bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacokinetics/Pharmacodynamics of Desloratadine and Fluoxetine in Healthy Volunteers
Authors: Gupta Samir , Banfield Christopher , Kantesaria Bhavna , Flannery Brian , Herron Jerry
Published: October 2004

The authors assessed the potential for a pharmacokinetic/pharmacodynamic interaction between desloratadine and fluoxetine. This randomized, placebo‐controlled, open‐label study was conducted in 54 healthy volunteers. Subjects received 1 of 3 treatments: desloratadine 5 mg plus fluoxetine 20 mg, desloratadine 5 mg plus placebo, or fluoxetine 20 mg plus placebo. Serial electrocardiograms (ECGs) were performed at baseline and day 35. Treatment effects on Cmax and AUC were assessed. During coadministration of desloratadine with fluoxetine, the ratio of the mean log‐transformed Cmax and AUC values for desloratadine following concomitant fluoxetine therapy revealed a small increase in Cmax values of 15% (90% confidence interval [CI], 95%–139%) but no increase for AUC values (90% CI, 82%–123%). Corresponding values for 3‐OH desloratadine demonstrated small increases in mean log‐transformed Cmax and AUC ratios: 17% (90% CI, 100%–136%) and 13% (90% CI, 96%–132%), respectively. Statistical evaluation of the ratio of the mean Cmax and AUC values for fluoxetine following concomitant desloratadine therapy revealed small decreases of 9% (90% CI, 72%–115%) and 11% (90% CI, 69%–113%), respectively. Corresponding values for norfluoxetine demonstrated modest increases in mean log‐transformed Cmax and AUC ratios: 22% (90% CI, 100%–139%) and 18% (90% CI, 101%–136%), respectively. Coadministration of desloratadine with a potent inhibitor of CYP2D6 did not result in clinically relevant changes in its pharmacokinetic parameters. Desloratadine administration was not associated with clinically important changes in the pharmacokinetics of fluoxetine, a drug metabolized by CYP2D6. The most common adverse event in all groups was headache (65%). Desloratadine plus fluoxetine caused no significant changes in ECGs or ventricular rate.
Production of hemo- and immunoregulatory cytokines by erythroblast antigen+ and glycophorin A+ cells from human bone marrow
Authors: Sennikov Sergey V , Injelevskaya Tatyana V , Krysov Sergey V , Silkov Alexandr N , Kovinev Igor B , Dyachkova Natalya J , Zenkov Anton N , Loseva Mary I , Kozlov Vladimir A
Published: October 2004

Background

Erythroid nuclear cells (ENC) of the bone marrow (BM) have not previously been considered as important producers of wide spectrum of haemo- and immunoregulatory cytokines. The aim of the current work was to confirm the production of the main hemo- and immunoregulatory cytokines in human ENC from BM.

Results

We used native human BM ENC in our experiments. We for the first time have shown, that the unstimulated erythroblasts (Gl A+ or AG-EB+) produced a wide spectrum of immunoregulatory cytokines. Human BM ENC produce cytokines such as interleukn (IL)-1β, IL-2, IL-4, IL-6, interferon (IFN)-γ, transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α and IL-10. They can be sub-divided into glycophorin A positive (Gl A+) and erythroblast antigen positive (AG-EB+) cells. To study potential differences in cytokine expression between these subsets, ENC were isolated and purified using specific antibodies to Gl A and AG-EB and the separated cells were cultivated for 24 hours. The cytokine contents of the supernatant were measured by electrochemiluminescence immunoassay. Quantitative differences in TGF-β1 and TNF-α production were found between Gl A+ and AG-EB+ BM ENC. Furthermore, in vitro addition of erythropoietin (EPO) reduced IFN-γ and IL-2 production specifically by the AG-EB+ ENC. Thus, Gl A+ and AG-EB+ ENC produce IL-1β, IL-2, IL-4, IL-6, IFN-γ, TGF-β1 and TNF-α. Gl A+ ENC also produce IL-10.

Conclusion

Cytokine production by erythroid nuclear cells suggests that these cells might be involved in regulating the proliferation and differentiation of hematopoietic and immunocompetent cells in human BM.
Patterns of use, dosing, and economic impact of biologic agent use in patients with rheumatoid arthritis: a retrospective cohort study
Authors: Gilbert Thomas D , Smith Daniel , Ollendorf Daniel A
Published: October 2004

Background

Variability in dosing and costs of biologics among patients with rheumatoid arthritis (RA) is of interest to healthcare descision-makers. We examined dosing and costs among RA patients newly treated with infliximab or etanercept under conditions of typical clinical practice.

Methods

Integrated pharmacy and medical claims data were obtained from 61 U.S. health plans. RA patients newly treated with infliximab or etanercept between July 1999–June 2002 were selected. A maintenance number of infliximab vials was determined after the "loading period" (2–3 infusions); those with ≥ 2 occurrences of an increase in vials or an interval between infusions of <49 days were considered to have had escalated. For etanercept patients, escalation was based on ≥ 2 instances of increased average daily dose. Multiple logistic regression analyses were conducted to assess variables associated with dose escalation. RA-related costs at one year post-initiation also were examined; comparisons were made using generalized linear models.

Results

A total of 1,548 patients were identified (n = 598 and 950 for infliximab and etanercept respectively). Infliximab recipients were somewhat older (50.5 vs. 46.6 years for etanercept). Nearly 60% of infliximab patients increased their dose at one year, compared to 18% for etanercept. Infliximab patients who escalated dose incurred a 25% increase in mean one-year costs ($20,915 vs. $16,713 for no increase; p < 0.0001). Costs among etanercept patients did not substantially differ based on dose escalation ($14,482 vs. $13,866 respectively).

Conclusions

Infliximab is associated with higher rates of dose escalation relative to etanercept, which contributes to substantially higher one-year medical costs.
Use of polyethyleneimine polymer in cell culture as attachment factor and lipofection enhancer
Authors: Vancha Ajith R , Govindaraju Suman , Parsa Kishore VL , Jasti Madhuri , González-García Maribel , Ballestero Rafael P
Published: October 2004

Background

Several cell lines and primary cultures benefit from the use of positively charged extracellular matrix proteins or polymers that enhance their ability to attach to culture plates. Polyethyleneimine is a positively charged polymer that has gained recent attention as a transfection reagent. A less known use of this cationic polymer as an attachment factor was explored with several cell lines.

Results

Polyethyleneimine compared favorably to traditional attachment factors such as collagen and polylysine. PC-12 and HEK-293 cells plated on dishes coated with polyethyleneimine showed a homogeneous distribution of cells in the plate, demonstrating strong cell adhesion that survived washing procedures. The polymer could also be used to enhance the adherence and allow axonal outgrowth from zebrafish retinal explants. The effects of this coating agent on the transfection of loosely attaching cell lines were studied. Pre-coating with polyethyleneimine had the effect of enhancing the transfection yield in procedures using lipofection reagents.

Conclusion

Polyethyleneimine is an effective attachment factor for weakly anchoring cell lines and primary cells. Its use in lipofection protocols makes the procedures more reliable and increases the yield of expressed products with commonly used cell lines such as PC-12 and HEK-293 cells.
Gastric T-cell lymphoma associated with hemophagocytic syndrome
Authors: Fukui Rika , Hata Fumitake , Yasoshima Takahiro , Denno Ryuuichi , Okazaki Minoru , Kasai Kiyoshi , Sato Masaaki , Homma Toshio , Ohno Keisuke , Yanai Yoshiyuki , Sogahata Katsuya , Nishimori Hidefumi , Hirata Koichi
Published: October 2004

Background

Lymphoma-associated hemophagocytic syndrome (LAHS) occurs in mostly extra nodal non-Hodgkin's lymphoma. LAHS arising from gastrointestinal lymphoma has never been reported. Here we report a case of gastric T-cell lymphoma-associated hemophagocytic syndrome.

Case presentation

A 51-year-old woman presented with pain, redness of breasts, fever and hematemesis. Hematological examination revealed anemia. Gastroscopy revealed small bleeding ulcers in the stomach and the computed tomography scan showed liver tumor. She underwent total gastrectomy for gastrointestinal bleeding and the histopathology revealed gastric T-cell lymphoma. She continued to bleed from the anastomosis and died on the 8th postoperative day. Autopsy revealed it to be a LAHS.

Conclusions

If Hemophagocytic syndrome (HPS) occurs in lymphoma of the gastrointestinal tract, bleeding from the primary lesion might be uncontrollable. Early diagnosis and appropriate treatment are needed for long-term survival.
Epidemiology in conflict – A call to arms
Authors: Tam Clarence C , Lopman Ben A , Bornemisza Olga , Sondorp Egbert
Published: October 2004

In this first special theme issue, Emerging Themes in Epidemiology publishes a collection of articles on the theme of Epidemiology in conflict. Violent conflict is an issue of great sensitivity within public health, but more structured research and reasoned discussion will allow us to better mitigate the public health impacts of war, and place the public health community in a more informed position in discussions about possible interventions in future conflicts.
Extravascular lung water assessed by transpulmonary single thermodilution and postmortem gravimetry in sheep
Authors: Kirov Mikhail Y , Kuzkov Vsevolod V , Kuklin Vladimir N , Waerhaug Kristine , Bjertnaes Lars J
Published: October 2004

Introduction

Acute lung injury is associated with accumulation of extravascular lung water (EVLW). The aim of the present study was to compare two methods for quantification of EVLW: transpulmonary single thermodilution (EVLWST) and postmortem gravimetric (EVLWG).

Methods

Eighteen instrumented and awake sheep were randomly assigned to one of three groups. All groups received Ringer's lactate (5 ml/kg per hour intravenously). To induce lung injury of different severities, sheep received Escherichia coli lipopolysaccharide 15 ng/kg per min intravenously for 6 hours (n = 7) or oleic acid 0.06 ml/kg intravenously over 30 min (n = 7). A third group (n = 4) was subjected to sham operation. Haemodynamic variables, including EVLWST, were measured using a PiCCOplus monitor (Pulsion Medical Systems, Munich, Germany), and the last measurement of EVLWST was compared with EVLWG.

Results

At the end of experiment, values for EVLWST (mean ± standard error) were 8.9 ± 0.6, 11.8 ± 1.0 and 18.2 ± 0.9 ml/kg in the sham-operated, lipopolysaccharide and oleic acid groups, respectively (P < 0.05). The corresponding values for EVLWIG were 6.2 ± 0.3, 7.1 ± 0.6 and 11.8 ± 0.7 ml/kg (P < 0.05). Ranges of EVLWIST and EVLWIG values were 7.5–21.0 and 4.9–14.5 ml/kg. Regression analysis between in vivo EVLWST and postmortem EVLWG yielded the following relation: EVLWST = 1.30 × EVLWG + 2.32 (n = 18, r = 0.85, P < 0.0001). The mean bias ± 2 standard deviations between EVLWST and EVLWG was 4.9 ± 5.1 ml/kg (P < 0.001).

Conclusion

In sheep, EVLW determined using transpulmonary single thermodilution correlates closely with gravimetric measurements over a wide range of changes. However, transpulmonary single thermodilution overestimates EVLW as compared with postmortem gravimetry.
Systemic lupus erythematosus in Pakistan
Authors: Rabbani M A , Siddiqui B K , Tahir M H , Ahmad B , Shamim A , Shah S MA , Ahmad A
Published: October 2004

Clinical features of systemic lupus erythematosus (SLE) have been described from different geographical regions in the world, with some clinical differences among different racial groups. Although data on the characteristics of SLE in Pakistan is scarce, it is not uncommon in the South East Asian region. The purpose of this study was, therefore, to delineate the clinical pattern and disease course in Pakistani patients with SLE and to compare it with international data on lupus patients. A total of 196 patients with SLE fulfilling the clinical and laboratory criteria of the American Rheumatism Association admitted to the hospital between 1986 and 2001 were studied by means of a retrospective review of their records. Demographically, it was seen that SLE is a disease predominantly of females in their third decade, which is consistent with worldwide data. The mean age of presentation was 31 years (range 14-76) and the mean duration of follow up was 34 (4-179) months. Generally, there was less cutaneous (46%), arthritic (38%), serositis (22%) and renal involvement (33%) but more neuropsychiatric symptoms (26%) in our population. Eighty-six percent of patients were ANA positive, whereas anti dsDNA was positive in 74% of patients. Infections, renal involvement, seizures and thrombocytopenia were associated with poor prognosis (P, 0.05). This study is the first of its kind in Pakistan. The clinical and laboratory characteristics of SLE patients in our study place our population in the middle of a spectrum between the Caucasians and other Asian populations. It has shown that the clinical characteristics of SLE patients in this country may be different to those of its neighbors.
Respiratory arrest in systemic lupus erythematosus due to phrenic nerve neuropathy
Authors: Omdal R , Roos P , Wildhagen K , Gunnarsson R
Published: October 2004

Diaphragmatic weakness in patients with systemic lupus erythematosus (SLE) is a controversial issue and is claimed to have a neuropathic, myopathic or unknown pathogenesis. In this patient a predominantly motor neuropathy with diaphragmatic paralysis due to axonal involvement of the phrenic nerve was discovered and successfully treated with immunosuppressive drugs.
The Logic of Feeling
Authors: Tuan Pham Michel
Published: October 2004

The contribution of the feelings‐as‐information hypothesis to our understanding of the role of affect in judgment and decision making is discussed. Basic principles and regularities in how affective feelings guide judgments and decisions are then identified. Based on these principles and regularities, it is argued that the role of feelings in judgment and decision making may be more adaptive than has been assumed in most academic circles. This adaptivity transpires (a) in the variety of goal‐relevant signals that feelings convey, (b) the flexibility with which feelings are interpreted, (c) the judgmental properties of feelings, and (d) the selectivity with which feelings are invoked. It is speculated that affective feelings may tap into a separate system of judgment and decision making with its unique strengths and weaknesses.
Hesgenes regulate size, shape and histogenesis of the nervous system by control of the timing of neural stem cell differentiation
Authors: Hatakeyama Jun , Bessho Yasumasa , Katoh Kazuo , Ookawara Shigeo , Fujioka Makio , Guillemot François , Kageyama Ryoichiro
Published: October 2004

Radial glial cells derive from neuroepithelial cells, and both cell types are identified as neural stem cells. Neural stem cells are known to change their competency over time during development: they initially undergo self-renewal only and then give rise to neurons first and glial cells later. Maintenance of neural stem cells until late stages is thus believed to be essential for generation of cells in correct numbers and diverse types, but little is known about how the timing of cell differentiation is regulated and how its deregulation influences brain organogenesis. Here, we report that inactivation of Hes1 and Hes5, known Notch effectors, and additional inactivation of Hes3 extensively accelerate cell differentiation and cause a wide range of defects in brain formation. In Hes-deficient embryos, initially formed neuroepithelial cells are not properly maintained, and radial glial cells are prematurely differentiated into neurons and depleted without generation of late-born cells. Furthermore,loss of radial glia disrupts the inner and outer barriers of the neural tube,disorganizing the histogenesis. In addition, the forebrain lacks the optic vesicles and the ganglionic eminences. Thus, Hes genes are essential for generation of brain structures of appropriate size, shape and cell arrangement by controlling the timing of cell differentiation. Our data also indicate that embryonic neural stem cells change their characters over time in the following order: Hes-independent neuroepithelial cells,transitory Hes-dependent neuroepithelial cells and Hes-dependent radial glial cells.
Inhibition of PrPSc formation by lentiviral gene transfer of PrP containing dominant negative mutations
Authors: Crozet Carole , Lin Yea-Lih , Mettling Clément , Mourton-Gilles Chantal , Corbeau Pierre , Lehmann Sylvain , Perrier Véronique
Published: October 2004

Currently, there is no treatment to cure transmissible spongiform encephalopathies. By taking advantage of the `prion-resistant' polymorphisms Q171R and E219K that naturally exist in sheep and humans, respectively, we have evaluated a therapeutic approach of lentiviral gene transfer. Here, we show that VSV-G (vesicular stomatitis virus G glycoprotein) pseudotyped FIV-(feline immunodeficiency virus) derived vectors carrying the mouse Prnp gene in which these mutations have been inserted, are able to inhibit prion replication in chronically prion-infected cells. Because lentiviral tools are able to transduce post-mitotic cells such as neurons or cells of the lymphoreticular system, this result might help the development of gene- or cell-therapy approaches to prion disease.
Metabolic influences of fiber size in aerobic and anaerobic locomotor muscles of the blue crab,Callinectes sapidus
Authors: Johnson L. K. , Dillaman R. M. , Gay D. M. , Blum J. E. , Kinsey S. T.
Published: October 2004

Diameters of some white locomotor muscle fibers in the adult blue crab, Callinectes sapidus, exceed 500 μm whereas juvenile white fibers are <100 μm. It was hypothesized that aerobically dependent processes,such as metabolic recovery following burst contractions, will be significantly impeded in the large white fibers. In addition, dark aerobic fibers of adults,which rely on aerobic metabolism for both contraction and recovery, grow as large as the white fibers. These large aerobic fibers are subdivided, however,thus decreasing the effective diameter of each metabolic functional unit and enabling aerobic contraction. The two goals of this study were: (1) to characterize the development of subdivisions in the dark levator muscle fibers and (2) to monitor post-contractile metabolism as a function of fiber size in aerobic and anaerobic levator muscles. Dark levator muscle fibers from crabs ranging from <0.1 g to >190 g were examined with transmission electron microscopy to determine the density of mitochondria and subdivision diameters. Across all size classes, there was a constant mitochondrial fractional area(25% of the total subdivision area) and subdivision size (mean diameter of 36.5±2.7 μm). Thus, blue crab dark levator fibers are unusual in having metabolic functional units (subdivisions) that do not increase in size during development while the contractile functional units (fibers) grow hypertrophically. The body mass scaling of post-contractile lactate dynamics was monitored during recovery from anaerobic, burst exercise in white and dark muscle, and in hemolymph. There were no differences among size classes in lactate accumulation during exercise in either muscle. However, in white fibers from large crabs, lactate continued to increase after exercise, and lactate removal from tissues required a much longer period of time relative to smaller crabs. Differences in lactate removal among size classes were less pronounced in dark fibers, and post-contractile lactate accumulation was significantly higher in white than in dark fibers from large animals. These data suggest that the large white fibers invoke anaerobic metabolism following contraction to accelerate certain phases of metabolic recovery that otherwise would be overly slow. This implies that, in addition to the typical mass-specific decrease in oxidative capacity that accompanies increases in animal mass, aerobic metabolic processes become increasingly limited by surface area to volume and intracellular diffusion constraints in developing white muscle fibers.
A base triple in the Tetrahymena group I core affects the reaction equilibrium via a threshold effect
Authors: KARBSTEIN KATRIN , TANG KUO-HSIANG , HERSCHLAG DANIEL
Published: October 2004

Previous work on group I introns has suggested that a central base triple might be more important for the first rather than the second step of self-splicing, leading to a model in which the base triple undergoes a conformational change during self-splicing. Here, we use the well-characterized L-21 ScaI ribozyme derived from the Tetrahymena group I intron to probe the effects of base-triple disruption on individual reaction steps. Consistent with previous results, reaction of a ternary complex mimicking the first chemical step in self-splicing is slowed by mutations in this base triple, whereas reaction of a ternary complex mimicking the second step of self-splicing is not. Paradoxically, mechanistic dissection of the base-triple disruption mutants indicates that active site binding is weakened uniformly for the 5′-splice site and the 5′-exon analog, mimics for the species bound in the first and second step of self-splicing. Nevertheless, the 5′-exon analog remains bound at the active site, whereas the 5′-splice site analog does not. This differential effect arises despite the uniform destabilization, because the wild-type ribozyme binds the 5′-exon analog more strongly in the active site than in the 5′-splice site analog. Thus, binding into the active site constitutes an additional barrier to reaction of the 5′-splice site analog, but not the 5′-exon analog, resulting in a reduced reaction rate constant for the first step analog, but not the second step analog. This threshold model explains the self-splicing observations without the need to invoke a conformational change involving the base triple, and underscores the importance of quantitative dissection for the interpretation of effects from mutations.
Optimizing an Empirical Scoring Function for Transmembrane Protein Structure Determination
Authors: Gray Genetha Anne , Kolda Tamara G. , Sale Ken , Young Malin M.
Published: October 2004

We examine the problem of transmembrane protein structure determination. Like many questions that arise in biological research, this problem cannot be addressed generally by traditional laboratory experimentation alone. Instead, an approach that integrates experiment and computation is required. We formulate the transmembrane protein structure determination problem as a bound-constrained optimization problem using a special empirical scoring function, called Bundler, as the objective function. In this paper, we describe the optimization problem and its mathematical properties, and we examine results obtained using two different derivative-free optimization algorithms.
Hope or Hype: On the Viability of Escrow Services as Trusted Third Parties in Online Auction Environments
Authors: Hu Xiaorui , Lin Zhangxi , Whinston Andrew B. , Zhang Han
Published: October 2004

Internet fraud has been on the rise in online consumer-to-consumer (C2C) auction markets, posing serious challenges to people's trust in electronic markets. Among various remedies to promote trust and reduce trader's risk, online escrow service has been proposed as a trusted third party to protect online transactions from Internet fraud. However, whether an escrow service constitutes a viable business model for a trusted third party to effectively block Internet fraud remains an open question. This research proposes a dynamic game model for online traders and a profit maximization model for the escrow service provider. Through the investigation of the optimal strategies of online traders, we explore the relationships among traders' decision making, escrow service fee rates, and adoption rates. We reveal the demand for escrow services and establish the optimal pricing rule for the escrow service provider. A numerical study based on the theoretical analysis is conducted to provide detailed guidelines of the model application for an escrow service provider and to explore if the escrow service is a viable business model in C2C auction markets.
The Plasminogen Activating System in Periodontal Health and Disease
Authors: Kinnby Bertil
Published: October 2004

The plasminogen activating system is important for extracellular proteolysis and plays a regulatory role in interactions with other tissue degrading systems. Studies on the plasminogen activating system in gingival crevicular fluid (GCF) as well as gingival tissue are reviewed. tPA, uPA, PAI-1 and PAI-2 have all been detected in GCF. Especially tPA and PAI-2 are found in high concentrations. In tissue studies fibrinolytic activity has been found in the gingival pocket epithelium in humans and in animal studies. tPA and PAI-2 have been detected there immunohistochemically. Local production of the PAs and PAIs has been verified with in situ hybridization. In inflammation, a more intense and widespread immunohistochemical staining of tPA and PAI-2 is seen. Higher concentrations of tPA and PAI-2 are found in GCF but the balance between them seems to be constant. A systemically disturbed balance of the plasminogen activating system in GCF has been observed during pregnancy, with a possible protective function of PAI-2. In studies of periodontitis, the production of PAI-2 seemed to be locally lowered at impaired sites. In a study of children, a higher inflammatory response to bacterial plaque was accompanied by a higher fibrinolytic ativity in GCF samples. Bacterial LPS has been found to change the ratio of tPA to PAI-2 in cultured gingival fibroblasts. Interactions between PAI-2 and a protease in the gingival epithelium has been verified through the immunohistochemical detection of relaxed PAI-2.
Inhibition of Receptor-Dependent Urokinase Signaling by Specific Ser to Glu Substitutions
Authors: Carriero Maria Vincenza , Franco Paola , Gargiulo Lucia , Vocca Immacolata , Cito Letizia , Fontana Laura , Iaccarino Ciro , Pozzo Giovanna Del , Guardiola John , Stoppelli Maria Patrizia
Published: October 2004

We have previously reported that phosphorylation of human urokinase on Ser138/303 abolishes its catalyticindependent motogen and proadhesive abilities, whereas receptor binding is not affected. Here we show that substitution of the two relevant serines with glutamic acid residues impairs the ability of urokinase to mobilize a variety of human and mouse cell lines as well as human primary T lymphocytes. Accordingly, urokinase receptordependent signaling, leading to cytoskeletal rearrangements and paxillin redistribution, does not occur in MCF-7 breast carcinoma cells exposed to phosphorylationlike urokinase. Unlike the wildtype form, disubstituted urokinase is unable to induce the physical association of urokinase receptor with αvβ5 vitronectin receptor, which is required for MCF-7 urokinasedependent cell migration. Finally, the disubstituted variant fails to activate p55fgr, a member of the Src tyrosine kinase family, which mediates cell migration and adhesion of U937 myelomonocytic cells. In conclusion, the finding that specific amino acid substitutions strongly interfere with the ability of urokinase to stimulate cell migration, and the associated intracellular events uncover a novel way to regulate urokinase receptordependent signaling.
Cultivation in Glucose-Deprived Medium Stimulates Mitochondrial Biogenesis and Oxidative Metabolism in HepG2 Hepatoma Cells
Authors: Weber K. , Ridderskamp D. , Alfert M. , Hoyer S. , Wiesner R.J.
Published: October 2004

In order to test the hypothesis that an imbalance between energy requirement and energy supply regulates mitochondrial genes and ultimately mitochondrial biogenesis, energy supply was challenged in HepG2 cells by withdrawal of glucose from the culture medium, making the cells exclusively dependent on mitochondrial ATP production. Such cells showed a 2-fold increase of cytochrome c oxidase activity, elevated levels of mitochondrial DNA, mitochondrial DNA encoded mRNAs and proteins, as well as the nuclear encoded mitochondrial transcription factor A. Lactate production was significantly reduced and glutamine was consumed as an alternative substrate for oxidative metabolism. Longterm adapted cells formed exclusively monolayers, while they normally grow in multilayers forming tumor spheroids. Also, longterm adapted cells proliferated significantly faster. No differences for the ATP/ADP ratio were observed, indicating that this is not the primary signal initiating the adaptative processes. These results show that mitochondrial biogenesis and oxidative metabolism are stimulated in HepG2 cells grown in the absence of fermentable glucose, probably in order to compensate for the diminished supply of glycolytic ATP.
Green Tea Extract Protects against Early Alcohol-Induced Liver Injury in Rats
Authors: Arteel G.E. , Uesugi T. , Bevan L.N. , Gäbele E. , Wheeler M.D. , McKim S.E. , Thurman R.G.
Published: October 2004

Oxidants have been shown to be involved in alcoholinduced
liver injury. This study was designed to test
the hypothesis that the antioxidant polyphenolic extract
of green tea, comprised predominantly of epigallocatechin
gallate, protects against early alcoholinduced
liver injury in rats. Male Wistar rats were fed
highfat liquid diets with or without ethanol (10 14 g
kg 1 day 1) and green tea (300 mg kg 1 day 1) continuously
for 4 weeks using an intragastric enteral feeding
protocol. Mean body weight gains (~4 g/day) were
not significantly different between treatment groups,
and green tea extract did not the affect average concentration
or the cycling of urine ethanol concentrations
(0 550 mg dl 1 day 1). After 4 weeks, serum ALT
levels were increased significantly about 4-fold over
control values (35±3 IU/l) by enteral ethanol (114±18);
inclusion of green tea extract in the diet significantly
blunted this increase (65±10). Enteral ethanol also
caused severe fatty accumulation, mild inflammation,
and necrosis in the liver. While not affecting fat accumulation
or inflammation, green tea extract significantly
blunted increases in necrosis caused by
ethanol. Furthermore, ethanol significantly increased
the accumulation of protein adducts of 4-hydroxynonenal,
a product of lipid peroxidation and an index
of oxidative stress; green tea extract blocked this effect
almost completely. TNFa protein levels were increased
in liver by alcohol; this phenomenon was also
blunted by green tea extract. These results indicate
that simple dietary antioxidants, such as those found
in green tea, prevent early alcoholinduced liver injury,
most likely by preventing oxidative stress.
Detection of Poly(ADP-Ribose) by Immunocytochemistry: A Sensitive New Method for the Early Identification of UVB- and H2O2-Induced Apoptosis in Keratinocytes
Authors: Chang H. , Sander C.S. , Müller C.S.L. , Elsner P. , Thiele J.J.
Published: October 2004

Apoptosis is an active form of cell death that is initiated
by a variety of stimuli, including reactive oxygen
species (ROS) and ultraviolet (UV) radiation. Poly
(ADPribose) (PAR) is formed upon activation of the
DNA repair enzyme poly(ADPribose) polymerase
(PARP), and therefore was suggested as a new marker
of apoptosis. Since DNA of epidermal cells represents
a wellknown chromophore for UVB irradiation,
and UVB is known to generate H2O2 in keratinocytes,
we hypothesized that PAR is a very sensitive marker
of UVB and H2O2-induced apoptosis in keratinocytes.
In order to test this hypothesis, human immortalized
keratinocytes (HaCaT) were UVBirradiated or
treated with H2O2, and subsequently apoptosis was
identified by comparing conventional parameters
such as morphological analysis, DNA laddering, and
TUNEL assay, with PAR formation. Both, UVB and
H2O2 treatment induced PAR formation in HaCaT cells
in a dosedependent manner, and its formation was
detected as early as 4 h after irradiation, and at lower
UVB doses (10 mJ/cm2) than observed by DNA laddering
and the TUNEL assay. In conclusion, the detection
of PAR formation is a very sensitive and early
method for the identification of apoptotic cells in
UVBinduced apoptosis of human keratinocytes.
Biochemical Features, Molecular Biology and Clinical Relevance of the Human 15-Domain Serine Proteinase Inhibitor LEKTI
Authors: Walden M. , Kreutzmann P. , Drögemüller K. , John H. , Forssmann W.-G. , Mägert H.-J.
Published: October 2004

Based on the isolation of a 55 amino acid peptide from human hemofiltrate, we cloned the cDNA for a novel human 15-domain serine proteinase inhibitor termed LEKTI. A trypsininhibiting activity was demonstrated for three different domains. High levels of expression of the corresponding gene were detected in oral mucosa, followed by the tonsils, parathyroid glands, thymus, and trachea. Hovnanian and coworkers recently found that certain mutations within the LEKTI gene are linked to the severe congenital disease Netherton syndrome and atopic manifestations (including asthma). Thus, a future therapeutic use of LEKTI is conceivable.
Zinc Ligands in an Astacin Family Metalloprotease Meprin A
Authors: Doll B. A. , Villa J. P. , Ishmael F. T. , Bond J. S.
Published: October 2004

A conserved tyrosine residue in the astacin family of metalloproteases is one of five ligands proposed to coordinate zinc at the active site. Sitedirected mutagenesis of the conserved Tyr (Y226) of recombinant mouse meprin α was used to test the hypothesis that this residue is essential for zinc binding and enzymatic activity. In addition, another proposed zinc binding ligand, H167, in the conserved (HEXXH) zinc binding motif of the meprin α protease domain was replaced by an alanine residue. Both mutants were expressed and secreted with the same subunit mass as wild type (90 kDa). The Y226F mutant retained the capacity to oligomerize to higher covalently and noncovalentlylinked oligomers as the wild type, whereas H167A was predominantly a monomer. The kcat/Km for Y226F against a fluorgenic bradykinin substrate analog was approximately 15% of the wild type, while the H167A mutant had no detectable activity. Both Y226F and H167A were more susceptible to extensive degradation by trypsin compared with the wildtype protein. The zinc content in the wildtype and Y226F mutant proteins were similar, one molecule of zinc per subunit. The results indicate that Y226 is not essential for zinc binding, but Y226 and H167 are essential for full enzymatic activity and stability of the metalloproteinase.
Aberrant Expression of Serpin Squamous Cell Carcinoma Antigen 2 in Human Tumor Tissues and Cell Lines: Evidence of Protection from Tumor Necrosis Factor-Mediated Apoptosis
Authors: Takeda A. , Kajiya A. , Iwasawa A. , Nakamura Y. , Hibino T.
Published: October 2004

Squamous cell carcinoma antigens (SCCA) 1 and 2 are highly homologous proteins of the serpin family, although they inhibit different types of proteinases. We investigated the expression of both SCCA mRNAs in tumor tissues, in various cell lines (A431, SAS, Ca9, HeLa, SKGIIIa, HSC-2, HSC-3, HSC-4 and KB) and in HSC cell lines in the presence of tumor necrosis factor α (TNFα). The expression of SCCA2 mRNA could be differentiated from that of SCCA1 in tumor tissues and cell lines by means of reverse transcriptionpolymerase chain reaction (RTPCR). The ratio between SCCA1 and SCCA2 mRNA expression showed selective expression of SCCA2 mRNA in SCC tissues from the uterine cervix compared to SCC tissues from the esophagus or skin. In addition, a significant level of SCCA2 mRNA expression was detected in the HSC-4 cell line, but not in Ca9, HeLa, SKGIIIa, or HSC-3 cells. In contrast, SCCA1 mRNA was detected in all samples tested. These results suggest that the level of expression of SCCA2 mRNA detected by RTPCR can be used to evaluate the status of SCC tumors. Next, we studied the effect of TNFα on SCCA1 and SCCA2 mRNA expression in HSC cell lines. SCCA1 mRNA expression was constantly increased in the three HSC cells examined with increasing time of exposure to TNFα. In contrast, SCCA2 mRNA expression was specific for HSC-4 cells. The survival rate of HSC-4 cells pretreated with TNFα (6.3 ng/ml) for 48 h was found to be 72%, compared with 42% and 9% for HSC-3 and HSC-2 cells, respectively, after apoptotic stimulation by TNFα (10 ng/ml) and cycloheximide (10 g/ml) for 18 h. Furthermore, selective expression of SCCA2 mRNA in HSC-4 pretreated with TNFα protected these cells from TNFαmediated apoptosis. Thus, SCCA2 overexpression in squamous tumor cells contributed to their survival by protecting them against TNFαinduced apoptosis.
Reconstitution of Phospholipid Flippase Activity from E. coli Inner Membrane: A Test of the Protein Translocon as a Candidate Flippase
Authors: Watkins W. E. , III III , Menon A.K.
Published: October 2004

Phospholipid flipping in biogenic membranes is a key feature of membrane bilayer assembly. Flipping is facilitated by proteinaceous transporters (flippases) that do not need metabolic energy to function. No flippase has yet been identified. The architecture of the E. coli protein translocon suggests that it could account for the flippase activity in the bacterial inner membrane. To test this possibility, we used E. coli cells depleted of SecYE or YidC to assay flipping in proteoliposomes reconstituted from detergent extracts of their inner membranes. We conclude that the protein translocon contributes minimally, if at all, to phospholipid flippase activity in the inner membrane.
Vascular Endothelial Growth Factor Receptor Family Genes: When Did the Three Genes Phylogenetically Segregate?
Authors: Shibuya M.
Published: October 2004

The vascular endothelial growth factor (VEGF) receptor family in mammals contains three members, VEGFR1(Flt-1), VEGFR2(KDR/Flk-1) and VEGFR3 (Flt-4), which directly regulate the formation of blood vessels and lymphatic vessels. These two circular systems are essential for the supply of O2 and nutrients to all tissues of the body as well as the drainage of excess fluids with waste metabolites from peripheral tissues. VEGF receptors have a characteristic structure with 7 Iglike domains in the extracellular domain and a cytoplasmic tyrosine kinase domain with a long kinase insert region. Recently, some novel findings on the phylogenetical conservation of VEGF receptor genes in animals were reported: the conservation of the VEGFR1/solubleVEGFR1 gene in birds, and the conservation of the VEGFRPDGFRlike receptor gene in nonvertebrates. Based on this new information as well as established observations, here the possibility is discussed that the three VEGFR genes phylogenetically segregated not at once when the vertebrates established, but in a stepwise manner: two genes first (the VEGFR1/R2 progenitor and the VEGFR3 gene), and subsequently the three genes VEGFR1, R2 and R3.
Pigment Epithelium-Derived Factor (PEDF), a Serpin with Potent Anti-Angiogenic and Neurite Outgrowth-promoting Properties
Authors: Gettins P. G. W. , Simonovic M. , Volz K.
Published: October 2004

Pigment epitheliumderived factor is a member of the serpin superfamily of proteins, but one that lacks inhibitory properties against either serine or cysteine proteinases. Nevertheless it possesses a number of physiological properties that make it a potentially important protein in regulation of angiogenesis, in neuronal cell survival and in protection of neurons from neurotoxic agents. It is also a protein that is highly up regulated in the G0 phase of earlypassage cells compared with rapidly proliferating cells or senescent cells, and so is also linked to both the cell cycle and cell senescence. The determination of a high resolution Xray crystal structure of native PEDF provides insight into regions of the protein that may be involved in one or more of these functions.
Characterization of Legumain
Authors: Schwarz G. , Brandenburg J. , Reich M. , Burster T. , Driessen C. , Kalbacher H.
Published: October 2004

The mammalian legumain, also called asparaginyl endopeptidase (AEP), is critically involved in the processing of bacterial antigens for MHC class II presentation. In order to investigate the substrate specificity of AEP in the P1 position, we created a peptide library and digested it with purified pig kidney AEP. Digestion was less efficient only when proline was in the P1 position. Maximum AEP activity was found in
lysosomal fractions of different types of antigen presenting cells (APC). When the multiple sclerosisassociated autoantigen myelin basic protein (MBP) was
digested with AEP, the immunodominant epitope
8399 was destroyed. Myoglobin as an alternative
substrate was AEP resistant. These results suggest
an important, but not necessarily critical role for AEP
in lysosomal antigen degradation.
Characterization of Different Reactive Lysines in Bovine Heart Mitochondrial Porin
Authors: jamal J.A. Al
Published: October 2004

Incubation of mitochondrial outer membrane porin with citraconic anhydride prior to treatment with fluorescein isothiocyanate (FITC) resulted in the labeling of a set of lysines located at a boundary between the water phase and lipid phase. The elution pattern of porin from the cation exchanger has been considered as indicative for the location of lysines. Electrical measurements after reconstitution of the modified protein in lipid bilayer membranes revealed that certain specific lysine residues are more susceptible to alterations. The innermost positive residues were only slightly influenced, while the outermost lysines exhibited a substantial change in channel properties. These results suggest the presence of critical charged residues in mitochondrial outer membrane porin that may be responsible for both the channels selectivity and its voltage dependence.
Remnant-Like Particle Cholesterol Levels in Korean Patients with Coronary Artery Disease and Non-Insulin Dependent Diabetes Mellitus
Authors: Song Junghan , Park Hyosoon , Hong Seung H. , Lee Hong K. , Park Young B. , Kim Jin Q
Published: October 2004

Several studies have provided evidence that the remnants of lipoproteins may be the atherogenic components of triglyceride-rich lipoproteins. The purpose of this study was to investigate whether the remnant-like particle cholesterol (RLP-C) is an independent risk factor for coronary artery disease (CAD) and non-insulin dependent diabetes mellitus (NIDDM) in the Korean population and to explore the relationship between RLP-C and other biochemical markers as well as the apolipoprotein (apo) E genotypes. Lipid and lipoproteins including RLP-C and apo E genotypes were analyzed in 98 normal adults (control group), 68 patients with CAD (CAD group), 88 patients with NIDDM (DM group), and 19 patients with both CAD and NDDM (CAD + DM group). RLP-C levels were significantly higher in the DM (p< 0.0001), CAD (p = 0.0012) and the CAD + DM groups (p = 0.0184) than in the controls. To determine which variable could discriminate most effectively and independently among the different groups, stepwise linear discriminant analysis was performed for all the variables that showed p< 0.15 by unvariate analysis. RLP-C was selected as an independent discriminator between the control and patient groups. RLP-C levels showed a strong positive correlation with trigylceride levels in the control, CAD and DM groups (r= 0.783, r = 0.610 and r= 0.746, respectively). In overall groups, apo ε4 and ε2 carrier genotypes showed a significant increase in RLP-C levels compared with ∊3/3 wild-type (p = 0.0085). After adjusting for the effect of apo E genotypes, a significant increase of the RLP-C levels in the disease groups remained. In conclusion, RLP-C was determined to be an independent risk factor in Korean patients with CAD and NIDDM and showed a strong correlation with triglyceride levels. We suggest that the increased cardiovascular risk associated with the ε4 and ε2 allele may be mediated by more atherogenic RLP-C.
Evaluation of a Newly Available Biochemical Analyzer: the Olympus AU 600
Authors: Blanc Marie-Céline , Neveux Nathalie , Laromiguière Muriel , Bérard Marie-Pierre , Cynober Luc
Published: October 2004

The performance of the Olympus AU 600, a newly available open multiparametric analyzer, available for routine biochemical analysis of biological samples, was evaluated. The analytical and technical performance of the apparatus and the quality of the Olympus reagents were both examined in a single site study. Electrolyte concentrations were determined with patented ion-selective electrodes; substrate concentrations and enzyme activities were determined by spectrophotometric measurement after coloured reaction or UV detection-based-reactions. The protocol of the evaluation and the acceptability criteria were those recommended by the French Society for Clinical Biology. For the parameters studied, the upper limits of linearity were equal to or higher than those claimed by the manufacturer. The CV values for within-run and between-run precision were lower than the target values with few exceptions. The comparison study gave satisfactory results for most of the parameters. Only expected interferences occurred. In summary, the results obtained for the 25 parameters studied and the characteristics of the apparatus were satisfactory. The analyzer is rapid (800 to 1200 tests per hour) and easy to use. In addition, the analyzer complies with good analytical practice and its flexibility enables users to plan work according to local laboratory constraints.
Stability of Brain Natriuretic Peptide (BNP) in Human Whole Blood and Plasma
Authors: Gobinet-Georges Agnès , Valli Nathalie , Filliatre Hélène , Dubernet Marie-France , Dedeystere Olivier , Bordenave Laurence
Published: October 2004

Brain natriuretic peptide is proposed as a biochemical marker which could provide a useful screening test to select patients for further cardiac investigations in heart failure. The applicability of such a biochemical test in clinics, hospital wards, and clinical laboratories is dependent on its ease of use and on the complexity of sample handling. The present study was undertaken to evaluate the stability of brain natriuretic peptide under a number of different handling conditions (sample collection, storage temperatures, freezing temperatures) assayed with a commercially available kit. The results clearly demonstrate that brain natriuretic peptide is stable at room temperature for 24 hours, or in up to 30°C for 12 hours in the presence and absence of aprotinin, on the condition that brain natriuretic peptide is assayed within one month (frozen at −20°C) after blood collection. The presence of aprotinin prevents brain natriuretic peptide degradation in samples preserved for more than 1 month at −20°C before assay.
Effect of Short- and Long-Term Storage on Human Serum and Recombinant Apolipoprotein E Concentration
Authors: Schiele Françoise , Vincent-Viry Monique , Herbeth Bernard , Visvikis Athanase , Siest Gérard
Published: October 2004

In order to assess the short- and long-term stability of apolipoprotein (apo) E concentration in serum, we compared the apo E concentrations measured in fresh human serum samples with those determined after storage at +4°C, −20°C or −80°C. The serum apo E concentration was measured by immunoturbidimetry using an anti-human apo E polyclonal antibody from goats. One week storage at +4°C did not significantly affect the serum apo E concentration. At −20°C or −80°C no significant change in apo E concentration occurred during up to three months of storage. Moreover, the concentration of apo E was not modified after long-term storage of serum samples kept at −196°C in liquid nitrogen for up to four years. In addition, 15 freeze-thaw cycles, over a 3-week period, did not affect the apo E concentration in serum. A similar freezethaw procedure applied to purified human recombinant apo E showed that apo E2 isoform was the most stable in comparison with the apo E3 and apo E4 isoforms.