bluuebunny/crossref_metadata_2025 huggingface dataset:
Large‐scale structures at various stages of separated boundary layer transition
Authors: Yang Zhiyin
Published: October 2002

Large‐eddy simulation is employed to investigate separated boundary layer transition induced by a change of curvature of the surface. The geometry is a flat plate with a semi‐circular leading edge. The Reynolds number based on the uniform inlet velocity and the leading edge diameter is 3450. The simulated mean and turbulence quantities compare well with the available experimental data. The LES data have been comprehensively analysed to elucidate the transition process leading to breakdown to turbulence. Large‐scale structures have been observed at various stage of the transition process. Understanding the formation of these large‐scale structures, their evolution and eventual break‐up into smaller structures may help to shed light on the transition mechanism. Copyright © 2002 John Wiley & Sons, Ltd.
Evaluation of Smagorinsky‐based subgrid‐scale models in a finite‐volume computation
Authors: Majander Petri , Siikonen Timo
Published: October 2002

Smagorinsky‐based models are assessed in a turbulent channel flow simulation at Reb=2800 and Reb=12500. The Navier–Stokes equations are solved with three different grid resolutions by using a co‐located finite‐volume method. Computations are repeated with Smagorinsky‐based subgrid‐scale models. A traditional Smagorinsky model is implemented with a van Driest damping function. A dynamic model assumes a similarity of the subgrid and the subtest Reynolds stresses and an explicit filtering operation is required. A top‐hat test filter is implemented with a trapezoidal and a Simpson rule. At the low Reynolds number computation none of the tested models improves the results at any grid level compared to the calculations with no model. The effect of the subgrid‐scale model is reduced as the grid is refined. The numerical implementation of the test filter influences on the result. At the higher Reynolds number the subgrid‐scale models stabilize the computation. An analysis of an accurately resolved flow field reveals that the discretization error overwhelms the subgrid term at Reb=2800 in the most part of the computational domain. Copyright © 2002 John Wiley & Sons, Ltd.
Numerical simulation and experimental validation of heat transfer within rotating flows for three‐dimensional non‐axisymmetric, turbulent conditions
Authors: Raimundo A. M. , Oliveira L. A. , Figueiredo A. R.
Published: October 2002

A control volume type numerical methodology for the analysis of steady three‐dimensional rotating flows with heat transfer, in both laminar and turbulent conditions, is implemented and experimentally tested. Non‐axisymmetric momentum and heat transfer phenomena are allowed for. Turbulent transport is alternatively represented through three existing versions of the k–ε model that were adjusted to take into account the turbulence anisotropy promoted by rotation, streamline curvature and thermal buoyancy. Their relative performance is evaluated by comparison of calculated local and global heat balances with those obtained through measurements in a laboratory device. A modified version of the Lam and Bremhorst, low Reynolds number model is seen to give the best results. A preliminary analysis focused on the flow structure and the transfer of heat is reported. Copyright © 2002 John Wiley & Sons, Ltd.
Optimization of the glucose feed rate profile for the production of tryptophan from recombinant E coli
Authors: Dodge Timothy C , Gerstner Jeffrey M
Published: October 2002

A strain of Escherichia coli was engineered to overproduce L‐tryptophan. A fed‐batch fermentation process was developed, producing 30.8 ± 1.4 g dm−3 with a yield on glucose of 0.132 ± 0.010 g g−1. Specific production rate did not appear to be limited by cloned enzyme activity, but by the carbon flux from central metabolism into the aromatic amino acid pathway. The glucose feed rate profile was modified in an attempt to increase the production rate. Tryptophan production was not affected, but led to glutamic acid excretion at high levels. The high specific glucose consumption rate at the low growth rate led to the high glutamate excretion. A new fermentation process involving modification of the feed profile to limit the formation of by‐products was discovered. The resulting final process increased tryptophan production to 42.3 ± 2.7 g dm−3 with yield on glucose of 0.176 ± 0.006 g g−1. The instantaneous yield realized the theoretical maximum for the majority of the fermentation.

© 2002 Society of Chemical Industry
The evolution of US pollution prevention, 1976–2001: a unique chemical engineering contribution to the environment – a review
Authors: Overcash Michael
Published: October 2002

The importance of pollution prevention or cleaner production has been substantial in changing the environmental approach within advanced industrialized countries. Since the critical factors for pollution prevention success relate to fundamental understanding of diverse industrial processes, chemical engineering has had a major and unique role in this environmental field. Expanding this new and evolutionary topic into the undergraduate curriculum of chemical engineering has also been vital to improving the sustainability of cleaner production. This article describes the fundamental concepts that led to active pollution prevention progress and then the historical progress toward this new paradigm.

© 2002 Society of Chemical Industry
Dilated coronary sinus in prenatal echocardiography; identification, associations and outcome
Authors: Machevin‐Surugue E. , David N. , Verspyck E. , Labadie G. , Blaysat G. , Durand I. , Ickowicz V. , Marpeau L.
Published: October 2002

The dilated coronary sinus (DCS) has only recently been clearly visualised in the fetus due to progress in prenatal echography. This is a retrospective study of 22 fetuses presenting with DCS revealed by prenatal echography. We report the circumstances leading to the detection of a DCS and the neonatal outcome of these fetuses. The coronary sinus was defined as dilated depending on its visualisation in cross‐section from the ‘4 chamber’ view, as well as a pseudo inter‐atrial septal defect from a more posterior view. In each case the gestational age, circumstances of detection, associated anomalies and postnatal outcome are reported. The circumstances were: evaluation of a clearly identified DCS in four cases and during detailed fetal echocardiography because of suspected congenital heart disease in 18 cases. Five cases were associated with a cardiac anomaly, three with an extracardiac anomaly, six with both cardiac and extracardiac anomaly and eight were isolated. Postnatal outcome was related to the associated anomaly. In conclusion, it is important that the echography image be correctly interpreted, as a DCS often implies possible associated defects and therefore affects prognosis. When not associated with other anomalies this condition is not considered serious. Copyright © 2002 John Wiley & Sons, Ltd.
Trans‐mediastinal Gunshot Wounds: Are "Stable" Patients Really Stable?
Authors: Nagy Kimberly K. , Roberts Roxanne R. , Smith Robert F. , Joseph Kimberly T. , An Gary C. , Bokhari Faran , Barrett John
Published: October 2002

Gunshot wounds that traverse the mediastinum frequently cause serious injury to the cardiac, vascular,
pulmonary, and digestive structures contained within. Most patients present with unstable vital signs signifying the need for emergency operation. An occasional patient will present with stable vital signs.
Work‐ups for such a patient may range from surgical exploration to radiographic and endoscopic testing to mere observation. We report our experience with diagnostic work‐up of the stable patient with a transmediastinal gunshot wound. All stable patients who present to our urban level I trauma center following a transmediastinal gunshot wound undergo diagnostic work‐up consisting of chest radiograph, cardiac ultrasound, angiography, esophagoscopy, barium swallow, and bronchoscopy. The work‐up is dependent on the trajectory of the missile. Information on these patients is kept in a prospective database maintained by the trauma attending physicians. This database was analyzed and comparisons were made using Student’s t‐test and the Fisher exact c2 as appropriate. Over a 68‐month period,
50 stable patients were admitted following a transmediastinal gunshot wound. All of these patients had a chest radiograph followed by one or more of the above tests. 8 patients (16%) were found to have a mediastinal injury (4 cardiac, 3 vascular, and 1 tracheo‐esophageal)
requiring urgent operation (group 1). The remaining 42 patients (84%)
did not have a mediastinal injury (group 2). There was no difference between groups with respect to blood pressure, pulse, respiratory rate,
pH, base deficit, or initial chest tube output. There was one death in each group, and three complications in group 2. Patients may appear stable following a transmediastinal gunshot wound, even when they have life‐threatening injuries. There is no difference in vital signs, blood gas, or hemothorax to indicate which patients have serious injuries. We advocate continued aggressive work‐up of these patients to avoid missing an injury with disastrous consequences.
Patient‐based Surgical Outcome Tool Demonstrating Alleviation of Symptoms following Parathyroidectomy in Patients with Primary Hyperparathyroidism
Authors: Pasieka Janice L. , Parsons Louise L. , Demeure Michael J. , Wilson Stuart , Malycha Peter , Jones Jean , Krzywda Beth
Published: October 2002

This study assessed the impact of parathyroidectomy on the preoperative symptoms of patients with primary hyperparathyroidism (1° HPT) using a surgical outcome tool designed specifically for HPT. The multicenter nature of this study allowed us to validate further this disease‐specific outcome tool. 1° HPT patients from Canada, the United States, and Australia filled out the questionnaire preoperatively and postoperatively on day 7 and at 3 and
12 months. The symptoms recorded by the patients were expressed as parathyroidectomy assessment of symptoms (PAS) scores: the higher the score, the more symptomatic is the patient. Quality of Life (QOL) and self‐rated health uni‐scales were included. Altogether, 203 patients with 1° HPT were enrolled; 27 from center A, 54 from center B, and 122 from center C; 58
nontoxic thyroid patients were enrolled for comparison. The comparison group had no significant change in their PAS scores throughout the study (scores 184, 215, 156, 186). All three centers demonstrated a significant reduction in symptoms following surgery. The median preoperative PAS score from center B patients was 282. Following surgery, PAS scores decreased significantly: 136, 58, 0
(p <0.05). Center C patients had a median preoperative PAS score of 344, decreasing postoperatively to 228 (p
<0.05) and continuing to decrease to 190, then 180. Center A also demonstrated a significant reduction in symptoms at 3 months, from 510
preoperatively to 209 (p <0.001). Both QOL and self‐rated health improved in the HPT patients, whereas no change was found in the comparison group following surgery. PAS scores are a reliable, disease‐specific measure of symptoms seen with HPT.
Parathyroidectomy significantly reduces these preoperative symptoms,
and this change translated into an improved health‐related QOL for the patients.
Loss of Heterozygosity on the Short Arm of Chromosome 1 in Pheochromocytoma and Abdominal Paraganglioma
Authors: Edström Elder Elisabeth , Nord Brita , Carling Tobias , Juhlin Claes , Bäckdahl Martin , Höög Anders , Larsson Catharina
Published: October 2002

Pheochromocytomas and abdominal paragangliomas are catecholamine‐producing tumors that arise from sympathetic paraganglia within and outside the adrenal medulla, respectively. Deletions of the short arm of chromosome 1 have been implicated as important genetic events in their tumorigenesis and suggest a common genetic etiology. The aim of this study was to define further the chromosomal regions on 1p that are involved in the development of these tumor types. We analyzed 46 pheochromocytomas (1 benign, 6
malignant, 9 hereditary) and 7 paragangliomas (3 benign, 4 malignant) from 50
patients for loss of heterozygosity (LOH) on 1p by genotyping 15 microsatellite markers spread over the chromosome arm. Overall, LOH was detected in 33 of 46
pheochromocytomas (72%) and in 6 of 7 (86%) paragangliomas. Three minimal regions of overlapping deletions were identified: one telomeric of D1S1612(1p36.2‐pter), one centromeric of D1S429 (1cen‐p13), and one in the 18 cM interval defined by D1S2134 andD1S1669 (1p32). The latter region harbors the leukocyte common antigen‐related (LAR) gene, which shows altered expression in sporadic rat pheochromocytomas. In conclusion, chromosome
1p may be the site of at least three putative tumor‐suppressor gene loci involved in the tumorigenesis of pheochromocytomas and abdominal paragangliomas. Further studies of these regions and of LARas a candidate gene would be valuable.
Quality of Life and Patient Satisfaction after Reoperation for Primary Hyperparathyroidism: Analysis of Long‐term Results
Authors: Hasse Christian , Sitter Helmut , Brune Melanie , Wollenteit Ina , Nies Christoph , Rothmund Matthias
Published: October 2002

orbidity after reoperation for persistent or recurrent primary hyperparathyroidism (pHPT) is higher than after primary surgery. According to our experience, there is a contrast between postoperative normalization of laboratory parameters and the quality of life/patient satisfaction after reoperation. Therefore the aim of the study was to analyze the outcomes of reoperations in comparison to primary surgery. We evaluated the patients’ reported quality of life using the SF‐36 (an accepted health status assessment tool) and complete prospectively documented perioperative and follow‐up data including postoperative complications. Additionally, we searched for reasons why primary surgical intervention did not succeed. In a prospective cohort study the perioperative data of 653 consecutive patients with pHPT, including 75 reoperated patients (11.5%) who underwent parathyroidectomy between 1987 and 1999, were evaluated by uni‐ and multivariate analysis. At a median 78 months (6–156 months) postoperatively, all patients underwent a planned follow‐up that included the SF‐36, physical examination, and laboratory investigations. A total of 51 reoperated patients were available for follow‐up. Postoperative alleviation of symptoms or being symptom‐free was reported by 70.6%. Patients after reoperation had lower SF‐36 scores in all health domains postoperatively than patients after a primary operation. Of the reoperated patients, 19.6% stated that after evaluating the development of their complaints they would not consent to reoperation again. Subgroup analysis showed that 80% of patients with postoperatively persistent pHPT, 60% of those who did not observe symptom alleviation, and 44% of those after sternotomy were in the group of dissatisfied patients. Surprisingly, none of the patients with more than one reoperation, only two of the five patients with permanent recurrent laryngeal nerve injury, and only one of the four patients with persistent hypoparathyroidism were dissatisfied overall. Parathyroidectomy resulted in normocalcemia in 90.2% of the reoperated patients, with an operative morbidity of 27.4% and no mortality. After an unsuccessful operation for pHPT, patients should be treated at an expert center to avoid persistent hypercalcemia. Reoperations necessitating sternotomy should be restricted to patients with severe symptoms and signs.
Histone H1 and the regulation of transcription by nuclear receptors
Authors: Oikarinen Jouko
Published: October 2002

Histone H1 is a eukaryotic repressor which recognizes specific DNA structures, and nucleotides regulate its interaction with DNA. Since their mode of action may be considered similar to that observed in the case of plasma membrane GTPases, H1 may be regarded as an ATP/GTPase involved in the action of nuclear receptors. A hypothesis is put forward here to suggest that transcriptional activators CTF/NF‐I and AP‐1 (fos/jun), for example, are effectors for H1. H1 and CTF/NF‐I may be members of a stimulatory regulatory cascade for nuclear receptor action that ends with selective activation of chromatin through histone modification and the disruption or a more subtle structural change of a specific nucleosome, while an opposite effect may be obtained through modification of fos/jun by H1.
Evidence of a tissue‐restricting DNA regulatory element in the mouse IRBP promoter
Authors: Boatright J.H. , Knox B.E. , Jones K.M. , Stodulkova E. , Nguyen H.T. , Padove S.A. , Borst D.E. , Nickerson J.M.
Published: October 2002

The expression of interphotoreceptor retinoid binding protein (IRBP) is limited to photoreceptor cells of the retina and pinealocytes of the pineal gland. We sought to define cis‐elements of the mouse IRBP 5′ flanking region that are required for this restricted activity. In vitro transient transfections of retinoblastoma and neuroblastoma cells and in vivo experiments with transgenic Xenopus laevis indicate that −1783/+101 and −156/+101 IRBP gene fragments directed expression predominantly to the retina and pineal, with minor neuronal expression elsewhere. In contrast, a −70/+101 fragment was less restrictive in controlling expression, exhibiting activity not only in retina, but also in forebrain, hindbrain, spinal cord, and motor neurons innervating gills.
Genomic organization and embryonic expression ofSuppressor of Fused, a candidate gene for the split‐hand/split‐foot malformation type 3
Authors: Grimm Thomas , Teglund Stephan , Tackels Darci , Sangiorgi Eugenio , Gurrieri Fiorella , Schwartz Charles , Toftgård Rune
Published: October 2002

The genes for human and mouseSuppressor of Fused(SU(FU)/Su(Fu)) in the Hedgehog signaling pathway were characterized and found to contain 12 exons. HumanSU(FU)localized on chromosome 10q24–25 between the markers D10S192 and AFM183XB12. We detected three additional SU(FU) isoforms, two of which have lost their ability to interact with the transcription factor GLI1. Expression analysis using whole mount in situ hybridization revealed strong expression ofSu(Fu)in various mouse embryonic tissues.SU(FU)was considered a candidate gene for the split‐hand/split‐foot malformation type 3 (SHFM3). However, no alterations in theSU(FU)gene were found in SHFM3 patients.
Sea urchin sperm cation‐selective channels directly modulated by cAMP
Authors: Sánchez Daniel , Labarca Pedro , Darszon Alberto
Published: October 2002

Components of the sea urchin outer egg jelly layer such as speract drastically change second messenger levels and membrane permeability in sperm. Ion channels are deeply involved in the sperm–egg dialogue in sea urchin and other species. Yet, due to the small size of sperm, studies of ion channels and their modulation by second messengers in sperm are scarce. In this report we offer the first direct evidence that cation‐selective channels upwardly regulated by cAMP operate in sea urchin sperm. Due to their poor selectivity among monovalent cations, channel activation in seawater could contribute to sperm membrane repolarization during the speract response.
Re‐examination of the mechanisms regulating nuclear inositol lipid metabolism
Authors: Martelli Alberto M. , Bortul Roberta , Tabellini Giovanna , Aluigi Michela , Peruzzi Daniela , Bareggi Renato , Narducci Paola , Cocco Lucio
Published: October 2002

Although inositol lipids constitute only a very minor proportion of total cellular lipids, they have received immense attention by scientists since it was discovered that they play key roles in a wide range of important cellular processes. In the late 1980s, it was suggested that these lipids are also present within the cell nucleus. Albeit the early reports about the intranuclear localization of phosphoinositides were met by skepticism and disbelief, compelling evidence has subsequently been accumulated convincingly showing that a phosphoinositide cycle is present at the nuclear level and may be activated in response to stimuli that do not activate the inositol lipid metabolism localized at the plasma membrane. Very recently, intriguing new data have highlighted that some of the mechanisms regulating nuclear inositol lipid metabolism differ in a substantial way from those operating at the cell periphery. Here, we provide an overview of recent findings regarding the regulation of both nuclear phosphatidylinositol 3‐kinase and phosphoinositide‐specific phospholipase C‐β1.
Probing into the function of the gene product responsible for glycogen storage disease type Ib
Authors: Xie Wensheng , van de Werve Gérald , Berteloot Alfred
Published: October 2002

This study aimed at directly assessing glucose 6‐phosphate (G6P) transport by intact rat liver microsomes. Tracer uptake from labeled G6P occurred with T
1/2 values that proved insensitive to unlabeled G6P or 100 μM vanadate, and could not be activated over background levels by intravesicular phosphate in the complete absence of G6P hydrolysis. [32P]Phosphate efflux was similarly unaffected by G6P or phosphate in the incubation medium. We conclude that the gene product responsible for glycogen storage disease type Ib is functionally distinct from the bacterial hexose phosphate transporter, which operates as an obligatory phosphate:phosphate or G6P:phosphate exchanger.
Apolipoprotein E isoform‐specific disruption of phosphoinositide hydrolysis: protection by estrogen and glutathione
Authors: Cedazo-Mı́nguez Angel , Cowburn Richard F
Published: October 2002

The mechanism(s) by which the E4 isoform of apolipoprotein E (apoE4) influences Alzheimer's disease (AD) are not fully known. We report that apoE4, but not apoE3, disrupts carbachol‐stimulated phosphoinositide (PI) hydrolysis in SH‐SY5Y neuroblastoma cells. Carbachol responses were also disrupted by β‐amyloid (Aβ) (1–42) and apoE4/Aβ(1–42) complexes, but not by apoE3/Aβ(1–42). Glutathione and estrogen protected against apoE4 and Aβ(1–42) effects, as well as those of H2O2. Estrogen protection was partially blocked by wortmannin, suggesting the involvement of phosphatidylinositol 3‐kinase. An apoE4‐induced disruption of acetylcholine muscarinic receptor‐mediated signalling may explain the lower effectiveness of cholinergic replacement treatments in apoE4 AD patients. Also, the beneficial effect of estrogen in AD may be partially due to its ability to protect against apoE4‐ and Aβ(1–42)‐mediated disruption of PI hydrolysis.
Hyperlipemia: a role in regulating UCP3 gene expression in skeletal muscle during cancer cachexia?
Authors: Busquets Sı́lvia , Carbó Neus , Almendro Vanessa , Figueras Maite , López-Soriano Francisco J. , Argilés Josep M.
Published: October 2002

Rats bearing the Yoshida AH‐130 ascites hepatoma showed an increased expression of both uncoupling protein‐2 (UCP2) (two‐fold) and UCP3 (three‐ to four‐fold) in skeletal muscle (both soleus and gastrocnemius). The increase in mRNA content was associated with increased circulating concentrations of fatty acids (two‐fold), triglyceride (two‐fold) and cholesterol (1.9‐fold). Administration of nicotinic acid to tumor‐bearing rats abolishes the hyperlipidemic increase associated with tumor burden. The vitamin treatment also resulted in a decreased UCP3 gene expression in soleus muscle but not in gastrocnemius. It is concluded that circulating fatty acids may be involved in the regulation of UCP3 gene expression in aerobic muscles during experimental cancer cachexia. Since the UCP3 protein could have a role in energy expenditure, it may be suggested that hypolipidemic agents may have a beneficial role in the treatment of the cachectic syndrome.
Bax‐dependent apoptosis induced by ceramide in HL‐60 cells
Authors: Kim Hae Jong , Mun Jung Yee , Chun Young Jin , Choi Kyung Hee , Kim Mie Young
Published: October 2002

Ceramide is an important lipid messenger involved in mediating a variety of cell functions including apoptosis. In this study, we show that antisense bax inhibits cytochrome c release, poly(ADP‐ribose)polymerase cleavage and cell death induced by ceramide in HL‐60 cells. In addition, ceramide induces translocation of Bax to mitochondria. The addition of the broad spectrum caspase inhibitor zVAD‐fmk prevented ceramide‐induced apoptotic cell death but did not inhibit translocation of Bax and mitochondrial cytochrome c release. Furthermore, ceramide inhibits the expression of the antiapoptotic protein Bcl‐xL with an increase in the ratio of Bax to Bcl‐xL. These data provide direct evidence that Bax plays an important role in regulating ceramide‐induced apoptosis.
Nitric oxide production in living neurons is modulated by sphingosine: a fluorescence microscopy study
Authors: Viani Paola , Giussani Paola , Ferraretto Anita , Signorile Alessandra , Riboni Laura , Tettamanti Guido
Published: October 2002

An investigation was carried out into the possible effect of sphingosine (Sph) on nitric oxide (NO) production in living neurons. Differentiated granule cells were used in a dynamic videoimaging analysis of single cells labeled, simultaneously, with FURA‐2 and the NO indicator 4,5‐diaminofluorescein. The results demonstrate that Sph exerts a potent inhibitory effect on the Ca2+‐dependent production of NO, without modifying the [Ca2+]i. The effect appears to be specific as neither ceramide nor Sph‐1‐phosphate had any effect on the NO and [Ca2+]i levels. The data demonstrate that Ca2+‐dependent NO production is a specific Sph target in living granule cells, suggesting that this bioactive sphingoid plays a relevant role in neuronal NO signaling.
The yeast cyclins Pc16p and Pc17p are involved in the control of glycogen storage by the cyclin‐dependent protein kinase Pho85p
Authors: Wang Zhong , Wilson Wayne A , Fujino Marie A , Roach Peter J
Published: October 2002

Pho85p is a yeast cyclin‐dependent protein kinase (Cdk) that can interact with 10 cyclins (Pcls) to form multiple protein kinases. The functions of most of the Pcls, including Pc16p and Pc17p, are poorly defined. We report here that Pc16p and Pc17p are involved in the metabolism of the branched storage polysaccharide glycogen under certain conditions and deletion of PCL6 and PCL7 restores glycogen accumulation to a snf1 pcl8 pcl10 triple mutant, paradoxically activating both glycogen synthase and phosphorylase. Pho85p thus affects glycogen accumulation through multiple Cdks composed of different cyclin partners.
Identification of an Arabidopsis cDNA encoding a lipoyltransferase located in plastids
Authors: Wada Miho , Yasuno Rie , Wada Hajime
Published: October 2002

In plant cells, the pyruvate dehydrogenase (PDH) complex that requires lipoic acid as an essential coenzyme is located in plastids and mitochondria. The enzyme complex has to be lipoylated in both organelles. However, the lipoyltransferase located in plastids has not been reported. In this study, an Arabidopsis thaliana LIP2p cDNA for a lipoyltransferase located in plastids has been identified. We have shown that this cDNA encodes a lipoyltransferase by demonstrating its ability to complement an Escherichia coli mutant lacking lipoyltransferase activity, and that LIP2p is targeted into chloroplasts. These findings suggest that LIP2p is located in plastids and responsible for lipoylation of the plastidial PDH complex.
Promoter structure and transcription initiation sites of the human death receptor 5/TRAIL‐R2 gene1
Authors: Yoshida Tatsushi , Maeda Ayaka , Tani Naoki , Sakai Toshiyuki
Published: October 2002

The death receptor 5 (DR5) is a receptor for tumor necrosis factor‐related apoptosis‐inducing ligand and is able to induce apoptosis in various tumor cells. The expression of DR5 is up‐regulated at the transcriptional level by p53, genotoxic stress and so on. To investigate the structure of the DR5 gene promoter, we screened and sequenced a genomic clone containing the 5′‐flanking region of the DR5 gene. RNase protection assays showed two major transcription start sites around −122 and −137 upstream of the translation initiation codon ATG. Transient transfections with serial 5′‐deletion mutants identified the minimal promoter element spanning −198 to −116. Site‐directed mutagenesis demonstrated that the DR5 gene promoter has no typical TATA‐box, but has two Sp1 sites responsible for the basal transcription activity of the DR5 gene promoter.
Cdc42‐independent activation and translocation of the cytostatic p21‐activated protein kinase γ‐PAK by sphingosine
Authors: Roig Joan , Tuazon Polygena T. , Traugh Jolinda A.
Published: October 2002

Autophosphorylation of p21‐activated protein kinase γ‐PAK is stimulated at 10 μM sphingosine in vitro and is maximal at 100 μM. Sites autophosphorylated on γ‐PAK in response to sphingosine are identical to those obtained with Cdc42(GTP). Autophosphorylation is paralleled by stimulation of γ‐PAK activity as measured with peptide and protein substrates. In 3T3‐L1 cells, sphingosine stimulates the autophosphorylation and activity of γ‐PAK associated with the membrane‐containing particulate fraction by 2.8‐fold, but does not stimulate the activity of the soluble enzyme. Thus, γ‐PAK is activatable via a Cdc42‐independent mechanism, suggesting sphingosine has a role in γ‐PAK activation under conditions of cell stress.
TNF‐α enhances intracellular glucocorticoid availability
Authors: Heiniger Christian D. , Rochat Mascha K. , Frey Felix J. , Frey Brigitte M.
Published: October 2002

For understanding the mechanism(s) relating inflammation to corticosteroid action, the effect of tumour necrosis factor‐α (TNF‐α) on 11β‐hydroxysteroid dehydrogenase type 2 (11β‐HSD2), the enzyme regulating access of 11β‐hydroxycorticosteroids to receptors, was studied in LLC‐PK1 cells. We observed (i) NAD‐dependent enzyme activity and mRNA for 11β‐HSD2, but not 11β‐HSD1, (ii) increasing 11β‐HSD2 activity with increasing degree of differentiation and (iii) a concentration‐dependent down‐regulation by TNF‐α, phorbol myristate acetate (PMA) or glucose of activity and mRNA of 11β‐HSD2. The decrease of activity and mRNA by glucose and PMA, but not that by TNF‐α, was abrogated by the protein kinase C inhibitor GF‐109203X. The effect of TNF‐α on 11β‐HSD2 was reversed by inhibiting the mitogen‐activated protein kinases ERK with PD‐098050 and p38 by SB‐202190, or by activating protein kinase A with forskolin. Overexpression of MEK1, an ERK activator, down‐regulated the 11β‐HSD2 activity. In conclusion, TNF‐α decreases 11β‐HSD2 activity and thereby enhances glucocorticoid access to glucocorticoid receptors to modulate the inflammatory response.
The MED‐7 transcriptional mediator encoded by let‐49 is required for gonad and germ cell development in Caenorhabditis elegans
Authors: Kwon Jae Young , Kim-Ha Jeongsil , Lee Byeong Jae , Lee Junho
Published: October 2002

Transcription mediators are evolutionarily conserved from yeast to human. We previously reported the specific in vivo roles of mediators during development. Transcriptional mediators including med‐6, med‐7, and med‐10 were shown to be involved in the regulated transcription of specific genes, but not in the transcription of ubiquitous genes. In this report we have identified and characterized the Caenorhabditis elegans med‐7 gene. A genetic mutation in the med‐7 gene was identified by comparing genetic and physical maps and determining the molecular lesion. let‐49 was found to have a nonsense mutation in the coding region of the med‐7 gene. The identification of let‐49 as the med‐7 gene was confirmed by rescue experiments. The phenotype of the let‐49 mutation indicated that the med‐7 gene is required for normal postembryonic development. RNAi experiments showed that med‐7 is also involved in embryogenesis and the gonad and germ cell development.
Residue size at position 87 of cytochrome P450 BM‐3 determines its stereoselectivity in propylbenzene and 3‐chlorostyrene oxidation
Authors: Li Qing-Shan , Ogawa Jun , Schmid Rolf D , Shimizu Sakayu
Published: October 2002

We report here oxidation of propylbenzene and 3‐chlorostyrene by wild‐type cytochrome P450 BM‐3 with high turnover (479 nmol 1‐phenyl‐1‐propanol/min/nmol P450 and 300 nmol 3‐chlorostyrene oxide/min/nmol P450). Furthermore, the residue size at position 87 of P450 BM‐3 was found to play critical roles in determining stereoselectivity in oxidation of propylbenzene and 3‐chlorostyrene. Replacement of Phe87 with Val, Ala and Gly resulted in decreases in optical purity of produced (R)‐(+)‐1‐phenyl‐1‐propanol from 90.0 to 37.4, 26.0 and −15.6% e.e., respectively, and in increases in those of produced (R)‐(+)‐3‐chlorostyrene oxide from −61.0 to −38.0, 67.0 and 94.6% e.e., respectively.
Calcium‐dependent cysteine reactivities in the neuronal calcium sensor guanylate cyclase‐activating protein 1
Authors: Hwang Ji-Young , Schlesinger Ramona , Koch Karl-Wilhelm
Published: October 2002

Guanylate cyclase‐activating protein 1 (GCAP‐1) is a Ca2+‐sensing protein in vertebrate photoreceptor cells. It activates a membrane‐bound guanylate cyclase. Three of four cysteines present in wild‐type GCAP‐1 were accessible to the thiol‐modifying reagent 5,5′‐dithio‐bis‐(2‐nitrobenzoic acid) in the presence of Ca2+. Only Cys106 became exposed to the solvent after Ca2+‐chelation. Since Cys106 is located in EF‐hand 3, we could determine an apparentKDof 2.9 μM for Ca2+binding to this site with a fast off‐rate (t∼2 ms). We conclude that the rapid dissociation of Ca2+from EF‐hand 3 in GCAP‐1 triggers activation of guanylate cyclase in rod cells.
S‐Adenosyl‐L‐homocysteine hydrolase is sequestered into actin rods in Dictyostelium discoideum spores
Authors: Kishi Yoshiro , Sugo Teruko , Mahadeo Dana , Cotter David , Sameshima Masazumi
Published: October 2002

Here we show evidence that S‐adenosyl‐L‐homocysteine hydrolase (SAHH) is linked to the actin cytoskeleton. Actin rods formed in Dictyostelium discoideum spores during the final stage of development are structurally composed of novel bundles of actin filaments. SAHH only accumulates with actin at this stage of development in the life cycle of D. discoideum. Recently SAHH is believed to be a target for antiviral chemotherapy and the suppression of T cells. Our finding may contribute to designing novel antiviral and immunosuppressive drugs.
Determination of amino acid sequence responsible for suppression of bone resorption by serum calcium‐decreasing factor (caldecrin)
Authors: Tomomura Akito , Yamada Hirotaka , Fujimoto Kengo , Inaba Akemi , Katoh Setsuko
Published: October 2002

We previously reported on the serum calcium‐decreasing activity of recombinant protein factor referred to as caldecrin [Tomomura et al. (1995) J. Biol. Chem. 270, 30315–30321]. To address the mechanism of this serum calcium‐decreasing activity, we investigated the effect of rat caldecrin on osteoclastic bone‐resorbing activity. Wild‐type caldecrin suppressed resorption pit formation by osteoclast on a dentine slice in a dose‐dependent manner. The suppressive effect on the bone resorption was not affected by treatment of caldecrin with phenylmethyl sulfonyl fluoride or by use of protease‐deficient mutant caldecrins. Recombinant procaldecrin (−13–239), and its fragments (−13–125), (1–111), (1–46), (47–111), and (126–239) were expressed as His‐tagged thioredoxin fusion proteins and investigated for their ability to suppress bone resorption. The proform (−13–239) and fragment (−13–125) did not affect the suppressive activity, whereas fragments (1–111) and (126–239) did suppress the bone resorption. The bone‐resorbing activity was also suppressed by fragment (47–111), not by fragment (1–46). Overlapping fragments (47–62), (47–79), (47–98), (56–111), (71–111), and (85–111) were compared for their suppressive activity. The fragments (47–62) and (85–111) did not affect the activity, but the other fragments suppressed the bone resorption. A synthetic peptide having the (71–79) sequence suppressed the bone resorption. These results suggest that amino acid sequence corresponding to rat caldecrin (aa 71–79) is responsible for the suppression of bone resorption by caldecrin.
N‐Acetylneuraminic acid uptake in Pasteurella (Mannheimia) haemolytica A2 occurs by an inducible and specific transport system
Authors: Solana Sonia , Reglero Ángel , Martı́nez-Blanco Honorina , Revilla-Nuin Beatriz , Bravo Ignacio G , Rodrı́guez-Aparicio Leandro B , Ferrero Miguel A
Published: October 2002

The N‐acetylneuraminic acid (NeuAc) transport system of Pasteurella (Mannheimia) haemolytica A2 was studied when this bacterium was grown in both complex and chemically defined media. Kinetic measurements were carried out at 37°C in 50 mM Tris–HCl buffer, pH 8.0, containing 50 μg/ml bovine serum albumin. Under these conditions, the uptake rate was linear for at least 3 min and the calculated K
m for NeuAc was 0.1 μM. The transport rate was increased by the addition of several cations and was inhibited by sodium arsenite (95%), N,N′‐dicyclohexyl‐carbodiimide (50%), and 2,4‐dinitrophenol (40%) at final concentration of 1 mM (each). These results support the notion that NeuAc uptake is an active sugar cation symporter. Study of specificities showed that glucosamine, mannose and mannosamine inhibited the transport of NeuAc in this bacterium. Analysis of expression revealed that the NeuAc transport system was induced by NeuAc and by the simultaneous presence of glucose and galactose in the growth medium.
Regulation of ribosome biogenesis within the nucleolus
Authors: Leary Daniel J , Huang Sui
Published: October 2002

Ribosome biogenesis is both necessary for cellular adaptation, growth, and proliferation as well as a major energetic and biosynthetic demand upon cells. For these reasons, ribosome biogenesis requires precise regulation to balance supply and demand. The complexity of ribosome biogenesis gives rise to many steps and opportunities where regulation could take place. For trans‐acting factors involved in ribosome biogenesis in the nucleolus, there may be a dynamic coordination, both spatially and temporally, that regulates their functions from the transcription of rDNA to the assembly and export of preribosomal particles. Here we summarize most of the described regulations on ribosome biogenesis in the nucleolus. However, these may represent only a small fraction of a larger picture. Further studies are required to determine the initial signals, signal transduction pathways utilized, and the specific targets of these regulatory modifications and how these are used to control ribosome biogenesis as a whole.
Effect of D609 on phosphatidylcholine metabolism in the nuclei of LA‐N‐1 neuroblastoma cells: a key role for diacylglycerol
Authors: Antony Pierre , Farooqui Akhlaq A. , Horrocks Lloyd A. , Freysz Louis
Published: October 2002

In our previous studies, TPA treatment of LA‐N‐1 cells stimulated the production of diacylglycerol in nuclei, probably through the activation of a phospholipase C. Stimulation of the synthesis of nuclear phosphatidylcholine by the activation of CTP:phosphocholine cytidylyltransferase was also observed. The present data show that both effects were inhibited by the pretreatment of the cells with D609, a selective phosphatidylcholine‐phospholipase C inhibitor, indicating that the diacylglycerol produced through the hydrolysis of phosphatidylcholine in the nuclei is reutilized for the synthesis of nuclear phosphatidylcholine and is required for the activation of CTP:phosphocholine cytidylyltransferase.
A novel metallo‐β‐lactamase, Mbl1b, produced by the environmental bacterium Caulobacter crescentus1
Authors: Simm Alan M. , Higgins Catherine S. , Pullan Steven T. , Avison Matthew B. , Niumsup Pannika , Erdozain Olivia , Bennett Peter M. , Walsh Timothy R.
Published: October 2002

Caulobacter crescentus 101123 possesses a gene (Mbl1b) encoding a metallo‐β‐lactamase with 32% amino acid identity to the L1 metallo‐β‐lactamase from Stenotrophomonas maltophilia. The gene was cloned into an expression vector and the enzyme, Mbl1b, was expressed in Escherichia coli. Mbl1b was purified. Catalytic properties for several antibiotics were determined. The enzyme exhibits Michaelis–Menten kinetics for imipenem, meropenem and nitrocefin but substrate inhibition kinetics with cefoxitin, cefaloridine, penicillin G and ampicillin. A homology model predicts Mbl1b has the same structural fold as other metallo‐β‐lactamases with a detailed structure very similar to L1 but whereas L1 is a homotetramer, Mbl1b is monomeric. The main differences between Mbl1 and L1 are in the N‐terminal region.
Bicyclic triterpenes as new main products of squalene–hopene cyclase by mutation at conserved tyrosine residues
Authors: Füll Christine
Published: October 2002

The catalytic cavity of the Alicyclobacillus acidocaldarius squalene–hopene cyclase is predominantly lined by aromatic amino acids. In mutant cyclases, the four tyrosine residues in the catalytic cavity were replaced by different amino acids. The mutants showed significant differences in catalytic behavior compared to the wild‐type and to each other. Mutants Y609L, Y609C and Y609S produced the bicyclic main product γ‐polypodatetraene, while Y495L and Y612L showed a wild‐type product pattern and produced hopene as the main product. Altered product patterns were also found with Y420 mutations.
RNA ligands generated against complex nuclear targets indicate a role for U1 snRNP in co‐ordinating transcription and RNA splicing
Authors: Tian Huicheng
Published: October 2002

RNA ligands were generated against various gene products present in HeLa nuclear extract. Functional profiling was performed to identify RNA ligands that modulate RNA polymerase II (pol II)‐mediated transcription. Unexpectedly, four of the eight inhibitor ligands identified by this screen contained an 11‐nucleotide sequence identical to the 5′‐splice site of eukaryotic pre‐mRNAs. Such ligands were shown to impede pre‐initiation complex assembly on a cytomegalovirus promoter. In addition, U1 small nuclear ribonucleoprotein particles (snRNP) and pol II had been co‐immunoprecipitated in the absence of transcription. These results suggest a role for U1 snRNP in co‐ordinating transcription and RNA splicing.
The amphiphilic character of glycogenin
Authors: Carrizo Marı́a E , Miozzo Marı́a C , Maggio Bruno , Curtino Juan A
Published: October 2002

This study describes for the first time the amphiphilicity of the protein moiety of proteoglycogen. Glycogenin but not proteoglycogen associates to phospholipid vesicles and forms by itself stable Gibbs and Langmuir monolayers at the air–buffer interface. The adsorption free energy (−6.7 kcal/mol) and the glycogenin collapse pressure (47 mN/m) are indicative of its high surface activity which can thermodynamically drive and retain the protein at the membrane interface to a maximum equilibrium adsorption surface pressure of 21 mN/m. The marked surface activity of glycogenin is further enhanced by its thermodynamically favorable penetration into zwitterionic and anionic phospholipids with a high cut‐off surface pressure point above 30 mN/m. The strong association to phospholipid vesicles and the marked surface activity of glycogenin correspond to a high amphiphilic character which supports its spontaneous association to membrane interfaces, in which the de novo biosynthesis of glycogen was proposed to initiate.
Sarcolemma phospholipid structure investigated by immunogold electron microscopy and 31P NMR spectroscopy with lanthanide ions
Authors: Moreau Céline , Cavalier Annie , Le Floch Marie , Segalen Jacqueline , Rocher Chantal , Traı̈kia Mounir , Leray Geneviève , Bondon Arnaud , Thomas Daniel , Le Rumeur Elisabeth
Published: October 2002

The biological functions of plasma membranes depend greatly on the biophysical properties resulting from protein and phospholipid structure. We investigated the phospholipid structure of the normal sarcolemma membrane, which is known to be highly dysfunctional in myopathies. Combining electron microscopy and 31P nuclear magnetic resonance (NMR) spectroscopy on isolated sarcolemma vesicles, we find that (i) the sarcolemma vesicles maintain the in‐vivo cellular sidedness, (ii) the phospholipid mobility is close to that observed in model membranes (similar lateral diffusion coefficients and spin–lattice T
1 relaxation times). Using broad‐band and magic angle spinning 31P NMR spectroscopy with lanthanide ions (Pr3+), it is possible to quantify the distribution of phospholipids between internal and external membrane layers, showing that the trans‐bilayer distribution is highly asymmetrical.
Decreased intracellular degradation of insulin‐like growth factor binding protein‐3 in cathepsin L‐deficient fibroblasts
Authors: Zwad Olaf , Kübler Bernd , Roth Wera , Scharf Jens-Gerd , Saftig Paul , Peters Christof , Braulke Thomas
Published: October 2002

Proteolysis of insulin‐like growth factor binding proteins (IGFBPs) is the major mechanism of releasing IGFs from their IGFBP complexes. Analysis of fibroblasts deficient for the lysosomal cysteine protease cathepsin L (CTSL) revealed an accumulation of IGFBP‐3 in the medium which was due neither to alterations in IGFBP‐3 mRNA expression nor to extracellular IGFBP‐3 protease activity. Incubation of CTSL‐deficient fibroblasts with radiolabeled IGFBP‐3 followed by subcellular fractionation indicates that both intact and fragmented IGFBP‐3 accumulate transiently in endosomal and lysosomal fractions of CTSL‐deficient cells. This suggests the involvement of CTSL in the intracellular degradation of IGFBP‐3 representing a new mechanism to regulate the extracellular concentration of IGFBP‐3.
Behavior of a photoactivatable analog of cholesterol, 6‐photocholesterol, in model membranes
Authors: Mintzer Evan A. , Waarts Barry-Lee , Wilschut Jan , Bittman Robert
Published: October 2002

6‐Photocholesterol, a new photoactivatable analog of cholesterol in which a diazirine functionality replaces the 5,6‐double bond in the steroid nucleus, was used recently to identify cholesterol‐binding proteins in neuroendocrine cells [Thiele, C., Hannah, M.J., Farenholz, F. and Huttner, W.B. (2000) Nat. Cell Biol. 2, 42–49], to track the distribution and transport of cholesterol in Caenorhabditis elegans [Matyash, V., Geier, C., Henske, A., Mukherjee, S., Hirsh, D., Thiele, C., Grant, B., Maxfield, F.R. and Kurzchalia, T.V. (2001) Mol. Biol. Cell 12, 1725–1736], and to probe lipid–protein interactions in oligodendrocytes [Simons, M., Kramer, E.M., Thiele, C., Stoffel, W. and Trotter, J. (2000) J. Cell Biol. 151, 143–154]. To determine whether 6‐photocholesterol is a faithful mimetic of cholesterol we analyzed the ability of this probe, under conditions in which it is not photoactivated to a carbene, to substitute for cholesterol in two unrelated assays: (1) to condense 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine monomolecular films and (2) to mediate the fusion of two alphaviruses (Semliki Forest and Sindbis) with liposomes. The results suggest that this analog is a suitable photoprobe of cholesterol.
The phosphatidylinositol 3‐phosphate‐binding FYVE finger
Authors: Stenmark Harald , Aasland Rein , Driscoll Paul C.
Published: October 2002

The FYVE zinc finger domain is conserved from yeast (five proteins) to man (27 proteins). It functions in the membrane recruitment of cytosolic proteins by binding to phosphatidylinositol 3‐phosphate (PI3P), which is found mainly on endosomes. Here we review recent work that sheds light on the targeting of FYVE finger proteins to PI3P‐containing membranes, and how these proteins serve to regulate multiple cellular functions.