided the same stimulation at one order less concentrations. Oxygen uptake rate increased promptly after NEM or PDM addition to thylakoids. The inhibitors of photosynthetic electron transport as well as catalase decreased this rate close to zero, whereas ascorbate increased it almost three‐fold. Dithiothreitol suppressed oxygen uptake stimulated by NEM. NEM stimulated light‐induced reduction of cytochrome c, and this stimulation was suppressed by superoxide dismutase. It was concluded that NEM and PDM being reduced can effectively reduce molecules O2 producing superoxide radicals.
Cryogenic absorption spectra of hydroperoxo‐ferric heme oxygenase, the active intermediate of enzymatic heme oxygenation
Authors: Denisov Ilia G , Ikeda-Saito Masao , Yoshida Tadashi , Sligar Stephen G
Published: December 2002

Using radiolysis with 32P enriched phosphate as an internal source of ionizing radiation, the formation of hydroperoxo‐ferric complex from oxy‐ferrous precursor with a high yield was monitored at 77 K in heme oxygenase (HO) by means of optical absorption spectroscopy. Well‐resolved absorption spectra (maxima at 421 nm, 530 nm, 557 nm) of hydroperoxo‐ferric intermediate of this heme enzyme were measured in 70% glycerol/buffer frozen glasses. After annealing at 210–215 K this complex converts to the product complex, α‐meso hydroxyheme‐HO. No heme degradation products were formed in control experiments with ferric HO or other heme proteins.
Do H+ ions obscure electrogenic Na+ and K+ binding in the E1 state of the Na,K‐ATPase?
Authors: Apell Hans-Jürgen , Diller Anna
Published: December 2002

In contrast to other P‐type ATPases, the Na,K‐ATPase binding and release of ions on the cytoplasmic side, to the state called E1, is not electrogenic with the exception of the third Na+. Since the high‐resolution structure of the closely related SR Ca‐ATPase in state E1 revealed the ion‐binding sites deep inside the transmembrane part of the protein, the missing electrogenicity in state E1 can be explained by an obscuring counter‐movement of H+ ions. Evidence for such a mechanism is presented by analysis of pH effects on Na+ and K+ binding and by electrogenic H+ movements in the E1 conformation of the Na,K‐ATPase.
N‐terminal acetyl group is essential for the inhibitory function of carboxypeptidase Y inhibitor (IC)
Authors: Mima Joji , Kondo Takahiro , Hayashi Rikimaru
Published: December 2002

Carboxypeptidase Y (CPY) inhibitor, IC, a yeast cytoplasmic inhibitor in which the N‐terminal amino acid is acetylated, was expressed in Escherichia coli and produced as an unacetylated form of IC (unaIC). Circular dichroism and fluorescence measurements showed that unaIC and IC were structurally identical and produce identical complexes with CPY. However, the K
i values for unaIC for anilidase and peptidase activity of CPY were much larger, by 700‐ and 60‐fold, respectively, than those of IC. The reactivities of phenylmethylsulfonyl fluoride and p‐chloromercuribenzoic acid toward the CPY–unaIC complex were considerably higher than those toward the CPY–IC complex. Thus, the N‐terminal acetyl group of IC is essential for achieving a tight interaction with CPY and for its complete inactivation.
DelGEF, a homologue of the Ran guanine nucleotide exchange factor RanGEF, binds to the exocyst component Sec5 and modulates secretion
Authors: Sjölinder Mikael , Uhlmann Jörg , Ponstingl Herwig
Published: December 2002

In order to identify the function of deafness locus putative guanine nucleotide exchange factor (DelGEF), a protein homologous to the nucleotide exchange factor for the small GTPase Ran, a cDNA library was screened for interacting proteins using a yeast two‐hybrid system. The human homologue of Sec5, a protein involved in vesicle transport and secretion, was identified as a binding partner. The interaction between DelGEF and Sec5 was found to be dependent on Mg2+ and stimulated by guanosine triphosphate (GTP) or deoxycytidine triphosphate (dCTP). Downregulation of endogenous DelGEF in HeLa cells induced increased extracellular secretion of proteoglycans indicating a possible role for DelGEF in the secretion process.
Small interfering RNA and gene silencing in transgenic mice and rats
Authors: Hasuwa Hidetoshi , Kaseda Kazuhiro , Einarsdottir Thorbjorg , Okabe Masaru
Published: December 2002

After short duplexes of synthetic 21–23 nt RNAs (siRNA) were reported to be effective in silencing specific genes, a vector‐based approach for siRNAs was demonstrated in mammalian cultured cell lines. However, the effect of RNA interference (RNAi) on various differentiated cells in live animals remains unknown. In this report, we demonstrate that transgenically supplied siRNA can silence ubiquitously expressed enhanced green fluorescent protein in every part of the mouse and rat body. These results suggest that transgenic RNAi could function as an alternative method of gene silencing by applying homologous recombination to embryonic stem (ES) cells, and should be successful even in species where ES cell lines remain unestablished.
BRCA1 and BRCA2 bind Stat5a and suppress its transcriptional activity
Authors: Vidarsson H , Mikaelsdottir E.K , Rafnar T , Bertwistle D , Ashworth A , Eyfjord J.E , Valgeirsdottir S
Published: December 2002

Germline mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, are thought to account for a large portion of familial breast cancer. The increased risk of breast cancer in women carrying such mutations suggests that these proteins play a critical role in the growth regulation of mammary epithelial cells. Another protein, Stat5a, is known to be essential for growth and terminal differentiation of breast epithelial cells. Here we show that Stat5a forms a complex with both BRCA1 and BRCA2 in breast epithelial cells upon stimulation with prolactin. In addition, we show that the activity of Stat5a on the β‐casein promoter is modulated by both BRCA1 and BRCA2. This interaction may be important during the expansion and terminal differentiation of breast epithelial cells, as happens during pregnancy and lactation.
Thermostability of membrane protein helix–helix interaction elucidated by statistical analysis
Authors: Schneider Dirk , Liu Yang , Gerstein Mark , Engelman Donald M
Published: December 2002

A prerequisite for the survival of (micro)organisms at high temperatures is an adaptation of protein stability to extreme environmental conditions. In contrast to soluble proteins, where many factors have already been identified, the mechanisms by which the thermostability of membrane proteins is enhanced are almost unknown. The hydrophobic membrane environment constrains possible stabilizing factors for transmembrane domains, so that a difference might be expected between soluble and membrane proteins. Here we present sequence analysis of predicted transmembrane helices of the genomes from eight thermophilic and 12 mesophilic organisms. A comparison of the amino acid compositions indicates that more polar residues can be found in the transmembrane helices of thermophilic organisms. Particularly, the amino acids aspartic acid and glutamic acid replace the corresponding amides. Cysteine residues are found to be significantly decreased by about 70% in thermophilic membrane domains suggesting a non‐specific function of most cysteine residues in transmembrane domains of mesophilic organisms. By a pair‐motif analysis of the two sets of transmembrane helices, we found that the small residues glycine and serine contribute more to transmembrane helix–helix interactions in thermophilic organisms. This may result in a tighter packing of the helices allowing more hydrogen bond formation.
NF‐κB activator Act1 associates with IL‐1/Toll pathway adaptor molecule TRAF6
Authors: Kanamori Mutsumi , Kai Chikatoshi , Hayashizaki Yoshihide , Suzuki Harukazu
Published: December 2002

NF‐κB activator 1 (Act1), also called CIKS, is a recently identified protein with NF‐κB and AP‐1 activation activities through its association with the IκB kinase complex. We identified and confirmed that Act1 interacts with tumor necrosis factor receptor‐associated factor 6 (TRAF6); notably, Act1 binds to TRAF6 only among TRAF family proteins. The amino‐terminal half of Act1 is required for its interaction with the TRAF domain. Act1‐mediated NF‐κB activation was inhibited by a dominant‐negative mutant of TRAF6 in a dose‐dependent manner, and IL‐1‐induced NF‐κB activation was inhibited by a high level of Act1 expression. Our results suggest that Act1 is involved in IL‐1/Toll‐mediated signaling through TRAF6.
The non‐ionic detergent Brij 58P mimics chaperone effects
Authors: Krause Matthias , Rudolph Rainer , Schwarz Elisabeth
Published: December 2002

The non‐ionic detergent Brij 58P is recommended as a stabilizing agent for protein storage; for example, the aggregation‐prone chaperone DnaJ can be maintained in solution by low concentrations of Brij 58P. During protein folding studies with α‐glucosidase, rhodanese and citrate synthase as model proteins, we discovered that the low concentrations of Brij 58P usually added with purified DnaJ to renaturation samples are sufficient to mimic chaperone effects with respect to prevention of protein aggregation. Furthermore, addition of Brij 58P to refolding α‐glucosidase and citrate synthase enhanced the yield of refolded protein by a factor of two. Thus, Brij 58P can mimic chaperone effects and care should be taken when the substance is used to stabilize chaperone preparations.
Paclitaxel targets mitochondria upstream of caspase activation in intact human neuroblastoma cells
Authors: André Nicolas , Carré Manon , Brasseur Gaël , Pourroy Bertrand , Kovacic Hervé , Briand Claudette , Braguer Diane
Published: December 2002

We previously reported that paclitaxel acted directly on mitochondria isolated from human neuroblastoma SK‐N‐SH cells. Here, we demonstrate that the direct mitochondrial effect of paclitaxel observed in vitro is relevant in intact SK‐N‐SH cells. After a 2 h incubation with 1 μM paclitaxel, the mitochondria were less condensed. Paclitaxel (1 μM, 1–4 h) also induced a 20% increase in respiration rate and a caspase‐independent production of reactive oxygen species by mitochondria. The paclitaxel‐induced release of cytochrome c was detected only after 24 h of incubation, was caspase‐independent and permeability transition pore‐dependent. Thus, paclitaxel targets mitochondria upstream of caspase activation, early during the apoptotic process in intact human neuroblastoma cells.
Stock price reactions to earnings announcements: evidence from Chinese markets
Authors: Su Dongwei
Published: December 2002

We examine the stock price reactions to changes in earnings per share (EPS) in the Chinese stock markets. We find that domestic A‐share investors do not correctly anticipate the changes in earnings and fail to adjust new earnngs information quickly, but international B‐share investors can predict earnings changes better than A‐share investors. As a result, abnormal returns (ARs) can be obtained by trading on the earnings information, but for A shares only. An explanation is that most A‐share holders are individuals with short‐term investment horizon while most B‐share holders are large institutions that trade on more detailed and accurate financial information not immediately available to A‐share holders.
A statistical study of traveling convection vortices using the Magnetometer Array for Cusp and Cleft Studies
Authors: Zesta E. , Hughes W. J. , Engebretson M. J.
Published: November 2002

The Magnetometer Array for Cusp and Cleft Studies (MACCS) is primarily a longitudinal magnetometer array located in the eastern Canadian arctic at cusp latitudes. We present a statistical study of traveling convection vortices (TCVs) as observed by MACCS from the first year of operations, August 1992 to July 1993. For this period, data are routinely available from three stations, extending more than 2 hours in magnetic local time (MLT) and located roughly on a line of constant geomagnetic latitude at 75°. With the selection criteria used, we find an average of 38 TCV events per month. We characterize the events as “isolated” and “nonisolated”. The isolated events have the typical, accepted signature of a TCV while the nonisolated ones are events that satisfy the selection criteria but occur among relatively strong magnetic activity. The diurnal distribution of these events shows a prenoon‐afternoon asymmetry, but the distribution has a single peak at 10 MLT and then falls off smoothly in the afternoon hours. It does not show the previously reported minimum of event occurrence around magnetic local noon except for isolated events occurring during higher Kp values. The most likely explanation for this discrepancy is the wide longitudinal extent of MACCS. The majority of the events have propagation velocities between 3 and 11 km/s, which can be a factor of 2 larger than the events reported in previous studies. The majority of the events move away from noon, as is expected, with a small fraction moving toward noon. Most (80%) of the studied events exhibit all the characteristics of a typical TCV, while the remaining 20% show some form of irregularity. We find four different types of irregularities. The isolated and nonisolated events have many similar morphological and propagation characteristics. However, quantitative comparison of the statistical distributions of the two types of TCVs indicates that they belong to different populations. We also find that nonisolated TCVs may occur for somewhat higher Kp values. We therefore suggest that the two classes of TCVs have different source mechanisms.