bluuebunny/crossref_metadata_2025 huggingface dataset:
Inner arm dynein 1 is essential for Ca++‐dependent ciliary reversals in Tetrahymena thermophila
Authors: Hennessey Todd M. , Kim Daniel Y. , Oberski Danial J. , Hard Robert , Rankin Scott A. , Pennock David G.
Published: October 2002

Cilia in many organisms undergo a phenomenon called ciliary reversal during which the cilia reverse the beat direction, and the cell swims backwards. Ciliary reversal is typically caused by a depolarizing stimulus that ultimately leads to a rise in intraciliary Ca++ levels. It is this increase in intraciliary Ca++ that triggers ciliary reversal. However, the mechanism by which an increase in intraciliary Ca++ causes ciliary reversal is not known. We have previously mutated the DYH6 gene of Tetrahymena thermophila by targeted gene knockout and shown that the knockout mutants (KO6 mutants) are missing inner arm dynein 1 (I1). In this study, we show that KO6 mutants do not swim backward in response to depolarizing stimuli. In addition to being unable to swim backwards, KO6 mutants swim forward at approximately one half the velocity of wild‐type cells. However, the ciliary beat frequency in KO6 mutants is indistinguishable from that of wild‐type cells, suggesting that the slow forward swimming of KO6 mutants is caused by an altered waveform rather than an altered beat frequency. Live KO6 cells are also able to increase and decrease their swim speeds in response to stimuli, suggesting that some aspects of their swim speed regulation mechanisms are intact. Detergent‐permeabilized KO6 mutants fail to undergo Ca++‐dependent ciliary reversals and do not show Ca++‐dependent changes in swim speed after MgATP reactivation, indicating that the axonemal machinery required for these responses is insensitive to Ca++ in KO6 mutants. We conclude that Tetrahymena inner arm dynein 1 is not only an essential part of the Ca++‐dependent ciliary reversal mechanism but it also may contribute to Ca++‐dependent changes in swim speed and to the formation of normal waveform during forward swimming. Cell Motil. Cytoskeleton 53:281–288, 2002. © 2002 Wiley‐Liss, Inc.
Assessing the efficiency of integrated pollution control regulation
Authors: Clinch J. Peter , Kerins David
Published: October 2002

A common criticism by economists of the command‐and‐control approach to environmental policymaking is that costs of compliance may be considerably higher than under flexible mechanisms. Unless all polluters face the same control costs, uniform emissions standards will not minimize the costs of reducing pollution. Integrated pollution control (IPC) licensing is an improvement on uniform regulation as, in principle, it takes into account the individual characteristics of the plant being allocated the licence. However, economists still tend to believe that the firm knows how to allocate resources best and that, because of the enormous information requirements, the regulator is unlikely to be able to achieve as low a cost of compliance as can a market corrected using market‐based instruments. This paper examines the efficiency of IPC regulation using the Irish system as a case study. In particular, it examines whether IPC regulation is likely to be cost minimizing. The results show that there appears to be the potential to reduce compliance costs if a flexible mechanism can be introduced which allows trading across sectors. Copyright © 2002 John Wiley & Sons, Ltd. and ERP Environment.
Buckling analysis for delaminated composites using plate bending elements based on higher‐order zig‐zag theory
Authors: Kim Jun‐Sik , Cho Maenghyo
Published: October 2002

A finite element based on the efficient higher‐order zig‐zag theory with multiple delaminations is developed. The bending part of the formulation is constructed from the concept of DKQ element. Unlike conventional elements, a developed element has its reference in the bottom surface which simplifies zig‐zag terms on formulation. Exact patch solutions are developed on elements which have the bottom reference system. The present element passes proper bending patch tests in the arbitrary mesh configurations in isotropic materials. Zig‐zag formulation is adopted to model laminated plates with multiple delaminations. To assess the accuracy and efficiency of the present element based on higher‐order zig‐zag theory with multiple delaminations, the linear buckling problem of laminated plates with multiple delaminations has been analysed. The results have been compared with three‐dimensional elasticity solutions. The present element works as an efficient tool for analysing the behaviour of the laminated composites with multiple delaminations. Copyright © 2002 John Wiley & Sons, Ltd.
Influence of maternal smoking on the birth prevalence of Down syndrome and on second trimester screening performance
Authors: Rudnicka Alicja R. , Wald Nicholas J. , Huttly Wayne , Hackshaw Allan K.
Published: October 2002

Objectives

To determine the influence of maternal smoking on the birth prevalence of Down syndrome and on second trimester screening performance.

Methods

First, a meta‐analysis of cohort and case‐control studies was performed to estimate the effect of maternal smoking on the live birth prevalence of Down syndrome. Then, data from 8779 women screened using the quadruple test (alphafetoprotein (AFP), unconjugated estriol (uE3), human chorionic gonadotrophin (free β‐hCG), and inhibin‐A levels with maternal age) were used to determine the effect of smoking on the serum markers. A Monte Carlo simulation was used to assess the impact of adjusting for smoking status on screening performance.

Results

The relative risk of Down syndrome in smokers (versus non‐smokers) was 0.95 (95% confidence interval (CI) 0.87 to 1.03). Serum marker levels were determined as multiples of the median for non‐smokers of the same gestational age and adjusted for maternal weight (MoM). The MoM values for AFP were 5% higher (95% CI 2–7%), uE3 4% lower (95% CI 2–5%), free β‐hCG 20% lower (95% CI 17–23%) and inhibin‐A 62% higher (95% CI 57–67%) in smokers compared with non‐smokers. Adjusting marker levels for smoking resulted in less than a 1 percentage point increase in the detection rate for a 5% false positive rate with the double, triple or quadruple tests.

Conclusions

There is no evidence of an association between the birth prevalence of Down syndrome and maternal smoking. The case for adjusting screening marker levels for smoking is not compelling. But if smoking data are collected routinely adjustment could be made and this would yield similar detection and false positive rates in smokers and non‐smokers. Copyright © 2002 John Wiley & Sons, Ltd.
Burrows–Wheeler compression with variable length integer codes
Authors: Fenwick Peter
Published: October 2002

The final coder in Burrows–Wheeler compression is usually either an adaptive Huffman coder (for speed) or a complex of arithmetic coders for better compression. This article describes the use of conventional pre‐defined variable length codes or universal codes and shows that they too can give excellent compression. The paper also describes a ‘sticky Move‐to‐Front’ modification which gives a useful improvement in compression for most files. Copyright © 2002 John Wiley & Sons, Ltd.
Papillary Thyroid Carcinoma Managed at the Mayo Clinic during Six Decades (1940–1999): Temporal Trends in Initial Therapy and Long‐term Outcome in 2444 Consecutively Treated Patients
Authors: Hay Ian D. , Thompson Geoffrey B. , Grant Clive S. , Bergstralh Eric J. , Dvorak Catherine E. , Gorman Colum A. , Maurer Megan S. , McIver Bryan , Mullan Brian P. , Oberg Ann L. , Powell Claudia C. , van Heerden Jon A. , Goellner John R.
Published: October 2002

It is uncertain whether more extensive primary surgery and increasing use of radioiodine remnant ablation
(RRA) for papillary thyroid carcinoma (PTC) have resulted in improved rates of cause‐specific mortality (CSM) and tumor recurrence (TR).
Details of the initial presentation, therapy, and outcome of 2444 PTC patients consecutively treated during 1940–1999 were recorded in a computerized database. Patients were followed for more than 43,000
patient‐years. The 25‐year rates for CSM and TR were 5% and 14%,
respectively. Temporal trends were analyzed for six decades. During the six decades, the proportion with initial MACIS (distant Metastasis, patient Age,
Completeness of resection, local Invasion, and tumor Size) scores <6 were 77%, 82%, 84%, 86%, 85%, and
82%, respectively (p = 0.06). Lobectomy accounted for
70% of initial procedures during 1940–1949 and 22% during
1950–1959; during 1960–1999 bilateral lobar resection (BLR) accounted for 91% of surgeries (p <0.001). RRA after BLR was performed during 1950–1969 in 3% but increased to 18%, 57%, and
46% in successive decades (p <0.001). The 40‐year rates for CSM and TR during 1940–1949 were significantly higher
(p = 0.002) than during 1950–1999. During the last 50
years the 10‐year CSM and TR rates for the 2286 cases did not significantly change with successive decades. Moreover, the 10‐year rates for CSM and TR were not significantly improved during the last five decades of the study, either for the 1917 MACIS <6 patients or the 369 MACIS ? 6 patients. Increasing use of RRA has not apparently improved the already excellent outcome, achieved before
1970, in low risk (MACIS <6) PTC patients managed by near‐total thyroidectomy and conservative nodal excision.
Targeted Parathyroidectomy in the Era of Intraoperative Parathormone Monitoring
Authors: Inabnet William B. , Dakin Gregory F. , Haber Richard S. , Rubino Francesco , Diamond Ed J. , Gagner Michel
Published: October 2002

A combination of preoperative localization and intraoperative parathormone (PTH) monitoring permits targeted parathyroidectomy. Multiple approaches have been developed,
ranging from unilateral neck exploration (UE) to radio‐guided parathyroidectomy (RP) to endoscopic parathyroidectomy (EP). The purpose of this study was to evaluate the efficacy of these approaches in the management of primary hyperparathyroidism. From June 1998 to November 2000 a total of 110 targeted parathyroid operations were performed at a university medical center. All patients underwent technetium‐99m‐sestamibi scanning, ultrasonography, or both prior to surgery. Intraoperative PTH monitoring was utilized in all cases.
Thirty‐seven patients underwent UE, 59 underwent RP, and 14 underwent EP. Follow‐up ranged from 1 to 24 months. All patients were cured following parathyroidectomy as predicted by a more than 50% reduction of the intraoperative PTH level following removal of all hypersecreting glands. Altogether, 103 patients had a solitary adenoma (95%), and 1
patient had a parathyroid carcinoma. Six patients (5%) had multigland disease, including four cases of hyperplasia and two patients with a double adenoma. Eighty‐three patients (75%) were discharged the day of surgery. The use of preoperative localization and intraoperative PTH monitoring permits a targeted approach to the treatment of primary hyperparathyroidism. Endocrine surgeons should be facile in all minimally invasive parathyroid techniques to individualize the operative approach.
Does Intraoperative Quick Parathyroid Hormone Assay Improve the Results of Parathyroidectomy?
Authors: Miura Daishu , Wada Nobuyuki , Arici Cumhur , Morita Eugene , Duh Quan‐Yang , Clark Orlo H.
Published: October 2002

Preoperative sestamibi (MIBI) and ultrasonography (US) are used to localize parathyroid tumors in patients with primary hyperparathyroidism (pHPT). The intraoperative quick PTH assay (qPTH) has been recommended to determine whether all hyperfunctioning parathyroid tissue has been removed. We questioned whether qPTH improves the results of parathyroidectomy in patients with pHPT. We analyzed 115 unselected patients with pHPT without a family history or multiple endocrine neoplasia but who had undergone parathyroidectomy. All 115 patients had successful operations without complications. Of these patients, 88 (77%) had solitary adenomas, 13
had double adenomas, 1 had triple adenomas, 12 had hyperplasia, and 1
had carcinoma. Overall, MIBI was correct in 72% (76/106), US in 49%
(49/99), and qPTH in 80% (92/115). For preoperative studies showing a single tumor, MIBI was correct in 83% (73/88), US was correct in 71%
(45/63), and combined MIBI and US were correct in 95% (37/39). Adding qPTH in this subgroup did not improve the successful focused approach:
70% for MIBI, 65% for US, and 87% for combined MIBI and US. However,
adding qPTH improved the overall success of parathyroidectomy (MIBI
92%, US 86%, combined MIBI and US 97%), but at the cost of unnecessary further exploration (MIBI 13%, US 6%, combined MIBI and US 8%). We conclude that when the same solitary tumor is identified by both MIBI and US, a focused exploration can be done with a 95% success rate. Adding qPTH to MIBI or US can improve the success rate but at a significant cost. General exploration of all parathyroid glands,
however, has the highest success rate (100%).
Vitamin D Status in Japanese Patients with Hyperparathyroidism: Seasonal Changes and Effect on Clinical Presentation
Authors: Yamashita Hiroyuki , Noguchi Shiro , Uchino Shinya , Watanabe Shin , Koike Eisuke , Murakami Tsukasa , Fujihira Takashi , Koga Yutaka , Masatsugu Toshihiro , Yamashita Hiroto
Published: October 2002

The disturbance of vitamin D metabolism plays an important role in determining the clinical presentation of hyperthyroidism. We studied 72 patients (65 women, 7 men) with primary hyperparathyroidism (pHPT). Clinical presentation, biochemical indices,
and bone mineral density (BMD) were compared in three patient groups classified according to their serum 25‐hydroxyvitamin D (25OHD)
levels: 23 patients whose 25OHD level was <25 nmol/L comprised the low group, 26 whose level was 25 to 40 nmol/L made up the intermediate group, and 23 whose level was > 40 nmol/L comprised the high group. The mean serum calcium level was 10.8 ± 0.9
mg/dl, and the mean weight of the resected parathyroids was 684 ±
749 mg. The mean serum 25OHD level was 36.5 ± 16.3 nmol/L (normal
25–100 nmol/L). Levels were below normal in 23 patients (32%). No between‐group differences existed for clinical presentation,
biochemistry, or BMD. Only differences in mean patient age were statistically significant between groups. Vitamin D deficiency is common among Japanese patients with pHPT, but the effects of HPT on clinical, biochemical, and densitometric indices are not pronounced.
Our study population was at an early stage of pHPT, so the vitamin D deficiency may not be associated with the effects of HPT.
Treatment of Small Intestinal Neuroendocrine Tumors: Is an Extended Multimodal Approach Justified?
Authors: Schindl Martin , Kaczirek Klaus , Passler Christian , Kaserer Klaus , Prager Gerhard , Scheuba Christian , Raderer Markus , Niederle Bruno
Published: October 2002

Neuroendocrine tumors (NETs) of the small bowel are known for their low malignant behavior. Although most cases are diagnosed in an advanced stage, natural life expectancy is significantly higher than with intestinal carcinomas. The question arises if there are any benefits to combining extended (radical or debulking) surgery, interventional treatment, and medical treatment with respect to life expectancy, and quality of life. A series of 58
patients (34 men, 24 women; mean age 61 ± 12 years, range 37–87
years) with NETs of the small bowel were retrospectively reviewed and prospectively followed over 63 ± 53 months. Clinical presentation, tumor characteristics, and postoperative medical treatment were documented. Quality of life was additionally analyzed using a questionnaire. Survival probability and quality of life were compared for tumor stages and type of treatment performed. In 47 of 58
(81%) patients the NET was diagnosed based on the presence of intestinal stenosis or endocrine symptoms. Of 16 patients without liver metastases, 15 (94%) were cured by radical surgery. Multiple liver metastases were evident in 40 of 58 (69%) patients and decreased the
5‐ and 10‐year cumulative survival to 64% ± 10% and 22% ± 10% (M0
vs. M1: p <0.05). The 5‐ and 10‐year survival rates after multimodal treatment (surgery, bio/immunotherapy, transarterial embolization of liver metastases) were 64% ± 11% and 28% ± 12%
compared to 61% ± 15% and 0% in patients without
(p = NS). In patients with small intestinal NETs, a consistent multimodal treatment helps to improve the overall survival and, in most patients, the quality of life.
Results of Laparoscopic Adrenalectomy for Large and Potentially Malignant Tumors
Authors: Henry Jean‐François , Sebag Frederic , Iacobone Maurizio , Mirallie Eric
Published: October 2002

ndoscopic adrenalectomy is the procedure of choice for patients with small functioning adrenal tumors. For most surgeons invasive adrenal carcinoma is an absolute contraindication for laparoscopic adrenalectomy (LA). Whether LA should be proposed for large (> 6 cm),
potentially malignant tumors is questionable. The aim of this study was to evaluate the risks and outcome of LA performed in our department in patients with tumors > 6 cm and potentially malignant. We performed a retrospective study of 216 patients who underwent 233 LAs in our department from 1994 to
2000. We selected 19 patients with a tumor > 6 cm and potentially malignant:
8 nonfunctional tumors, 4 cortisol‐secreting tumors, 1 virilizing tumor, and 6
pheochromocytomas. In none of these patients did preoperative investigations demonstrated invasive carcinoma. The median tumor size was 70 mm. LA was performed by a transperitoneal flank approach. Conversion to open adrenalectomy was performed in two patients owing to intraoperative evidence of invasive carcinoma. The median operating time was 150 minutes (range 95–240
minutes). Capsular disruption occurred during the dissection of two pheochromocytomas. There was no postoperative morbidity. Six patients had an adrenocortical carcinoma on pathologic diagnosis: three of the eight nonfunctional tumors, one of the four cortisol‐secreting tumors, and one virilizing tumor. One patient presented with liver metastases 6 months after surgery and died. The five other patients are disease‐free with a follow‐up ranging from 8 to 83 months. The 13 patients with benign lesions (6 cortical adenomas, 1 ganglioneuroma, 6 pheochromocytomas) are disease‐free with a median follow‐up of 47 months (range 10–81 months). In experienced hands LA can be proposed for large, potentially malignant tumors. Conversion to open adrenalectomy should be performed if local invasion is observed during surgery.
At present the risk of intraabdominal recurrence is unknown.
FMN is covalently attached to a threonine residue in the NqrB and NqrC subunits of Na+‐translocating NADH‐quinone reductase from Vibrio alginolyticus
Authors: Hayashi Maki , Nakayama Yuji , Yasui Mikako , Maeda Masahiro , Furuishi Kazuchika , Unemoto Tsutomu
Published: October 2002

The Na+‐translocating NADH‐quinone reductase (NQR) from Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). We previously demonstrated that both NqrB and NqrC subunits contain a flavin cofactor covalently attached to a threonine residue. Fluorescent peptide fragments derived from the NqrB and NqrC subunits were applied to a matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometer, and covalently attached flavin was identified as FMN in both subunits. From post‐source decay fragmentation analysis, it was concluded that FMN is attached by a phosphate group to Thr‐235 in the NqrB subunit and to Thr‐223 in the NqrC subunit. The phosphoester binding of FMN to a threonine residue reported here is a new type of flavin attachment to a polypeptide.
Carbohydrate transporters of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS)
Authors: Siebold Christian , Flükiger Karin , Beutler Rudolf , Erni Bernhard
Published: October 2002

The glucose transporter of Escherichia coli couples translocation with phosphorylation of glucose. The IICBGlc subunit spans the membrane eight times. Split, circularly permuted and cyclized forms of IICBGlc are described. The split variant was 30 times more active when the two proteins were encoded by a dicistronic mRNA than by two genes. The stability and activity of circularly permuted forms was improved when they were expressed as fusion proteins with alkaline phosphatase. Cyclized IICBGlc and IIAGlc were produced in vivo by RecA intein‐mediated trans‐splicing. Purified, cyclized IIAGlc and IICBGlc had 100% and 30% of wild‐type glucose phosphotransferase activity, respectively. Cyclized IIAGlc displayed increased stability against temperature and GuHCl‐induced unfolding.
Topogenesis of membrane proteins: determinants and dynamics
Authors: Goder Veit , Spiess Martin
Published: October 2002

For targeting and integration of proteins into the mammalian endoplasmic reticulum, two types of signals can be distinguished: those that translocate their C‐terminal sequence (cleavable signals and signal‐anchors) and those that translocate their N‐terminus (reverse signal‐anchors). In addition to the well established effect of flanking charges, also the length and hydrophobicity of the apolar core of the signal as well as protein folding and glycosylation contribute to orienting the signal in the translocon. In multi‐spanning membrane proteins, topogenic determinants are distributed throughout the sequence and may even compete with each other. During topogenesis, segments of up to 60 residues may move back and forth through the translocon, emphasizing unexpected dynamic aspects of topogenesis.
Superoxide dismutase mutations of familial amyotrophic lateral sclerosis and the oxidative inactivation of calcineurin
Authors: Volkel Helge , Scholz Maike , Link Johannes , Selzle Martin , Werner Peter , Tunnemann Ralf , Jung Guenter , Ludolph Albert C. , Reuter Alexander
Published: October 2002

Approximately 10% of all familial cases of amyotrophic lateral sclerosis (fALS) are linked to mutations in the SOD1 gene, which encodes the copper/zinc superoxide dismutase (CuZnSOD). Recently, wild‐type CuZnSOD was shown to protect calcineurin, a calcium/calmodulin‐regulated phosphoprotein phosphatase, from inactivation by reactive oxygen species. We asked whether the protective effect of CuZnSOD on calcineurin is affected by mutations associated with fALS. For this, we monitored calcineurin activity in the presence of mutant and wild‐type SOD. We found that the degree of protection against inactivation of calcineurin by different SOD mutants correlates with the severity of the phenotype associated with the different mutations, suggesting a potential role for calcineurin–SOD1 interaction in the etiology of fALS.
Two‐dimensional crystals: a powerful approach to assess structure, function and dynamics of membrane proteins
Authors: Stahlberg Henning , Fotiadis Dimitrios , Scheuring Simon , Rémigy Hervé , Braun Thomas , Mitsuoka Kaoru , Fujiyoshi Yoshinori , Engel Andreas
Published: October 2002

Electron crystallography and atomic force microscopy allow the study of two‐dimensional membrane protein crystals. While electron crystallography provides atomic scale three‐dimensional density maps, atomic force microscopy gives insight into the surface structure and dynamics at sub‐nanometer resolution. Importantly, the membrane protein studied is in its native environment and its function can be assessed directly. The approach allows both the atomic structure of the membrane protein and the dynamics of its surface to be analyzed. In this way, the function‐related conformational changes can be assessed, thus providing a detailed insight on the molecular mechanisms of essential biological processes.
Multiple phosphorylation sites in RGS16 differentially modulate its GAP activity
Authors: Chen Canhe , Wang Huashan , Fong Chee Wai , Lin Sheng-Cai
Published: October 2002

Regulators of G‐protein signaling (RGS) are GTPase‐activating proteins (GAP) for activated Gα subunits. We found that mouse RGS16, when expressed in HEK293T cells, is phosphorylated constitutively at serine 194 based on in vivo orthophosphate labeling experiments, while serine 53 is phosphorylated in a ligand‐dependent manner upon stimulation by epinephrine in cells expressing the α2A adrenergic receptor. Phosphorylation on both sites impairs its GAP activity and subsequent attenuation on heterotrimeric G‐protein‐stimulated extracellular signal‐regulated protein kinase activity. This is the first report of RGS functional downregulation by phosphorylation via a G‐protein‐coupled receptor.
Subunit positioning and transmembrane helix organisation in the core dimer of photosystem II
Authors: Hankamer Ben , Morris Ed , Nield Jon , Carne Alex , Barber James
Published: October 2002

Recently 3D structural models of the photosystem II (PSII) core dimer complexes of higher plants (spinach) and cyanobacteria (Synechococcus elongatus) have been derived by electron [Rhee et al. (1998) Nature 396, 283–286; Hankamer et al. (2001) J. Struct. Biol., in press] and X‐ray [Zouni et al. (2001) Nature 409, 739–743] crystallography respectively. The intermediate resolutions of these structures do not allow direct identification of side chains and therefore many of the individual subunits within the structure are unassigned. Here we review the structure of the higher plant PSII core dimer and provide evidence for the tentative assignment of the low molecular weight subunits. In so doing we highlight the similarities and differences between the higher plant and cyanobacterial structures.
Detection of a conformational change in Gγ upon binding Gβ in living cells
Authors: Dues Gisbert , Müller Silke , Johnsson Nils
Published: October 2002

Interaction induced changes in the conformation of proteins are frequently the molecular basis for the modulation of their activities. Although proteins perform their functions in cells, surrounded by many potential interaction partners, the studies of their conformational changes have been mainly restricted to in vitro studies. Ste4p (Gβ) and Ste18p (Gγ) are the subunits of a heterotrimeric G‐protein in the yeast Saccharomyces cerevisiae. A split‐ubiquitin based conformational sensor was used to detect a major structural rearrangement in Ste18p upon binding to Ste4p. Based on these in vivo results and the solved structure of the mammalian Gβγ, we propose that Gγ of yeast adopts an equally extended structure, which is only induced upon association with Gβ.
A region encompassing the FERM domain of Jak1 is necessary for binding to the cytokine receptor gp130
Authors: Hilkens Catharien M.U. , Is'harc Hayaatun , Lillemeier Björn F. , Strobl Birgit , Bates Paul A. , Behrmann Iris , Kerr Ian M.
Published: October 2002

The terminal portion of the Janus kinases (Jaks) contains a divergent FERM (our‐point‐one, zrin, adixin, oesin) homology domain comprising 19 conserved hydrophobic regions. To determine the role of this domain in governing recruitment of Jak1, but not Jak3, to the gp130 subunit of the interleukin‐6 family of cytokine receptors, the interaction of three Jak1/Jak3 chimeras with gp130 was investigated. Chimeras 1, 2 and 3 (Jak1 FERM regions 1–19, 1–18 and 1–8/Jak3, respectively) were all enzymically active. Chimeras 1 and 2 interacted with the cytoplasmic domain of gp130, although less efficiently than Jak1. Only chimera 2, however, restored gp130 signalling in Jak1‐negative cells. The data are consistent with recruitment of Jak1 to gp130 through the Jak1 FERM domain, but also emphasise the likely requirement for precise Jak/receptor orientation to sustain function.
NMR indirect detection of glutamate to measure citric acid cycle flux in the isolated perfused mouse heart
Authors: Burgess Shawn C. , Babcock Evelyn E. , Jeffrey F.Mark H. , Sherry A.Dean , Malloy Craig R.
Published: October 2002

13C‐edited proton nuclear magnetic resonance (NMR) spectroscopy was used to follow enrichment of glutamate C3 and C4 with a temporal resolution of ∼20 s in mouse hearts perfused with 13C‐enriched substrates. A fit of the NMR data to a kinetic model of the tricarboxylic acid (TCA) cycle and related exchange reactions yielded TCA cycle (V
tca) and exchange (V
x) fluxes between α‐ketoglutarate and glutamate. These fluxes were substrate‐dependent and decreased in the order acetate (V
tca=14.1 μmol g−1 min−1; V
x=26.5 μmol g−1 min−1)>octanoate (V
tca=6.0 μmol g−1 min−1; V
x=16.1 μmol g−1 min−1)>lactate (V
tca=4.2 μmol g−1 min−1; V
x=6.3 μmol g−1 min−1).
Activation of the MKK4‐JNK pathway during erythroid differentiation of K562 cells is inhibited by the heat shock factor 2‐β isoform
Authors: Hietakangas Ville , Elo Iina , Rosenström Heidi , Coffey Eleanor T. , Kyriakis John M. , Eriksson John E. , Sistonen Lea
Published: October 2002

In this study we report the activation of c‐Jun N‐terminal kinase (JNK) in human K562 erythroleukemia cells undergoing hemin‐mediated erythroid differentiation, which occurs concomitantly with activation of heat shock factor 2 (HSF2) and leads to a simultaneous in vivo phosphorylation of c‐Jun. The activation of JNK occurs through activation of mitogen‐activated protein kinase kinase (MKK) 4 and not by activation of MKK7 or inhibition of JNK‐directed phosphatases. We have previously shown that overexpression of the HSF2‐β isoform inhibits the activation of HSF2 upon hemin‐induced erythroid differentiation. Here we demonstrate that HSF2‐β overexpression blocks the hemin‐induced activation of the MKK4‐JNK pathway, suggesting an erythroid lineage‐specific JNK activation likely to be regulated by HSF2.
Mimicking phosphorylation at Ser‐48 strongly reduces surface expression of human macrophage scavenger receptor class A: implications on cell motility
Authors: Heider Harald , Wintergerst Eva S
Published: October 2002

The role of human macrophage scavenger receptor A1 (SRA1) in the development of atherosclerotic lesions is still scarcely defined. Substituting serine 48 in human SRA1 by an aspartate demonstrated that (1) surface expression of the mutated receptor was 13‐fold decreased; (2) the amount of cell‐associated Texas red‐labeled acetylated low density lipoprotein (LDL) in mutant receptor‐expressing cells was almost three‐fold reduced; (3) the migration of mutant receptor‐transfected cells towards surfaces coated with oxidized LDL decreased by almost 60% compared to cells that were transfected with the wild type receptor. Phosphorylation of the cytoplasmic part of SRA1 may help to modulate the residence time of macrophages in atherosclerotic lesions.
Involvement of protein dynamics in enzyme stability
Authors: Haouz Ahmed , Glandières Jean Marie , Alpert Bernard
Published: October 2002

Dynamics of glucose oxidase immobilized and in solution were compared through their tryptophan fluorescence spectra, decay times and quenching by acrylamide. Energy barrier for thermal inactivation and melting temperature of both soluble and immobilized enzyme were also measured. Data show that the fluctuation amplitude is at the origin of protein instability.
Interaction between two adapter proteins, PAG and EBP50: a possible link between membrane rafts and actin cytoskeleton
Authors: Brdičková Naděžda , Brdička Tomáš , Anděra Ladislav , Špička Jiřı́ , Angelisová Pavla , Milgram Sharon L. , Hořejšı́ Václav
Published: October 2002

Phosphoprotein associated with GEMs (PAG), also known as Csk‐binding protein (Cbp), is a broadly expressed palmitoylated transmembrane adapter protein found in membrane rafts, also called GEMs (glycosphingolipid‐enriched membrane microdomains). PAG is known to bind and activate the essential regulator of Src‐family kinases, cytoplasmic protein tyrosine kinase Csk. In the present study we used the yeast 2‐hybrid system to search for additional proteins which might bind to PAG. We have identified the abundant cytoplasmic adapter protein EBP50 (ezrin/radixin/moesin (ERM)‐binding phosphoprotein of 50 kDa), also known as NHERF (Na+/H+ exchanger regulatory factor), as a specific PAG‐binding partner. The interaction involves the C‐terminal sequence (TRL) of PAG and N‐terminal PDZ domain(s) of EBP50. As EBP50 is known to interact via its C‐terminal domain with the ERM‐family proteins, which in turn bind to actin cytoskeleton, the PAG–EBP50 interaction may be important for connecting membrane rafts to the actin cytoskeleton.
A simple estimation of peroxisomal degradation with green fluorescent protein – an application for cell cycle analysis
Authors: Arai Kunizo , Ohkuma Shoji , Matsukawa Toru , Kato Satoru
Published: October 2002

When nutrients are depleted from the environment, mammalian cells begin to degrade their own cytosol and organelles. This bulk protein degradation is mediated by autophagy. In this study, peroxisomes in living CHO‐K1 cells were visualized by targeting the green fluorescent protein (GFP) tagged with a type 1 peroxisomal targeting signal. The nutrient‐starved condition induced a decay of GFP fluorescence in the peroxisomes and autophagic inhibitors such as 3‐methyladenine suppressed the decay of GFP fluorescence (13–60% of starvation). By double labeling the nuclear DNA and peroxisomal GFP, the autophagy specifically occurred at the G1 phase of the cell cycle and the autophagic inhibitors suppressed the G1 arrest. The vital stain technique with GFP is a very simple and useful marker to quantitatively estimate or to further study peroxisomal degradation.
Focal adhesion kinase and Src mediate integrin regulation of insulin receptor phosphorylation
Authors: El Annabi Slim , Gautier Nadine , Baron Véronique
Published: October 2002

We show here that phosphorylation of the insulin receptor and insulin receptor substrate‐1 is increased when suspended cells are replated on fibronectin. This is not due to decreased numbers of cell surface receptors, alteration of insulin binding, or stimulation of a phosphatase activity in non‐adherent cells. Expression of Src together with focal adhesion kinase (FAK) in suspended cells restores insulin‐induced receptor autophosphorylation to levels observed in fibronectin‐attached cells. Conversely, expression of dominant‐negative mutants of either Src or FAK abolishes potentiation of insulin receptor phosphorylation by cell adhesion. The results suggest that both Src and FAK participate in integrin‐mediated regulation of insulin receptor signal.
A look into kinesin's powerhouse
Authors: Woehlke Guenther
Published: October 2002

Kinesins are microtubule‐dependent motors that serve a multitude of cellular purposes. The conserved motor domain provides the energy required for these processes. Shortly after the solution of the first kinesin motor domain crystal structures the similarity to myosin and G‐proteins was noted. By analogy, it was suspected that regions flanking the γ‐phosphate group of the nucleotide (in particular the so‐called switch I and II regions) play important roles in the catalytic mechanism and the communication between the nucleotide cleft and the microtubule binding site. Since then, mutational analyses have supported this notion. Moreover, additional high‐resolution structures have demonstrated that the switch regions can assume variable conformations. In one case, a comparison of an ADP state and an ATP‐like state indicates a crucial involvement of the helix flanking switch II in modulating microtubule affinity. High‐resolution structures of a kinesin‐related protein mutated in the switch regions confirm the correlation between structural features in the switch vicinity and coupling of microtubule binding and nucleotide state.
Histamine increases the expression of LOX‐1 via H2 receptor in human monocytic THP‐1 cells
Authors: Tanimoto Akihide , Murata Yoshitaka , Nomaguchi Masako , Kimura Satoshi , Arima Nobuyuki , Xu Hui , Hamada Tetsuo , Sasaguri Yasuyuki
Published: October 2002

Lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1) is a member of the scavenger receptor family, and is known to be expressed in monocytes/macrophages. We investigated the effect of histamine on the expression of LOX‐1 in cells of the human monocytic leukemia cell line THP‐1. Histamine as well as forskolin and dibutyryl cyclic AMP (Bt2‐cAMP) stimulated the THP‐1 monocytes to express the LOX‐1 gene at the transcription level. This histamine effect on LOX‐1 gene expression, via the histamine H2 receptor‐mediated cAMP signal transduction pathway, was reduced after differentiation of the cells into macrophages, even though forskolin and Bt2‐cAMP still enhanced the gene expression. The alteration of the responsiveness of LOX‐1 expression to histamine was related to suppressed expression of the H2 receptor in THP‐1 macrophages. The switch of the predominant class of histamine receptors between H1 and H2 would modulate the effects of histamine on LOX‐1 gene expression in monocytes and macrophages, and therefore, would play a certain role in the inflammatory aspects of atherogenesis.
Mechanism of Kex2p inhibition by its proregion
Authors: Lesage Guillaume , Tremblay Mélanie , Guimond Julie , Boileau Guy
Published: October 2002

Many proteases are produced as zymogens bearing an N‐terminal proregion acting both as intramolecular chaperone and as enzyme inhibitor. We studied here the inhibition mechanism of the yeast proprotein convertase Kex2p by its proregion. A recombinant secreted and soluble form of Kex2p was produced in Pichia pastoris and its enzymatic properties toward a fluorogenic synthetic peptide were characterized. Recombinant Escherichia coli‐produced Kex2p proregion specifically and potently inhibited the enzyme, with an IC50 of 160 nM. Exploration of the inhibition mechanism revealed that the proregion behaved as a mixed inhibitor.
Urokinase‐type plasminogen activator up‐regulates the expression of its cellular receptor through a post‐transcriptional mechanism
Authors: Montuori Nunzia , Mattiello Amalia , Mancini Alessandro , Santoli Mariano , Taglialatela Paola , Caputi Mario , Rossi Guido , Ragno Pia
Published: October 2002

We have recently reported that the urokinase‐type plasminogen activator (uPA) up‐regulates the cell surface expression of its own receptor (uPAR) in several cell types, independently of its enzymatic activity. uPA has no effect on kidney 293 cells which do not express uPAR and then cannot bind uPA. Kidney cells, transfected with the coding region of uPAR cDNA, express very large amounts of uPAR and respond to uPA stimulation by regulating uPAR both at mRNA and protein levels. uPA effect occurs also in the presence of the transcriptional inhibitor dichloro‐ribobenzimidazole, whereas it is abolished by the protein synthesis inhibitor cycloheximide. Moreover, uPA‐dependent uPAR up‐regulation correlates with the increase of a complex between the coding region of uPAR mRNA and an unknown cellular factor. We then propose that uPA regulates uPAR expression at a post‐transcriptional level, by promoting the binding of uPAR mRNA to a stabilizing factor.
Ca2+‐induced folding of a family I.3 lipase with repetitive Ca2+ binding motifs at the C‐terminus
Authors: Amada Kei , Kwon Hyun-Ju , Haruki Mitsuru , Morikawa Masaaki , Kanaya Shigenori
Published: October 2002

In order to understand a role of the Ca2+ ion on the structure and function of a Ca2+‐dependent family I.3 lipase from Pseudomonas sp. MIS38, apo‐PML, holo‐PML, holo‐PML*, and the N‐terminal domain alone (N‐fragment) were prepared and biochemically characterized. Apo‐PML and holo‐PML represent refolded proteins in the absence and presence of the Ca2+ ion, respectively. Holo‐PML* represents a holo‐PML dialyzed against 20 mM Tris–HCl (pH 7.5). The results suggest that the C‐terminal domain of PML is almost fully unfolded in the apo‐form and its folding is induced by Ca2+ binding. The folding of this C‐terminal domain may be required to make a conformation of the N‐terminal catalytic domain functional.
α‐Synuclein metabolism and aggregation is linked to ubiquitin‐independent degradation by the proteasome
Authors: Tofaris George K , Layfield Robert , Spillantini Maria Grazia
Published: October 2002

α‐Synuclein has been implicated in the pathogenesis of Parkinson's disease based on mutations in familial cases of the disease and its presence in Lewy bodies. Here we show that over‐expression of wild‐type human α‐synuclein is sufficient to induce inclusion formation in SH‐SY5Y cells. In this cellular model, proteasome inhibition leads to an increase of α‐synuclein accumulation in vivo without ubiquitylation. In accordance, we find that in vitro, unmodified α‐synuclein can be directly degraded by the 20S proteasome. These findings suggest an ubiquitin‐independent mechanism of proteasomal degradation for α‐synuclein and other natively unfolded proteins.
Citrulline, a novel compatible solute in drought‐tolerant wild watermelon leaves, is an efficient hydroxyl radical scavenger
Authors: Akashi Kinya , Miyake Chikahiro , Yokota Akiho
Published: October 2002

Drought‐tolerant wild watermelon accumulates high levels of citrulline in the leaves in response to drought conditions. In this work, the hydroxyl radical‐scavenging activity of citrulline was investigated in vitro. The second‐order rate constant for the reaction between citrulline and hydroxyl radicals was found to be 3.9×109 M−1 s−1, demonstrating that citrulline is one of the most efficient scavengers among compatible solutes examined so far. Moreover, citrulline effectively protected DNA and an enzyme from oxidative injuries. Liquid chromatography‐mass spectrometry analysis revealed that at least four major products were formed by the reaction between citrulline and hydroxyl radicals. Activities of metabolic enzymes were not inhibited by up to 600 mM citrulline, indicating that citrulline does not interfere with cellular metabolism. We reasoned, from these results, that citrulline contributes to oxidative stress tolerance under drought conditions as a novel hydroxyl radical scavenger.
Asn78 and His81 form a destabilizing locus within the Max HLH‐LZ homodimer
Authors: Tchan Michel C , Weiss Anthony S
Published: October 2002

The human Max protein lies at the center of the Myc/Max/Mad family of transcription factors. Its role at the center of this regulatory network is dependent on the helix‐loop‐helix leucine zipper (HLH‐LZ) dimerization domain. The Max LZ contains three residues that deviate from the pattern of hydrophobic amino acids normally present at the interface of LZ dimers: Asn78, His81 and Asn92. In contrast to interfacial Asn residues in other LZ proteins, we have shown that Asn92 does not act to destabilize the homodimer. Here we describe thermal denaturation experiments performed on Asn78 and His81 mutants demonstrating that these residues are involved in actively destabilizing the Max homodimer.
The effect of β‐carotene on the expression of interleukin‐6 and heme oxygenase‐1 in UV‐irradiated human skin fibroblasts in vitro
Authors: Obermüller-Jevic Ute C. , Schlegel Beate , Flaccus Andrea , Biesalski Hans Konrad
Published: October 2002

β‐Carotene is discussed as an anti‐oxidant micronutrient and singlet oxygen quencher in human skin, protecting against UV light‐induced damage. However, we recently demonstrated that β‐carotene has a pro‐oxidant potential in cultured human skin fibroblasts because it enhances the UVA induction of heme oxygenase‐1 (HO‐1). Herein, we further show that β‐carotene also strongly promotes the UVA induction of pro‐inflammatory interleukin‐6 (IL‐6) in skin fibroblasts in vitro. Singlet oxygen quencher sodium azide abrogated up‐regulation of IL‐6, and likewise also of HO‐1. In UVB‐irradiated cells, β‐carotene did not modulate levels of IL‐6 and HO‐1. The observed effects might be relevant for UV‐induced inflammatory processes.
Multiple interactions of the cytosolic polyproline region of the CD95 ligand: hints for the reverse signal transduction capacity of a death factor1
Authors: Wenzel Jennifer , Sanzenbacher Ralf , Ghadimi Markus , Lewitzky Marc , Zhou Qingchun , Kaplan David R. , Kabelitz Dieter , Feller Stephan M. , Janssen Ottmar
Published: October 2002

The CD95/Fas/Apo‐1 ligand is expressed on activated lymphocytes, NK cells, platelets, certain immune‐privileged cells and some tumor cells and induces apoptosis through the death receptor CD95/Fas/Apo‐1. In murine T cells, membrane‐bound CD95L (Fas ligand) also acts as a costimulatory receptor to coordinate activation and function in vivo. The molecular basis for this reverse signal transduction is yet unknown. In the present report, we identify individual interaction domains of enzymes and adapter molecules that selectively interact with full‐length CD95L from transfectants and human T cells. These results may help to explain the costimulatory capacity of CD95L.
Modulation of HIV‐1 enhancer activity and virus production by cAMP
Authors: Banas Brigitte , Eberle Josef , Banas Bernhard , Schlöndorff Detlef , Luckow Bruno
Published: October 2002

The effect of cAMP on the transcriptional activity of the HIV‐1 long terminal repeat/enhancer was investigated and compared to the effect of cAMP on virus replication. In culture cAMP repressed virus replication in vivo using different cell types. Transient transfection studies with HIV‐1 enhancer‐derived luciferase reporter gene constructs identified the minimal DNA sequence mediating the negative regulatory effect of cAMP on HIV‐1 transcription. A single nuclear factor κB element from the HIV‐1 enhancer mediates the repressive effect on transcription. AP‐2 is not involved in cAMP repression. Stable transfection of Jurkat T cells with the co‐activators CREB binding protein (CBP) and p300 completely abolished the cAMP repressive effect, supporting the hypothesis that elevation of intracellular cAMP increases phosphorylation of CREB, which then competes with phosphorylated p65 and Ets‐1 for limiting amounts of CBP/p300 thereby mediating the observed repressive effect on transcription. These findings suggest an important role of cAMP on HIV‐1 transcription.
Transferrin binds insulin‐like growth factors and affects binding properties of insulin‐like growth factor binding protein‐3
Authors: Storch Stephan , Kübler Bernd , Höning Stefan , Ackmann Michael , Zapf Jürgen , Blum Werner , Braulke Thomas
Published: October 2002

In the circulation, most of the insulin‐like growth factors (IGFs) are bound to a ternary 150 kDa complex with IGF‐binding protein (IGFBP)‐3 and the acid labile subunit. In the current study, we identify transferrin (Tf) by mass spectrometry, and immunoprecipitation as a component of a major IGF‐binding fraction separated from human plasma. IGF ligand blotting, cross‐linkage experiments and surface plasmon resonance spectrometry have been used to demonstrate the capability of Tf to bind IGFs specifically. In combination with Tf, IGFBP‐3 showed a 5‐fold higher affinity for IGF‐II than IGFBP‐3 alone. The data suggest that Tf may play an important role in regulating IGF/IGFBP‐3 functions.
Simplicity in complexity: the photosynthetic reaction center performs as a simple 0.2 V battery
Authors: van Rotterdam Bart J. , Crielaard Wim , van Stokkum Ivo H.M. , Hellingwerf Klaas J. , Westerhoff Hans V.
Published: October 2002

The photosynthetic reaction center is one of the most complicated molecular complexes. Transducing photon energy to a transmembrane electrochemical potential difference for protons, it is the direct or indirect energy source for virtually all life. We show here that it operates in a simple, battery‐like manner, with a maximum potential of 0.20 V. Intriguingly this is only one fifth of the energy of the absorbed photon.
Cytosolic thioredoxin peroxidase I is essential for the antioxidant defense of yeast with dysfunctional mitochondria
Authors: Demasi Ana P.D. , Pereira Gonçalo A.G. , Netto Luis E.S.
Published: October 2002

The specific role of cytosolic thioredoxin peroxidase I (cTPx I), encoded by TSA1 (thiol‐specific antioxidant), was investigated in the oxidative stress response of Saccharomyces cerevisiae. In most cases, deletion of TSA1 has showed only a slight effect on hydrogen peroxide sensitivity. However, when the functional state of the mitochondria was compromised, the necessity of TSA1 in cell protection against this oxidant was much more evident. All the procedures used to disrupt the mitochondrial respiratory chain promoted increases in the generation of H2O2 in cells, which could be related to their elevated sensitivity to oxidative stress. In fact, TSA1 is highly expressed when cells with respiratory deficiency are exposed to H2O2. In conclusion, our results indicate that cTPx I is a key component of the antioxidant defense in respiratory‐deficient cells.