bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of a putative progenitor subpopulation of alveolar epithelial type 2 cells
Authors: Reddy Raghava , Buckley Sue , Doerken Melissa , Barsky Lora , Weinberg Kenneth , Anderson Kathryn D. , Warburton David , Driscoll Barbara
Published: March 2004

Alveolar epithelial type 2 cells (AEC2) isolated from hyperoxia-treated animals exhibit increases in both proliferation and DNA damage in response to culture. AEC2 express the zonula adherens proteins E-cadherin, α-, β- and γ-catenin, desmoglein, and pp120, as demonstrated by Western blotting. Immunohistochemical analysis of cultured AEC2 showed expression of E-cadherin on cytoplasmic membranes varying from strongly to weakly staining. When cultured AEC2 placed in suspension were labeled with fluorescent-tagged antibodies to E-cadherin, cells could be sorted into at least two subpopulations, either dim or brightly staining for this marker. With the use of antibody to E-cadherin bound to magnetic beads, cells were physically separated into E-cadherin-positive and -negative subpopulations, which were then analyzed for differences in proliferation and DNA damage. The E-cadherin-positive subpopulation contained the majority of damaged cells, was quiescent, and expressed low levels of telomerase activity, whereas the E-cadherin-negative subpopulation was undamaged, proliferative, and expressed high levels of telomerase activity.
Conditional expression of transforming growth factor-α in adult mouse lung causes pulmonary fibrosis
Authors: Hardie William D. , Le Cras Timothy D. , Jiang Kenny , Tichelaar Jay W. , Azhar Mohamad , Korfhagen Thomas R.
Published: March 2004

To determine whether overexpression of transforming growth factor (TGF)-α in the adult lung causes remodeling independently of developmental influences, we generated conditional transgenic mice expressing TGF-α in the epithelium under control of the doxycycline (Dox)-regulatable Clara cell secretory protein promoter. Two transgenic lines were generated, and following 4 days of Dox-induction TGF-α levels in whole lung homogenate were increased 13- to 18-fold above nontransgenic levels. After TGF-α induction, transgenic mice developed progressive pulmonary fibrosis and body weight loss, with mice losing 15% of their weight after 6 wk of TGF-α induction. Fibrosis was detected within 4 days of TGF-α induction and developed initially in the perivascular, peribronchial, and pleural regions but later extended into the interstitium. Fibrotic regions were composed of increased collagen and cellular proliferation and were adjacent to airway and alveolar epithelial sites of TGF-α expression. Fibrosis progressed in the absence of inflammatory cell infiltrates as determined by histology, without changes in bronchiolar alveolar lavage total or differential cell counts and without changes in proinflammatory cytokines TNF-α or IL-6. Active TGF-β in whole lung homogenate was not altered 1 and 4 days after TGF-α induction, and immunostaining was not increased in the peribronchial/perivascular areas at all time points. Chronic epithelial expression of TGF-α in adult mice caused progressive pulmonary fibrosis associated with increased collagen and extracellular matrix deposition and increased cellular proliferation. Induction of pulmonary fibrosis by TGF-α was independent of inflammation or early activation of TGF-β.
Inhibition of LPS- and CpG DNA-induced TNF-α response by oxidized phospholipids
Authors: Ma Zheng , Li Jiang , Yang Lijuan , Mu Ying , Xie Wen , Pitt Bruce , Li Song
Published: March 2004

Lipid oxidation is commonly seen in the innate immune response, in which reactive oxygen intermediates are generated to kill pathogenic microorganisms. Although oxidation products of phospholipids have generally been regarded to play a role in a number of chronic inflammatory processes, several studies have shown that oxidized phospholipids inhibit the LPS-induced acute proinflammatory response in cultured macrophages and endothelial cells. We report in this study that oxidized 1-palmitoyl-2-arachidonoyl- sn-glycero-3-phosphorylcholine (PAPC), but not nonoxidized PAPC, significantly inhibits the LPS-induced TNF-α response in intact mice. Oxidized PAPC also inhibits the 2′-deoxyribo(cytidine-phosphate-guanosine) (CpG) DNA-induced TNF-α response in cultured macrophages and intact mice. To elucidate the mechanisms of action, we show that oxidized PAPC, but not nonoxidized PAPC, inhibits the LPS- and CpG-induced activation of p38 MAPK and the NF-κB cascade. These results suggest a role for oxidized lipids as a negative regulator in controlling the magnitude of the innate immune response. Further studies on the mechanisms of action may lead to development of a new type of anti-inflammatory drug for treatment of acute inflammatory diseases such as sepsis.
Roles of iNOS and nNOS in sepsis-induced pulmonary apoptosis
Authors: Rudkowski Jill C. , Barreiro Esther , Harfouche Rania , Goldberg Peter , Kishta Osama , D'Orleans-Juste Pedro , Labonte Julie , Lesur Olivier , Hussain Sabah N. A.
Published: March 2004

Apoptosis(programmed cell death) is induced in pulmonary cells and contributes to the pathogenesis of acute lung injury in septic humans. Previous studies have shown that nitric oxide (NO) is an important modulator of apoptosis; however, the functional role of NO derived from inducible NO synthase (iNOS) in sepsis-induced pulmonary apoptosis remains unknown. We measured pulmonary apoptosis in a rat model of Escherichia coli lipopolysaccharide (LPS)-induced sepsis in the absence and presence of the selective iNOS inhibitor 1400W. Four groups were studied 24 h after saline (control) or LPS injection in the absence and presence of 1400W pretreatment. Apoptosis was evaluated using DNA fragmentation, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and caspase activation. LPS administration significantly augmented pulmonary cell apoptosis and caspase-3 activity in airway and alveolar epithelial cells. Pretreatment with 1400W significantly enhanced LPS-induced pulmonary apoptosis and increased caspase-3 and -7 activation. The antiapoptotic effect of iNOS was confirmed in iNOS-/-mice, which developed a greater degree of pulmonary apoptosis both under control conditions and in response to LPS compared with wild-type mice. By comparison, genetic deletion of the neuronal NOS had no effect on LPS-induced pulmonary apoptosis. We conclude that NO derived from iNOS plays an important protective role against sepsis-induced pulmonary apoptosis.
Retinoic acid inhibits interleukin-4-induced eotaxin production in a human bronchial epithelial cell line
Authors: Takamura Kei , Nasuhara Yasuyuki , Kobayashi Motoko , Betsuyaku Tomoko , Tanino Yoko , Kinoshita Ichiro , Yamaguchi Etsuro , Matsukura Satoshi , Schleimer Robert P. , Nishimura Masaharu
Published: March 2004

Retinoic acid (RA) is known to accelerate wound healing and induce cell differentiation. All- trans RA (ATRA) exerts its effect by binding retinoic acid receptors, which are members of the nuclear receptor family. We investigated whether RA can alter expression of eotaxin, a potent eosinophil chemoattractant that is regulated by the transcription factors signal transducer and activator of transcription 6 (STAT6) and NF-κB. We examined the effects of RA on eotaxin expression in a human bronchial epithelial cell line BEAS-2B. ATRA and its stereodimer 9- cis retinoic acid (9- cis RA) inhibited IL-4-induced release of eotaxin at 10-6M by 78.0 and 52.0%, respectively ( P < 0.05). ATRA and 9- cis RA also significantly inhibited IL-4-induced eotaxin mRNA expression at 10-6M by 52.3 and 53.5%, respectively ( P < 0.05). In contrast, neither ATRA nor 9- cis RA had any effects on TNF-α-induced eotaxin production. In transfection studies using eotaxin promoter luciferase plasmids, the inhibitory effect of ATRA on IL-4-induced eotaxin production was confirmed at the transcriptional level. Interestingly, ATRA had no effects on IL-4-induced tyrosine phosphorylation, nuclear translocation, or DNA binding activity of STAT6. Activating protein-1 was not involved in ATRA-mediated transrepression of eotaxin with IL-4 stimulation. The mechanism of the inhibitory effect of ATRA on IL-4-induced eotaxin production in human bronchial epithelial cells has not been elucidated but does not appear to be due to an effect on STAT6 activation. These findings raise the possibility that RA may reduce eosinophilic airway inflammation, one of the prominent pathological features of allergic diseases such as bronchial asthma.
Positron emission tomography with [18F]fluorodeoxyglucose to evaluate neutrophil kinetics during acute lung injury
Authors: Chen Delphine L. , Schuster Daniel P.
Published: March 2004

We measured neutrophil glucose uptake with positron emission tomographic imaging and [18F]fluorodeoxyglucose ([18F]FDG-PET) in anesthetized dogs after intravenous oleic acid-induced acute lung injury (ALI; OA group, n = 6) or after low-dose intravenous endotoxin (known to activate neutrophils without causing lung injury) followed by OA (Etx + OA group, n = 7). The following two other groups were studied as controls: one that received no intervention ( n = 5) and a group treated with Etx only ( n = 6). PET imaging was performed ∼1.5 h after initiating experimental interventions. The rate of [3H]deoxyglucose ([3H]DG) uptake was also measured in vitro in cells recovered from bronchoalveolar lavage (BAL) performed after PET imaging. Circulating neutrophil counts fell significantly in animals treated with Etx but not in the other two groups. The rate of [18F]FDG uptake, measured by the influx constant Ki, was significantly elevated ( P < 0.05) in both Etx-treated groups (7.9 ± 2.6 × 10-3ml blood·ml lung-1·min-1in the Etx group, 9.3 ± 4.8 × 10-3ml blood·ml lung-1·min-1in the Etx + OA group) but not in the group treated only with OA (3.4 ± 0.8 × 10-3ml blood·ml lung-1·min-1) when compared with the normal control (1.6 ± 0.4 × 10-3ml blood·ml lung-1·min-1). [3H]DG uptake was increased (73 ± 7%) in BAL neutrophils recovered from the Etx + OA group ( P < 0.05) but not in the OA group. Kiand [3H]DG uptake rates were linearly correlated ( R2= 0.65). We conclude that the rate of [18F]FDG uptake in the lungs during ALI reflects the state of neutrophil activation. [18F]FDG-PET imaging can detect pulmonary sequestration of activated neutrophils, despite the absence of alveolar neutrophilia. Thus [18F]FDG-PET imaging may be a useful tool to study neutrophil kinetics during ALI.
Leptin prevents obesity induced by a high-fat diet after diet-induced weight loss in the marsupial S. crassicaudata
Authors: Wittert Gary A. , Turnbull Helen , Hope Perdita , Morley John E. , Horowitz Michael
Published: March 2004

The aims of this study were to determine in the marsupial Sminthopsis crassicaudata, the effects of leptin on food intake, body weight, tail width (a reflection of fat stores), and leptin mRNA, after caloric restriction followed by refeeding ad libitum with either a standard or high-fat preferred diet. S. crassicaudata ( n = 32), were fed standard laboratory diet (LabD; 1.01 kcal/g, 20% fat) ad libitum for 3 days. On days 4-10, animals received LabD at 75% of basal intake and then ( days 11-25) were fed either LabD or a choice of LabD and mealworms (MW; 2.99 kcal/g, 30% fat); during this time, half the animals ( n = 8) in each group received either leptin (2.5 mg/kg) or PBS intraperitoneally two times daily. On day 26, animals were killed and fat was removed for assay of leptin mRNA. At baseline, body weight, tail width, and food intake were similar in each group. After caloric restriction, body weight ( P < 0.001) and tail width ( P < 0.001) decreased. On return to ad libitum feeding in the PBS-treated animals, body weight and tail width returned to baseline in the LabD-fed animals ( P < 0.001) and increased above baseline in the MW-fed animals ( P < 0.001). In the LabD groups, tail width ( P < 0.001) and body weight ( P < 0.001) decreased after leptin compared with PBS. In the MW groups, the increase in tail width ( P < 0.001) and body weight ( P = 0.001) were attenuated after leptin compared with PBS. The expression of leptin mRNA in groups fed MW were greater in PBS than in leptin-treated animals ( P < 0.05). Therefore, after diet-induced weight loss, leptin prevents a gain in fat mass in S. crassicaudata; this has potential implications for the therapeutic use of leptin.
Changes in dietary sodium consumption modulate GLUT4 gene expression and early steps of insulin signaling
Authors: Okamoto Maristela Mitiko , Sumida Dóris Hissako , Carvalho Carla Roberta Oliveira , Vargas Alessandra Martins , Heimann Joel Cláudio , Schaan Beatriz D'Agord , Machado Ubiratan Fabres
Published: March 2004

Previous studies have shown that chronic salt overload increases insulin sensitivity, while chronic salt restriction decreases it. In the present study we investigated the influence of dietary sodium on 1) GLUT4 gene expression, by Northern and Western blotting analysis; 2) in vivo GLUT4 protein translocation, by measuring the GLUT4 protein in plasma membrane and microsome, before and after insulin injection; and 3) insulin signaling, by analyzing basal and insulin-stimulated tyrosine phosphorylation of insulin receptor (IR)-β, insulin receptor substrate (IRS)-1, and IRS-2. Wistar rats were fed normal-sodium (NS-0.5%), low-sodium (LS-0.06%), or high-sodium diets (HS-3.12%) for 9 wk and were killed under pentobarbital anesthesia. Compared with NS rats, HS rats increased ( P < 0.05) the GLUT4 protein in adipose tissue and skeletal muscle, whereas GLUT4 mRNA was increased only in adipose tissue. GLUT4 expression was unchanged in LS rats compared with NS rats. The GLUT4 translocation in HS rats was higher ( P < 0.05) both in basal and insulin-stimulated conditions. On the other hand, LS rats did not increase the GLUT4 translocation after insulin stimulus. Compared with NS rats, LS rats showed reduced ( P < 0.01) basal and insulin-stimulated tyrosine phosphorylation of IRS-1 in skeletal muscle and IRS-2 in liver, whereas HS rats showed enhanced basal tyrosine phosphorylation of IRS-1 in skeletal muscle ( P < 0.05) and of IRS-2 in liver. In summary, increased insulin sensitivity in HS rats is related to increased GLUT4 gene expression, enhanced insulin signaling, and GLUT4 translocation, whereas decreased insulin sensitivity of LS rats does not involve changes in GLUT4 gene expression but is related to impaired insulin signaling.
Feeding and temperature responses to intravenous leptin infusion are differential predictors of obesity in rats
Authors: Ruffin Marie-Pierre , Adage Tiziana , Kuipers Folkert , Strubbe Jan H. , Scheurink Anton J. W. , van Dijk Gertjan
Published: March 2004

Obesity is frequently associated with leptin resistance. The present study investigated whether leptin resistance in rats is present before obesity develops, and thus could underlie obesity induced by 16 wk exposure to a liquid, palatable, high-energy diet (HED). Before HED exposure, male Wistar rats (weighing between 330 and 360 g) received intravenous infusions of 20 μg leptin 2 h before dark (∼57 μg/kg rat). Relative to saline infusion, this caused a highly variable effect on food intake (ranging between -94 and +129%), with food intake suppression that appeared negatively correlated with HED-induced increases in body weight gain, caloric intake, adiposity, and plasma leptin levels. In contrast, leptin's thermogenic response was positively correlated to body weight gain linked to weights of viscera, but not to adiposity. Before HED exposure, leptin unexpectedly increased food intake in some rats (fi+, n = 8), whereas others displayed the normal reduction in food intake (fi-, n = 7). HED-exposed fi+ rats had higher plasma leptin levels, retroperitoneal fat pad weight, HED intake, and body weight gain than fi- and chow-fed rats. These parameters were also higher in HED-exposed fi- rats relative to chow rats, except for plasma leptin concentrations. It is concluded that leptin's reduced efficacy to suppress food intake could predict obesity on an HED. An unexpected orexigenic effect of leptin might potentially contribute to this as well.
Effects of insulin on renal interstitial hydrostatic pressure and natriuretic response to volume expansion in diabetic rats
Authors: Tang Daiyi , Yu Tianzheng , Khraibi Ali A.
Published: March 2004

Diabetes mellitus (DM) is characterized by alterations in fluid balance and blood volume homeostasis. Renal interstitial hydrostatic pressure (RIHP) has been shown to play a critical role in mediating sodium and water excretion under various conditions. The objective of this study was to determine the effects of immediate and delayed initiation of insulin treatment on the restoration of the relationship between RIHP, natriuretic, and diuretic responses to acute saline volume expansion (VE) in diabetic rats. Diabetes was induced by an intraperitoneal injection of streptozotocin (STZ; 65 mg/kg body wt). Four groups of female Sprague-Dawley rats were studied: normal control group (C), untreated diabetic group (D), immediate insulin-treated diabetic group (DI; treatment with insulin for 2 wk was initiated immediately when diabetes was confirmed, which was 2 days after STZ injection), and delayed insulin-treated diabetic group (DDI; treatment with insulin for 2 wk was initiated 2 wk after STZ injection). RIHP and sodium and water excretions were measured before and during VE (5% body wt/30 min) in the four groups of anesthetized rats. VE significantly increased RIHP, fractional excretion of sodium (FENa), and urine flow rate (V) in all groups of rats. Basal RIHP, RIHP response to VE (ΔRIHP), and FENa and V responses to VE (ΔFENa and ΔV) were significantly lower in the D group compared with the C group of rats. ΔRIHP was significantly higher in both DI and DDI groups compared with D group but was similar to that of the C group of rats. While in the DI group the ΔFENa response to VE was restored, ΔFENa was significantly increased in DDI compared with D group, but it remained lower than that of the C group. In conclusion, insulin treatment initiated immediately after the onset of diabetes restores basal RIHP and RIHP, natriuretic, and diuretic responses to VE; however, delayed insulin treatment restores the basal RIHP and RIHP response to VE but does not fully restore the natriuretic response to VE.
Acipimox enhances spontaneous growth hormone secretion in obese women
Authors: Kok Petra , Buijs Madelon M. , Kok Simon W. , van Ierssel Inge H. A. P. , Frölich Marijke , Roelfsema Ferdinand , Voshol Peter J. , Meinders A. Edo , Pijl Hanno
Published: March 2004

We hypothesized that a high circulating free fatty acid (FFA) concentration is involved in the pathogenesis of hyposomatotropism associated with obesity. To evaluate this hypothesis, 10 healthy premenopausal women (body mass index 33.8 ± 1.0 kg/m2) were studied in the follicular phase of their menstrual cycle at two occasions with a time interval of at least 8 wk, where body weight remained stable. Subjects were randomly assigned to treatment with either acipimox (an inhibitor of lipolysis, 250 mg orally 4 times daily) or placebo in a double-blind crossover design, starting 1 day before admission until the end of the blood sampling period. Blood samples were taken during 24 h with a sampling interval of 10 min for assessment of growth hormone (GH) concentrations, and GH secretion was estimated by deconvolution analysis. Identical methodology was used to study GH secretion in a historical control group of age-matched normal weight women. GH secretion was clearly blunted in obese women (total daily release 66 ± 10 vs. lean controls: 201 ± 23 mU·lVd-1·24 h-1, P = 0.005, where lVd is liter of distribution volume). Acipimox considerably enhanced total (113 ± 50 vs. 66 ± 10 mU·lVd-1·24 h-1, P = 0.02) and pulsatile GH secretion (109 ± 49 vs. 62 ± 30 mU·lVd-1·24 h-1, P = 0.02), but GH output remained lower compared with lean controls. Further analysis did not show any relationship between the effects of acipimox on GH secretion and regional body fat distribution. In conclusion, acipimox unleashes spontaneous GH secretion in obese women. It specifically enhances GH secretory burst mass. This might mean that lowering of systemic FFA concentrations by acipimox modulates neuroendocrine mechanisms that orchestrate the activity of the somatotropic ensemble.
Studies on the genus Paraphanolaimus Micoletzky, 1923 (Nematoda: Aphanolaimidae) with description of P. paraguayensis sp. n.
Authors: Holovachov Oleksandr , Sturhan Dieter
Published: March 2004

Two known and one new species of Paraphanolaimus are described, viz. P.
behningi from Germany, P. anisitsi from Paraguay and Germany and P.
paraguayensis sp. n. from Paraguay. Paraphanolaimus paraguayensis sp. n. is
characterised by 0.84-0.94 mm long body, presence of 37-44 lateral epidermal
glands, strongly sclerotised and broad cylindrical stoma, swollen tail tip;
female reproductive system with developed spermathecae, straight vagina;
male without neck setae, 16-19 tubular supplements, two precloacal and nine
to ten caudal setae, spicules 32-33 μm long, arcuate, with oval manubrium
and fusiform shaft, platelike gubernaculum. Paraphanolaimus indicus and
P.micoletzkyi are considered as species inquirendae. An emended diagnosis
and a revised classification of Paraphanolaimus are proposed and a key to
the species of the genus is provided. Aphanonchus bayensis (Keppner, 1988)
comb. n. is proposed for Paraphanolaimus bayensis Keppner, 1988.
Purification of two substances from bulbs of onion (Allium cepa L.) with nematicidal activity against Meloidogyne exigua Goeldi
Authors: Amaral Daniel Rufino , Rocha Oliveira Fernanda Emanuele da , Oliveira Denilson Ferreira , Campos Vicente Paulo
Published: March 2004

To contribute to the development of a new method for Meloidogyne exigua
Goeldi control, a nematode widely disseminated through Brazilian coffee
fields, this work aimed to purify the nematicidal substances produced by
onion (Allium cepa L.). Thus, the methanolic extract of onion bulbs was
concentrated under reduced pressure and submitted to a purification process
guided by in vitro assays with second-stage juveniles (J2) of M. exigua.
After several steps, two nematicidal substances that seemed to be
interconverting when in solution were isolated. When exposed to 500 ppm
aqueous solutions of substances 1 and 2, 55 and 38% of the J2, respectively,
were dead after 24 h. Very preliminary nuclear magnetic resonance
spectroscopy studies suggested that these substances correspond to a pair of
carbohydrates.
Pre-parasitic juveniles of Strelkovimermis spiculatus Poinar & Camino, 1986 (Nematoda: Mermithidae) predated upon by freshwater copepods (Crustacea: Copepoda)
Authors: Achinelly María , Micieli María , García Juan
Published: March 2004

Five copepod species, Acantocyclops robustus, Macrocyclops albidus,
Mesocyclops annulatus, Thermocyclops sp. (all Cyclopoidea) and
Argyrodiaptomus bergi (Calanoidea), were evaluated under laboratory
conditions as potential predators of preparasites of the mermithid nematode
Strelkovimermis spiculatus. Adults of all five copepod species consumed
70-100% of the preparasites within 24 h. Copepodids and adults of M.
annulatus predated upon 93 and 100% of the nematode pre-parasites,
respectively. An average of 197 pre-parasites was daily predated upon by an
adult M. annulatus. Copepod density affected prevalence and intensity of S.
spiculatus on mosquito larvae. Prevalence and intensity of S. spiculatus on
Aedes aegypti larvae was reduced from 98% and 2.8 nematodes per larva in
control containers free of copepods to 61.4% and 1.4 nematodes per larva in
containers with 50 M. annulatus females per l.
Distribution and pathogenicity of bacteria species carried by Bursaphelenchus xylophilus in China
Authors: Zhao Bo Guang , Wang Hu Li , Han Su Fen , Han Zheng Ming
Published: March 2004

Bacteria carried by the pine wood nematode (PWN), Bursaphelenchus xylophilus,
from both healthy and diseased Pinus thunbergii and P. massoniana were
studied in five main pine wilt disease epidemic provinces in P. R. China. No
bacteria were found in healthy pines but were found on PWN from all samples
collected from diseased trees. Twenty-four bacteria strains were isolated
from the nematodes and were identified by a combination of classical and
automatic testing bacteriology (ATB) expression methods. Bioassay showed
that 17 of the 24 identified strains produced phytotoxins. Eleven of these
17 phytotoxin producers belonged to the genus Pseudomonas. Glasshouse and
field inoculation tests using sterile techniques showed that both PWN and
the toxin-producing bacteria carried were necessary to induce disease. We
hypothesise that pine wilt disease is a complex, induced by both PWN and the
bacteria it carries.
A Novel Dysfunctional Growth Hormone Variant (Ile179Met) Exhibits a Decreased Ability to Activate the Extracellular Signal-Regulated Kinase Pathway
Authors: Lewis Mark D. , Horan Martin , Millar David S. , Newsway Vicky , Easter Tammy E. , Fryklund Linda , Gregory John W. , Norin Martin , Del Valle Cristóbal-Jorge , López-Siguero Juan Pedro , Cañete Ramón , López-Canti Luis Fernando , Díaz-Torrado Nieves , Espino Rafael , Ulied Angels , Scanlon Maurice F. , Procter Annie M. , Cooper David N.
Published: March 2004

The pituitary-expressed GH1 gene was screened for mutation in a group of 74 children with familial short stature. Two novel mutations were identified: an Ile179Met substitution and a −360A→G promoter variant. The Ile179Met variant was shown to exhibit a similar degree of resistance to proteolysis as wild-type GH, indicating that the introduction of Met does not cause significant misfolding. Secretion of Ile179Met GH from rat pituitary cells was also similar to that of wild type. Although receptor binding studies failed to show any difference in binding characteristics, molecular modeling studies suggested that the Ile179Met substitution might nevertheless perturb interactions between GH and the GH receptor loop containing the hotspot residue Trp169, thereby affecting signal transduction. The ability of the Ile179Met variant to activate a signal transducer and activator of transcription (STAT) 5-responsive luciferase reporter gene and induce phosphorylation of STAT 5 and ERK was therefore studied. In contrast to its ability to activate STAT 5 normally, activation of ERK by the Ile179Met variant was reduced to half that observed with wild type. Although differential effects on the activation of distinct signaling pathways by a mutant receptor agonist are unprecedented, these findings also suggest that the ERK pathway could play a role in mediating the action of GH.
Failure of Mathematical Indices to Accurately Assess Insulin Resistance in Lean, Overweight, or Obese Women with Polycystic Ovary Syndrome
Authors: Diamanti-Kandarakis Evanthia , Kouli Chryssa , Alexandraki Krystallenia , Spina Giovanna
Published: March 2004

Insulin resistance is a common metabolic feature of polycystic ovary syndrome (PCOS). In this study, we examined the validity of the mathematical indices [the quantitative insulin sensitivity check index (QUICKI) and the homeostasis model of assessment (HOMA)] that calculate insulin sensitivity and their correlation to glucose utilization with the insulin infusion rate in 40 mU/m2·min by the euglycemic clamp (M) in women with PCOS.

We studied 59 women with PCOS (20 lean, 16 overweight, and 23 obese subjects). Euglycemic clamp testing was performed, and QUICKI, HOMA, total testosterone, fasting insulin, fasting glucose, and glucose-to-insulin ratio were estimated.

No difference was found in testosterone and glucose levels among the three groups. Lean or overweight women compared with obese women differed in insulin levels, glucose-to-insulin ratio, QUICKI, and HOMA (P < 0.01). No statistical difference was found between lean and overweight women in the above parameters. M differed when lean women were compared with overweight (P < 0.002) or obese women (P < 0.0001); however, no statistical difference was observed between overweight and obese women. No significant correlation was found between M and QUICKI or HOMA.

We conclude that mathematical indices should be applied with caution in different insulin-resistant populations and should not be considered a priori equivalent to the euglycemic clamp technique.
Promoter-Specific Repression of Hepatocyte Nuclear Factor (HNF)-1β and HNF-1α Transcriptional Activity by an HNF-1β Missense Mutant Associated with Type 5 Maturity-Onset Diabetes of the Young with Hepatic and Biliary Manifestations
Authors: Kitanaka Sachiko , Miki Yuko , Hayashi Yasuhide , Igarashi Takashi
Published: March 2004

Mutations in the hepatocyte nuclear factor (HNF)-1β lead to type 5 maturity-onset diabetes of the young (MODY5). HNF-1β forms a homodimer or a heterodimer with HNF-1α and regulates various target genes. HNF-1β mutations are rare, and no functional analysis has been performed in conjunction with HNF-1α. HNF-1β is expressed in the liver and biliary system and controls liver-specific and bile acid-related genes. Moreover, liver-specific Hnf-1β knockout mice present with severe jaundice. However, no patients with HNF-1β mutations have biliary manifestations. In this report, we found a novel missense mutation in the HNF-1β gene in a patient with neonatal cholestasis and liver dysfunction together with the common features of MODY5. Functional analysis revealed that the mutant HNF-1β had diminished transcriptional activity by loss of the DNA binding activity. The mutant had a promoter-specific dominant-negative transcriptional effect on wild-type HNF-1β and inhibited its DNA binding. Moreover, the mutant had a promoter- and cell-specific transcriptional repressive effect on HNF-1α and a promoter-specific inhibitory effect on HNF-1α DNA binding. From these results, we considered that the different phenotype of patients with HNF-1β mutations might be caused by the different HNF-1β activity in conjunction with the different repression of HNF-1α activity in selected promoters and tissues.
Increased Diagnosis of Primary Aldosteronism, Including Surgically Correctable Forms, in Centers from Five Continents
Authors: Mulatero Paolo , Stowasser Michael , Loh Keh-Chuan , Fardella Carlos E. , Gordon Richard D. , Mosso Lorena , Gomez-Sanchez Celso E. , Veglio Franco , Young William F.
Published: March 2004

Primary aldosteronism (PA) is a common form of endocrine hypertension previously believed to account for less than 1% of hypertensive patients. Hypokalemia was considered a prerequisite for pursuing diagnostic tests for PA. Recent studies applying the plasma aldosterone/plasma renin activity ratio (ARR) as a screening test have reported a higher prevalence. This study is a retrospective evaluation of the diagnosis of PA from clinical centers in five continents before and after the widespread use of the ARR as a screening test. The application of this strategy to a greater number of hypertensives led to a 5- to 15-fold increase in the identification of patients affected by PA. Only a small proportion of patients (between 9 and 37%) were hypokalemic. The annual detection rate of aldosterone-producing adenoma (APA) increased in all centers (by 1.3–6.3 times) after the wide application of ARR. Aldosterone-producing adenomas constituted a much higher proportion of patients with PA in the four centers that employed adrenal venous sampling (28–50%) than in the center that did not (9%). In conclusion, the wide use of the ARR as a screening test in hypertensive patients led to a marked increase in the detection rate of PA.
Oncodevelopmental α-Fetoprotein Acts as a Selective Proangiogenic Factor on Endothelial Cell from the Fetomaternal Unit
Authors: Liang Olin D. , Korff Thomas , Eckhardt Jessica , Rifaat Jasmin , Baal Nelli , Herr Friederike , Preissner Klaus T. , Zygmunt Marek
Published: March 2004

The molecular coordination between angiogenesis and vascular remodeling is a critical step for the development of a functional vasculature in the placenta and the uterus during pregnancy. The oncodevelopmental albumin homolog α-fetoprotein (AFP) is mainly synthesized in the developing fetus, and its expression has been found to be associated with highly vascularized tumors in the adult. In this study, we investigated the angiogenic activity of AFP and its possible role in the fetomaternal unit. Immunohistochemical studies revealed that the AFP-binding protein(s) is expressed in blood vessels of chorionic villi from placentae of the second and the third but not of the first trimester during pregnancy. At low concentrations, AFP directly stimulates or enhances, respectively, vascular endothelial growth factor-induced proliferation and sprout formation of endothelial cells isolated from the placenta and the uterus possibly by a MAPK-dependent pathway. Furthermore, AFP enhances blood vessel formation in a chick chorioallantoic membrane assay in vivo. Interestingly, AFP has no proliferative or migratory effects on endothelial cells isolated from the umbilical vein in the absence of vascular endothelial growth factor. These data indicate that AFP may act as a specific proangiogenic factor of endothelial cells within the fetomaternal unit during advanced stages in pregnancy.
Prediction of scratch resistance of cobalt chromium alloy bearing surface, articulating against ultra-high molecular weight polyethylene, due to third-body wear particles
Authors: Mirghany M , Jin Z M
Published: March 2004

The entrapment of abrasive particles within the articulation between a cobalt chromium alloy (CoCrMo) femoral component and an ultra-high molecular weight polyethylene (UHMWPE) cup of artificial hip joints or tibial inserts of artificial knee joints usually scratches the metallic bearing surface and consequently increases the surface roughness. This has been recognized as one of the main causes of excessive polyethylene wear, leading to osteolysis and loosening of the prosthetic components. The purpose of this study was to use the finite element method to investigate the resistance of the cobalt chromium alloy bearing surface to plastic deformation, as a first approximation to causing scratches, due to various entrapped debris such as bone, CoCrMo and ZrO2 (contained in radiopaque polymethyl methacrylate cement). A simple axisymmetric micro contact mechanics model was developed, where a spherical third-body wear particle was indented between the two bearing surfaces, modelled as two solid cylinders of a given diameter, under the contact pressure determined from macro-models representing either hip or knee implants. The deformation of both the wear particle and the bearing surfaces was modelled and was treated as elastic-plastic. The indented peak-to-valley height on the CoCrMo bearing surface from the finite element model was found to be in good agreement with that reported in a previous study when the third-body wear particle was assumed to be rigid. Under the physiological contact pressure experienced in both hip and knee implants, ZrO2 wear particles were found to be fully embedded within the UHMWPE bearing surface, and the maximum von Mises stresses within the CoCrMo bearing surface reached the corresponding yield strength. Consequently, the CoCrMo bearing surface was deformed plastically and the corresponding peak-to-valley height (surface roughness) was found to increase with both the hardness and the size of the wear particle. Even in the case of CoCrMo wear particles, with similar mechanical properties to those of the CoCrMo bearing surface, a significant plastic deformation of the bearing surface was also noted; this highlighted the importance of considering the deformation of the wear particles. These findings support the hypotheses made by clinical studies on the contribution of entrapped debris to increased surface roughness of CoCrMo femoral bearing surfaces.
Haemodynamic CHIVA correction surgery versus compression for primary venous ulcers: first year results
Authors: Zamboni P , Cisno C , Marchetti F , Mazza P , Fogato L , Carandina S , De Palma M , Liboni A
Published: March 2004

Objective: To compare two different treatments for primary venous ulcers: a minimally invasive surgical technique for the haemodynamic correction of reflux, versus a traditional compression treatment.

Method: From a cohort of 87 lower extremities affected by the first episode of venous ulcers, 45 mobile patients affected by primary chronic venous insufficiency were randomized to receive either the haemodynamic correction procedure (CHIVA) or compression treatment.

Results: Mean follow up lasted one year. The rate of healing in the surgical group was 100% in a mean time of 29 days with a velocity of 2.86 mm2/day,and in the conservative group the rate was 96% in 61 days, with a velocity of 1.66 mm2/day ( P<0.02). All air plethysmographic parameters, with the exception of ejection fraction, significantly improved at six months in the surgical group. Finally, quality of life significantly improved in both groups, but in the surgical group the following domains were significantly different compared with the compression group: RP, role limitations due to physical problems; VT, energy/vitality; SF, social functioning; RE, role limitations due to emotional problems; and MH, mental health.

Conclusions: Surgical haemodynamic correction of reflux has been demonstrated to improve venous function, time to ulcer healing and quality of life when compared with compression treatment.
Wearing graduated compression stockings: the reality of everyday deep vein thrombosis prophylaxis
Authors: Barker S G E , Hollingsworth S J
Published: March 2004

Objective: Current hospital policy states that all patients should use graduated compression stockings (GCS) for prophylaxis against deep vein thrombosis (DVT).

Methods: A total of 218 patients on 16 mixed-speciality surgical wards were surveyed on a single 'snapshot' day, for compliance with this policy.

Results: Of these patients, 119 (54%) were not wearing GCS. Of the 99 (46%) patients who were wearing GCS, 85 wore below-knee products and 14 wore above-knee products. Incorrect use was far more common with above-knee GCS.

Conclusions: Non-compliance appreciably alters the risk of a patient suffering a DVT and leaves hospitals open potentially to litigation. Regular reminders of set hospital policy (and occasional reviews of policy) should help aid compliance. There is a 'natural selection' for below-knee GCS products.
Differential effects of growth factors and cytokines on the synthesis of SPARC, DNA, fibronectin and alkaline phosphatase activity in human periodontal ligament cells
Authors: Fujita Tsuyoshi , Shiba Hideki , Van Dyke Thomas E. , Kurihara Hidemi
Published: March 2004

Growth factors and cytokines play an important role in tissue development and repair. However, it remains unknown how they act on proliferation and differentiation of periodontal ligament cells. In this study, we investigated the effects of several growth factors and cytokines on the synthesis of DNA, alkaline phosphatase (ALPase), fibronectin, and secreted protein acidic and rich in cysteine (SPARC) in human periodontal ligament (HPL) cells. Transforming growth factor‐β (TGF‐β) increased the synthesis of DNA, fibronectin and SPARC, whereas it decreased ALPase activity. Basic fibroblast growth factor (bFGF), platelet‐derived growth factor (PDGF) and tumor necrosis factor‐α (TNF‐α) decreased SPARC and ALPase levels, whereas these peptides increased DNA synthesis and did not affect fibronectin synthesis. Epidermal growth factor (EGF) up‐regulated the synthesis of DNA and fibronectin and inhibited SPARC and ALPase levels. Interleukin‐1β (IL‐1β) decreased the synthesis of DNA, ALPase, fibronectin and SPARC. These findings demonstrate that TGF‐β, bFGF, EGF, PDGF, TNF‐α and IL‐1β have characteristically different patterns of action on DNA, SPARC, fibronectin and ALPase synthesis by HPL cells. The differences in regulation of function of periodontal ligament cells by these peptides may be involved in the regeneration and repair of periodontal tissue.
Morphometry of nuclear and nucleolar structures in a CaCo‐2 cell line
Authors: Karalyan Z.A. , Djaghatspanyan N.G. , Gasparyan M.H. , Hakobyan L.A. , Abroyan L.O. , Magakyan Y.H. , Ter‐Pogossyan Z.R. , Kamalyan L.A. , Karalova E.M.
Published: March 2004

The number of the nucleoli in a CaCo‐2 cell nucleus does not generally depend on the quantity of DNA in the nucleus, but nucleolar DNA content is directly proportional to total nuclear DNA. However, in multinucleolar cells (three or more nucleoli), the nucleolar DNA content increases after 96 h incubation in culture without concomitant quantitative changes in nuclear DNA. The percentage of multinucleolar cells and the average number of nucleoli per nucleus increase with increasing incubation time. After 72 and 96 h in culture, multinucleolar cells show distinctive morphologies. The ratio of the sum of nucleolar perimeters to the nuclear perimeter increases linearly when the number of nucleoli in a nucleus increases, but there is no concomitant increase in total nucleolar area or DNA content, except in the 72 and 96 h populations. When the number of nucleoli in CaCo‐2 cells increases after 48 and 60 h in culture, the amount of DNA per nucleolus decreases.
Effect of Rifampin on the Pharmacokinetics of Rosiglitazone in Healthy Subjects
Authors: Park Ji‐Young , Kim Kyoung‐Ah , Kang Mun‐Ho , Kim Su‐Lyun , Shin Jae‐Gook
Published: March 2004

Background and objective

Rifampin (INN, rifampicin) causes several drug interactions with coadministered antidiabetic drugs. Rosiglitazone is a novel thiazolidinedione antidiabetic drug, but little is known about the drug interaction between rifampin and rosiglitazone. Our objective was to investigate the effect of rifampin on the pharmacokinetics of rosiglitazone in humans.

Method

In an open‐label, randomized, 2‐way crossover study, 10 healthy Korean male subjects were treated once daily for 6 days with 600 mg rifampin or with placebo. On day 7, a single dose of 8 mg rosiglitazone was administered orally. Plasma rosiglitazone concentrations were measured.

Results

Rifampin significantly decreased the mean area under the plasma concentration–time curve for rosiglitazone by 65% (2947.9 ng · h/mL versus 991.5 ng · h/mL, P < .001) and the mean elimination half‐life from 3.9 to 1.5 hours (P < .001). The peak plasma concentration of rosiglitazone was significantly decreased by rifampin (537.7 ng/mL versus 362.3 ng/mL, P < .01). The apparent oral clearance of rosiglitazone increased about 3‐fold after rifampin treatment (2.8 L/h versus 8.5 L/h, P < .001).

Conclusion

This study showed that rifampin affected the disposition of rosiglitazone in humans, probably by the induction of cytochrome P450 (CYP) 2C8 and, to a lesser extent, CYP2C9. Therefore caution should be exercised during the coadministration of rifampin and rosiglitazone.

Clinical Pharmacology & Therapeutics (2004) 75, 157–162; doi: 10.1016/j.clpt.2003.10.003
A novel functional polymorphism in the uridine diphosphate–glucuronosyltransferase 2B7 promoter with significant impact on promoter activity
Authors: Duguay Yannick , Báár Cecilie , Skorpen Frank , Guillemette Chantal
Published: March 2004

To clarify the molecular determinants of the metabolic variability of morphine, we searched for genetic polymorphisms in the gene for uridine diphosphate–glucuronosyltransferase 2B7 (UGT2B7) and evaluated their functional impact in vitro and in patients with cancer receiving long‐term morphine therapy. Genetic analysis revealed the existence of 8 single‐nucleotide polymorphisms (SNPs), 6 of which are tightly linked and are at positions −1248, −1241, −1054, −842, −268, and −102 relative to the hepatic start site. In contrast, an SNP at position −66 occurs independently, whereas a novel variation at position −79 appears to be in linkage disequilibrium with the codon 268 SNP (UGT2B7*2). At least 4 haplotypes were observed in white subjects included in the initial SNP screening. On functional in vitro characterization, promoter‐reporter gene constructs with the −79 variation displayed 2.5‐ to 7‐fold less activity compared with the wild‐type construct in Caco‐2 colon cells and HepG2 hepatoma cells, respectively (P = .015 and P < .001, respectively). To investigate a possible effect of the −79 variation in vivo, serum morphine and morphine glucuronide concentrations were measured by liquid chromatography–mass spectrometry in patients with cancer who received long‐term oral morphine therapy, and subjects were then genotyped for the −79 polymorphism. Among 175 patients with normal hepatic and renal function, 6 were heterozygous for the −79 variation, and the morphine‐6‐glucuronide (M6G)/morphine and morphine‐3‐glucuronide (M3G)/morphine ratios versus those in the 169 noncarriers were 5.9 ± 3.5 versus 7.1 ± 7.0 for M6G/morphine (P = .96) and 31.2 ± 17.1 versus 42.9 ± 31.2 for M3G/morphine (P = .53), respectively. Further studies in larger samples are needed to make conclusions about the possible clinical relevance of the −79 polymorphism in the UGT2B7 gene.

Clinical Pharmacology & Therapeutics (2004) 75, 223–233; doi: 10.1016/j.clpt.2003.10.006
Cytochrome P450 3A4 and P‐glycoprotein Expression in Human Small Intestinal Enterocytes and Hepatocytes: A Comparative Analysis in Paired Tissue Specimens
Authors: von Richter Oliver , Burk Oliver , Fromm Martin F. , Thon Klaus P. , Eichelbaum Michel , Kivistö Kari T.
Published: March 2004

Objectives

Our objectives were to determine the content of cytochrome P450 (CYP) 3A4, CYP3A5, and P‐glycoprotein and to measure CYP3A4‐dependent catalytic activity in paired human small intestinal and liver specimens.

Methods

Samples of duodenum or proximal jejunum and liver wedge biopsy specimens were obtained from 15 patients undergoing a gastrointestinal operation. Enterocytes were isolated from the intestinal samples. The contents of CYP3A4, CYP3A5, and P‐glycoprotein and CYP3A4‐mediated catalytic activities were determined in homogenized enterocyte and liver samples.

Results

The CYP3A4 protein content was about 3 times (P < .01) and the P‐glycoprotein content about 7 times (P < .0001) higher in the enterocyte homogenates than in the liver homogenates. CYP3A5 protein was detected in all samples, but the levels were too low in most cases to allow quantification. The 2 cases with a quantifiable hepatic CYP3A5 content had the CYP3A5*1/*3 genotype; all other cases were homozygous for the CYP3A5*3 allele. No intraindividual correlations between the intestine and liver with respect to CYP3A4 content, P‐glycoprotein content, or the measured catalytic activities were present. Values for the maximum rate of metabolism (Vmax) of verapamil N‐dealkylation (formation of D‐617) and N‐demethylation (formation of norverapamil) activities correlated with the CYP3A4 protein content in both organs.

Conclusions

This work demonstrated a much higher content of both CYP3A4 protein and P‐glycoprotein in enterocytes isolated from human duodenal or jejunal mucosa than in paired specimens of liver tissue. These results lend support to the view that biotransformation in the gut wall substantially contributes to the overall first‐pass metabolism of many CYP3A4 substrates. Furthermore, the high content of P‐glycoprotein on the apical surface of enterocytes supports the theory that this efflux transporter may act in concert with CYP3A4 to limit oral drug bioavailability. Finally, these results indicate that neither CYP3A4 nor MDR1 (P‐glycoprotein) is coordinately regulated in the liver and intestine.

Clinical Pharmacology & Therapeutics (2004) 75, 172–183; doi: 10.1016/j.clpt.2003.10.008