bluuebunny/crossref_metadata_2025 huggingface dataset:
Long-run Stock Performance of Equity-Issuing Firms: The Case of Private Placements in Singapore
Authors: Chen Sheng-Syan , Ho Kim Wai , Lee Cheng-Few , Yeo Gillian H. H.
Published: December 2002

We find that Singapore listed firms which have conducted private placements subsequently experience long-run stock underperformance. The long-run underperformance is more severe for small firms and firms with a higher book-to-market ratio. This suggests that small firms and firms with poorer growth prospects are more likely to time the issue when the stock is temporarily overvalued. Further more, we find a positive relation between the long-run stock performance and the change in ownership concentration of the issuing firms, which is consistent with the alignment-of-interests hypothesis. We do not find evidence supporting the earnings-management hypothesis.
Option Implied Moments: An Application to Nikkei 225 Futures Options
Authors: Ang Kian-Ping , Rahman Shafiqur , Tan Kok-Hui
Published: December 2002

This paper proposes an integrated process to recover the moments of the risk-neutral distribution using a Gram-Charlier expansion series and Rubinstein's implied binomial tree approach. The advantage of using the implied tree approach is that it accounts for the possibility of early exercise of American options. We apply the method to American-style options on Nikkei 225 futures. We then demonstrate how to use the implied moments for trading options.
A Study of Mispricing and Parity in the Hang Seng Futures and Options Markets
Authors: Lai Kelvin Wai Lung , Marshall Andrew
Published: December 2002

This paper examines mispricing, volatility and parity on the Hang Seng Index (HSI) options and futures market. Most of the previous research has focused on futures contracts; we update this research and extend it by considering also option contracts. It is also important to examine these issues post 1997 Asian crisis. We find mispricing of HSI futures and option contracts if no transaction costs were considered. However, by incorporating transaction costs, the HSI futures are bounded within the arbitrage free region and most of the mispricing of the HSI options disappears. Additional tests on the mispricing series reveals that most of the derivative HSI contracts are positively autocorrelated and that the mispricing series for both derivative contracts are not identical among the different contract months. From our results we cannot conclude that there is causal relationship between the mispricing and the spot index volatility. Finally, our empirical results show that for HSI derivative contracts future and option parity holds, supporting our mispricing test that the HSI derivative market is efficient and has not been adversely affected by the Asian economic crisis.
NCX-4016 (NO-Aspirin) Inhibits Lipopolysaccharide-Induced Tissue Factor Expression In Vivo
Authors: Fiorucci Stefano , Mencarelli Andrea , Meneguzzi Alessandra , Lechi Alessandro , Morelli Antonio , del Soldato Piero , Minuz Pietro
Published: December 2002

Background—

NCX-4016 is an acetylsalicylic acid (ASA) derivative containing a nitric oxide–releasing moiety. Compared with ASA, NCX-4016 has a broader spectrum of antithrombotic and antiinflammatory activities. We hypothesized that NCX-4016 might inhibit in vivo lipopolysaccharide (LPS)-induced expression of tissue factor (TF).

Methods and Results—

Rats were administered 90 mg/kg NCX-4016 orally for 5 days. Placebo, 50 mg/kg ASA, and 80 mg/kg isosorbide-5-mononitrate (ISMN) were used in control groups. On day 5, rats were injected intraperitoneally with 100 μg/kg LPS and killed 6 hours later. The expression of TF in monocytes was measured by flow cytometry and Western blot analysis. Reverse transcriptase–polymerase chain reaction was performed to assess expression of TF and cyclooxygenase-2 (COX-2) genes. Plasma concentrations of interleukin-1β and tumor necrosis factor-α were measured. Urine samples were collected to evaluate the excretion of the thromboxane metabolite 11-dehydro-thromboxane (TX)B
2
. Gastric mucosa was inspected. LPS injection was followed by synthesis TF and COX-2 mRNAs in circulating monocytes, which were blunted by NCX-4016 but not by ASA or ISMN. Both NCX-4016 and ISMN reduced TF expression on surface of circulating monocyte. LPS increased the excretion 11-dehydro-TXB
2
, and this was prevented by NCX-4016 and ASA. Unlike ASA, NCX-4016 reduced plasma interleukin-1β and tumor necrosis factor-α. In addition, NCX-4016 almost completely prevented mucosal damage, whereas ASA increased the extension of gastric lesions in LPS-injected rats.

Conclusions—

NCX-4016 prevents monocyte TF expression; this is accompanied by inhibition of TX and cytokine biosynthesis. These additive effects of nitric oxide release and COX inhibition may help explain efficacy and tolerability of NCX-4016.
Nonselective Cation Currents Regulate Membrane Potential of Rabbit Coronary Arterial Cell
Authors: Terasawa Kuniko , Nakajima Toshiaki , Iida Haruko , Iwasawa Kuniaki , Oonuma Hitoshi , Jo Taisuke , Morita Toshihiro , Nakamura Fumitaka , Fujimori Yoshiharu , Toyo-oka Teruhiko , Nagai Ryozo
Published: December 2002

Background—

The effects of lysophosphatidylcholine (LPC) on electrophysiological activities and intracellular Ca
2+
concentration ([Ca
2+
]
i
) were investigated in coronary arterial smooth muscle cells (CASMCs).

Methods and Results—

The patch clamp techniques and Ca
2+
measurements were applied to cultured rabbit CASMCs. The membrane potential was −46.0±5.0 mV, and LPC depolarized it. Replacement of extracellular Na
+
with NMDG
+
hyperpolarized the membrane and antagonized the depolarizing effects of LPC. In Na
+
-, K
+
-, or Cs
+
-containing solution, the voltage-independent background current with reversal potential (E
r
) of approximately +0 mV was observed. Removal of Cl
−
failed to affect it. When extracellular cations were replaced by NMDG
+
, E
r
was shifted to negative potentials. La
3+
and Gd
3+
abolished the background current, but nicardipine and verapamil did not inhibit it. In Na
+
-containing solution, LPC induced a voltage-independent current with E
r
of approximately +0 mV concentration-dependently. Similar current was recorded in K
+
- and Cs
+
-containing solution. La
3+
and Gd
3+
inhibited LPC-induced current, but nicardipine and verapamil did not inhibit it. In cell-attached configurations, single-channel activities with single-channel conductance of ≈32pS were observed when patch pipettes were filled with LPC. LPC increased [Ca
2+
]
i
as the result of Ca
2+
influx, and La
3+
completely antagonized it.

Conclusions—

These results suggest that (1) nonselective cation current (I
NSC
) contributes to form membrane potentials of CASMCs and (2) LPC activates I
NSC
, resulting in an increase of [Ca
2+
]
i
. Thus, LPC may affect CASMC tone under various pathophysiological conditions such as ischemia.
Pioglitazone, a Peroxisome Proliferator–Activated Receptor-γ Agonist, Attenuates Left Ventricular Remodeling and Failure After Experimental Myocardial Infarction
Authors: Shiomi Tetsuya , Tsutsui Hiroyuki , Hayashidani Shunji , Suematsu Nobuhiro , Ikeuchi Masaki , Wen Jing , Ishibashi Minako , Kubota Toru , Egashira Kensuke , Takeshita Akira
Published: December 2002

Background—

Peroxisome proliferator–activated receptor-γ activators have recently been implicated as regulators of cellular proliferation and inflammatory response such as cytokine expression. Because proinflammatory cytokines play a critical role in left ventricular (LV) remodeling after myocardial infarction (MI), we examined the effects of pioglitazone treatment in an experimental model of chronic heart failure.

Methods and Results—

Mice with extensive anterior MI were treated with placebo or pioglitazone (3 mg · kg
−1
· d
−1
) as a dietary supplement for 4 weeks starting 6 hours after surgery. Infarct size and glucose levels were similar among all groups. LV cavity dilatation and dysfunction by echocardiography were significantly attenuated in MI mice given pioglitazone. LV end-diastolic pressure was increased in MI mice and was significantly reduced by pioglitazone treatment. Pioglitazone partially normalized LV dP/dt
max
and dP/dt
min
, indices of LV contractile function, which were significantly reduced in MI mice. Improvement of LV function by pioglitazone was accompanied by a decrease in myocyte hypertrophy and interstitial fibrosis and a reduced expression of tumor necrosis factor-α, transforming growth factor-β, and monocyte chemoattractant protein-1 genes in the noninfarcted LV from MI mice. LV inducible nitric oxide synthase and gelatinase B protein levels were increased in MI and were not altered by pioglitazone treatment.

Conclusions—

Pioglitazone improved LV remodeling and function in mice with post-MI heart failure. This effect was associated with an attenuated LV expression of inflammatory cytokines and chemokines. Peroxisome proliferator–activated receptor-γ ligands have promise as preventive and therapeutic agents against heart failure.
Lack of Benefit From Intravenous Platelet Glycoprotein IIb/IIIa Receptor Inhibition as Adjunctive Treatment for Percutaneous Interventions of Aortocoronary Bypass Grafts
Authors: Roffi Marco , Mukherjee Debabrata , Chew Derek P. , Bhatt Deepak L. , Cho Leslie , Robbins Mark A. , Ziada Khaled M. , Brennan Danielle M. , Ellis Stephen G. , Topol Eric J.
Published: December 2002

Background—

Despite widespread use of platelet glycoprotein (GP) IIb/IIIa receptor inhibitors for percutaneous coronary interventions (PCI) of bypass grafts, data supporting this strategy are lacking.

Methods and Results—

A pooled analysis of 5 randomized intravenous GP IIb/IIIa inhibitor trials (EPIC, EPILOG, EPISTENT, IMPACT II, and PURSUIT) was performed, and outcomes of graft interventions were assessed at 30 days and 6 months. Compared with PCI of native circulation (n=13 158), graft interventions (n=627) were associated with worse outcomes and in particular with a doubling of mortality at 30 days (2.1% versus 1.0%,
P
=0.006) and 6 months (4.7% versus 2.0%,
P
<0.001). Revascularization of a graft was identified as an independent predictor of death, myocardial infarction, or revascularization at 6 months (hazard ratio, 1.42; 95% CI, 1.24 to 1.63;
P
<0.001). Among patients undergoing graft PCI, the incidence of the triple end point at 30 days was 16.5% in the platelet GP IIb/IIIa inhibitor group and 12.6% in the placebo group (odds ratio, 1.38; 95% CI, 0.85 to 2.24;
P
=0.18). At 6 months, 39.4% of patients randomized to GP IIb/IIIa inhibitors and 32.7% of patients allocated to placebo had an ischemic event (hazard ratio, 1.29; 95% CI, 0.97 to 1.72;
P
=0.07).

Conclusions—

Intravenous platelet GP IIb/IIIa receptor inhibition does not improve outcomes after PCI of bypass grafts. In the absence of mechanical emboli protection, this procedure is associated with high incidence of death and nonfatal ischemic events.
Exercise Anaerobic Threshold and Ventilatory Efficiency Identify Heart Failure Patients for High Risk of Early Death
Authors: Gitt Anselm K. , Wasserman Karlman , Kilkowski Caroline , Kleemann Thomas , Kilkowski Andreas , Bangert Matthias , Schneider Steffen , Schwarz Armin , Senges Jochen
Published: December 2002

Background—

The maximal oxygen uptake (peak V̇
o
2
) is used in risk stratification of patients with chronic heart failure (CHF). Peak V̇
o
2
might be lower than maximally possible if exercise is stopped early because of lack of patient motivation or premature cessation by the investigator. In contrast, the anaerobic threshold (V̇
o
2
AT) and the ventilatory efficiency (V̇
e
versus V̇
co
2
slope) are less subject to these influences. Thus, we compared these parameters with peak V̇
o
2
in identifying patients with CHF at increased risk for death within 6 months after evaluation.

Methods and Results—

We performed cardiopulmonary exercise tests with gas exchange measurements in 223 consecutive patients with CHF (114 coronary artery disease, 92 dilated cardiomyopathy, 17 others) at the Herzzentrum Ludwigshafen between 1995 and 1998. We measured peak V̇
o
2
, V̇
o
2
AT and V̇
e
versus V̇
co
2
slope. We selected peak V̇
o
2
of ≤14 mL/kg per minute, V̇
o
2
AT of <11 mL/kg per minute, and V̇
e
versus V̇
co
2
slope of >34 as threshold values for high risk of death. The median follow-up time was 644 days. Patients with peak V̇
o
2
of ≤14 mL/kg per minute had a >3-fold-increased risk (OR=3.4; CI, 1.3 to 9.1), with V̇
o
2
AT <11 mL/min per kg or V̇
e
versus V̇
co
2
slope >34 a 5-fold increased risk for early death (OR=5.3; CI, 1.5 to 19.0; OR=4.8; CI, 1.7 to 13.8, respectively). In patients with both V̇
o
2
AT <11 mL/kg per minute and V̇
e
versus V̇
co
2
slope >34, the risk of early death was 10-fold higher (OR=9.6; CI, 2.1 to 44.7). After correction for age, sex, left ventricular ejection fraction, and New York Heart Association class in a multivariate analysis, the combination of V̇
o
2
AT <11 mL/kg per minute and V̇
e
versus V̇
co
2
slope >34 was the best predictor of 6-month mortality (RR=5.1,
P
=0.001).

Conclusions—

V̇
o
2
AT of <11 mL/kg per minute and slope of V̇
e
versus V̇
co
2
>34, combined, better identified patients at high risk for early death from CHF than did peak V̇
o
2
and should therefore be considered when prioritizing patients for heart transplantation.
Acetylated Low-Density Lipoprotein Stimulates Human Vascular Smooth Muscle Cell Calcification by Promoting Osteoblastic Differentiation and Inhibiting Phagocytosis
Authors: Proudfoot D. , Davies J.D. , Skepper J.N. , Weissberg P.L. , Shanahan C.M.
Published: December 2002

Background—

Vascular smooth muscle cells (VSMCs) in atherosclerotic lesions display an osteogenic phenotype, and calcification commonly occurs in association with lipid. We therefore tested the hypothesis that lipid components in atherosclerotic lesions influenced VSMC phenotype and calcification using an in vitro model of calcification.

Methods and Results—

In situ hybridization of human atherosclerotic plaques (n=10) collected from patients undergoing carotid endarterectomy demonstrated that subsets of lipid-filled VSMCs adjacent to sites of calcification expressed alkaline phosphatase, bone Gla protein, and bone sialoprotein, suggesting an osteogenic phenotype. Treatment of VSMCs in culture with acetylated low-density lipoprotein (acLDL) or lipoprotein-deficient serum altered the time course of bone-associated protein gene expression and calcification. AcLDL increased nodule calcification 3-fold, whereas lipoprotein-deficient serum significantly inhibited it. Reverse transcriptase–polymerase chain reaction and Western analysis demonstrated the presence of the acLDL receptor, SRA1, exclusively in calcifying nodular VSMCs, and blockade of SRA with polyinosinic acid inhibited acLDL-induced calcification. Because apoptotic bodies can serve as nucleation sites for calcification, we investigated whether acLDL could stimulate apoptosis in nodules. Apoptosis of nodular VSMCs was unaltered, but the number of apoptotic bodies per nodule increased ≈3-fold, implying a defect in phagocytosis. Consistent with these observations, binding of apoptotic bodies to VSMCs was decreased in the presence of acLDL.

Conclusions—

These studies suggest that modified lipoproteins stimulate calcification by enhancing osteogenic differentiation of VSMCs and by a novel mechanism whereby acLDL interacts with SRA on VSMCs and blocks phagocytic removal of apoptotic bodies.
Vascular Oxidative Stress and Endothelial Dysfunction in Patients With Chronic Heart Failure
Authors: Landmesser Ulf , Spiekermann Stephan , Dikalov Sergey , Tatge Helma , Wilke Ragna , Kohler Christoph , Harrison David G. , Hornig Burkhard , Drexler Helmut
Published: December 2002

Background—

Impaired flow-dependent, endothelium-mediated vasodilation (FDD) in patients with chronic heart failure (CHF) results, at least in part, from accelerated degradation of nitric oxide by oxygen radicals. The mechanisms leading to increased vascular radical formation, however, remain unclear. Therefore, we determined endothelium-bound activities of extracellular superoxide dismutase (ecSOD), a major vascular antioxidant enzyme, and xanthine-oxidase, a potent radical producing enzyme, and their relation to FDD in patients with CHF.

Methods and Results—

ecSOD and xanthine-oxidase activities, released from endothelium into plasma by heparin bolus injection, were determined in 14 patients with CHF and 10 control subjects. FDD of the radial artery was measured using high-resolution ultrasound and was assessed before and after administration of the antioxidant vitamin C (25 mg/min; IA). In patients with CHF, endothelium-bound ecSOD activity was substantially reduced (5.0±0.7 versus 14.4±2.6 U · mL
−1
· min
−1
;
P
<0.01) and closely related to FDD (
r
=0.61). Endothelium-bound xanthine-oxidase activity was increased by >200% (38±10 versus 12±4 nmol O
2
·−
· μL
−1
;
P
<0.05) and inversely related to FDD (
r
=−0.35) in patients with CHF. In patients with low ecSOD and high xanthine-oxidase activity, a greater benefit of vitamin C on FDD was observed, ie, the portion of FDD inhibited by radicals correlated negatively with ecSOD (
r
=−0.71) but positively with xanthine-oxidase (
r
=0.75).

Conclusions—

These results demonstrate that both increased xanthine-oxidase and reduced ecSOD activity are closely associated with increased vascular oxidative stress in patients with CHF. This loss of vascular oxidative balance likely represents a novel mechanism contributing to endothelial dysfunction in CHF.
Vascular Structural and Functional Changes in Type 2 Diabetes Mellitus
Authors: Schofield Ian , Malik Rayaz , Izzard Ashley , Austin Clare , Heagerty Anthony
Published: December 2002

Background—

To further investigate vascular morphology and function in type 2 (non–insulin-dependent) diabetes mellitus (type 2D), small arteries were examined in vitro from carefully defined cohorts of patients with or without concomitant hypertension and the results compared with those from selected normotensive nondiabetic control subjects and a group of untreated patients with essential hypertension (EH).

Methods and Results—

Blood vessels were studied through the use of pressure myography to determine vascular morphology, mechanics, and myogenic responsiveness, together with testing of constrictor and dilator function. Small arteries from patients with EH demonstrated eutrophic inward remodeling and an increased distensibility. Vessels from type 2D patients demonstrated hypertrophy, a further increase in distensibility, and a highly significant loss of myogenic responsiveness compared with patients with EH and control patients. Vasoconstrictor function to norepinephrine was normal in patients with type 2D and type 2D+H and EH. Endothelium-dependent dilation was normal in patients with EH but abnormal in patients with type 2D and type 2D+H. There was a significant correlation between dilator impairment and the degree of dyslipidemia recorded in all groups.

Conclusions—

These results demonstrate vascular hypertrophy in small arteries from patients with type 2D. This could be a consequence of impaired myogenic responsiveness, which will increase wall stress for a given intraluminal pressure, which may be a stimulus for vascular hypertrophy. A substantial proportion of endothelial dysfunction can be attributed to an effect of the abnormal lipid profile seen in such patients.
Increased Expression of Membrane Type 3-Matrix Metalloproteinase in Human Atherosclerotic Plaque
Authors: Uzui Hiroyasu , Harpf Alice , Liu Ming , Doherty Terence M. , Shukla Arun , Chai Ning-Ning , Tripathi Pinky V. , Jovinge Stefan , Wilkin Douglas J. , Asotra Kamlesh , Shah Prediman K. , Rajavashisth Tripathi B.
Published: December 2002

Background—

Matrix metalloproteinases (MMPs) are thought to play a prominent role in atherogenesis and destabilization of plaque. Pericellularly localized membrane-type (MT)-MMPs activate secreted MMPs. We investigated the hypothesis that MT3-MMP is expressed in human atherosclerotic plaques and is regulated by locally produced inflammatory cytokines and oxidized low-density lipoprotein (Ox-LDL).

Methods and Results—

Expression and cellular localization of MT3-MMP in normal and atherosclerotic human coronary arteries were examined using specific antibodies. Abundant MT3-MMP expression was noted in medial smooth muscle cells (SMCs) of normal arteries. In atherosclerotic arteries, MT3-MMP expression was observed within complex plaques and colocalized with SMCs and macrophages (Mφ). Cultured human monocyte-derived Mφ constitutively expressed MT3-MMP mRNA and proteolytically active protein, as demonstrated by mRNA analyses, immunoblotting, and gelatin zymography, respectively. Ox-LDL, tumor necrosis factor-α, or macrophage colony-stimulating factor caused dose- and time-dependent increases in steady-state levels of MT3-MMP mRNA in cultured Mφ. This correlated with a 2- to 4-fold increase in levels of MT3-MMP immunoreactive protein and enzymatic activity in Mφ membranes. Confocal microscopy and flow cytometry confirmed induction and spatial distribution of MT3-MMP protein from intracellular domains to the Mφ plasma membrane by Ox-LDL, tumor necrosis factor-α, or macrophage colony-stimulating factor.

Conclusions—

MT3-MMP is expressed by SMCs and Mφ in human atherosclerotic plaques. Proinflammatory molecules cause a progressive increase in the expression of MT3-MMP in cultured Mφ. Our results suggest a mechanism by which inflammatory molecules could promote Mφ-mediated degradation of extracellular matrix and thereby contribute to plaque destabilization.
Low-Density Lipoprotein Upregulates Low-Density Lipoprotein Receptor-Related Protein Expression in Vascular Smooth Muscle Cells
Authors: Llorente-Cortés Vicenta , Otero-Viñas Marta , Sánchez Sonia , Rodríguez Cristina , Badimon Lina
Published: December 2002

Background—

Low-density lipoprotein (LDL) receptor-related protein (LRP) is highly expressed in vascular smooth muscle cells (VSMCs) of both normal and atherosclerotic lesions. However, little is known about LRP regulation in the vascular wall.

Methods and Results—

We analyzed the regulation of LRP expression in vitro in human VSMCs cultured with native LDL (nLDL) or aggregated LDL (agLDL) by semiquantitative reverse transcriptase-polymerase chain reaction, real-time polymerase chain reaction, and Western blot and in vivo during diet-induced hypercholesterolemia by in situ hybridization. LRP expression in human VSMCs is increased by nLDL and agLDL in a time- and dose-dependent manner. Maximal induction of LRP mRNA expression was observed after 24 hours of exposure to LDL. However, agLDL induced higher LRP mRNA expression (3.0-fold) than nLDL (1.76-fold). LRP mRNA upregulation was associated with an increase on LRP protein expression with the greatest induction by agLDL. VSMC-LRP upregulation induced by nLDL or agLDL was reduced by an inhibitor of sterol regulatory element binding protein (SREBP) catabolism (
N
-acetyl-leucyl-leucyl-norleucinal). In situ hybridization analysis indicates that there is a higher VSMC-LRP expression in hypercholesterolemic than in normocholesterolemic pig aortas.

Conclusions—

These results indicate that LRP expression in VSMCs is upregulated by intravascular and systemic LDL.
Prevalence and Severity of “Benign” Mutations in the β-Myosin Heavy Chain, Cardiac Troponin T, and α-Tropomyosin Genes in Hypertrophic Cardiomyopathy
Authors: Van Driest Sara L. , Ackerman Michael J. , Ommen Steve R. , Shakur Rameen , Will Melissa L. , Nishimura Rick A. , Tajik A. Jamil , Gersh Bernard J.
Published: December 2002

Background—

Genotype-phenotype correlative studies have implicated 8 particular mutations that cause hypertrophic cardiomyopathy (HCM) as “benign defects,” associated with near-normal survival: N232S, G256E, F513C, V606M, R719Q, and L908V of β-myosin heavy chain (
MYH7
); S179F of troponin T (
TNNT2
); and D175N of α-tropomyosin (
TPM1
). Routine genetic screening of HCM patients for specific mutations is anticipated to provide important diagnostic and prognostic information. The frequency and associated phenotype of these mutations in a large, unselected cohort of HCM is unknown.

Methods and Results—

A total of 293 unrelated HCM patients were genotyped for the presence of a benign mutation. DNA was obtained after informed consent; specific
MHY7, TNNT2
, and
TPM1
fragments were amplified by polymerase chain reaction; and the mutations were detected by denaturing high-performance liquid chromatography and automated DNA sequencing. Only 5 (1.7%) of the 293 patients possessed a benign mutation. Moreover, all 5 subjects with an ascribed benign mutation had already manifested clinically severe expression of HCM, with all 5 requiring surgical myectomy, 3 of the 5 having a family history of sudden cardiac death, and 1 adolescent requiring an orthotopic heart transplant.

Conclusions—

These findings demonstrate the rarity of specific mutations in HCM and challenge the notion of mutation-specific clinical outcomes. Fewer than 2% of the subjects harbored a benign mutation, and those patients with a benign mutation experienced a very serious clinical course.
Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI)
Authors: Assmus Birgit , Schächinger Volker , Teupe Claudius , Britten Martina , Lehmann Ralf , Döbert Natascha , Grünwald Frank , Aicher Alexandra , Urbich Carmen , Martin Hans , Hoelzer Dieter , Dimmeler Stefanie , Zeiher Andreas M.
Published: December 2002

Background—

Experimental studies suggest that transplantation of blood-derived or bone marrow–derived progenitor cells beneficially affects postinfarction remodeling. The safety and feasibility of autologous progenitor cell transplantation in patients with ischemic heart disease is unknown.

Methods and Results—

We randomly allocated 20 patients with reperfused acute myocardial infarction (AMI) to receive intracoronary infusion of either bone marrow–derived (n=9) or circulating blood–derived progenitor cells (n=11) into the infarct artery 4.3±1.5 days after AMI. Transplantation of progenitor cells was associated with a significant increase in global left ventricular ejection fraction from 51.6±9.6% to 60.1±8.6% (
P
=0.003), improved regional wall motion in the infarct zone (−1.5±0.2 to −0.5±0.7 SD/chord;
P
<0.001), and profoundly reduced end-systolic left ventricular volumes (56.1±20 mL to 42.2±15.1 mL;
P
=0.01) at 4-month follow-up. In contrast, in a nonrandomized matched reference group, left ventricular ejection fraction only slightly increased from 51±10% to 53.5±7.9%, and end-systolic volumes remained unchanged. Echocardiography revealed a profound enhancement of regional contractile function (wall motion score index 1.4±0.2 at baseline versus 1.19±0.2 at follow-up;
P
<0.001). At 4 months, coronary blood flow reserve was significantly (
P
<0.001) increased in the infarct artery. Quantitative F-18-fluorodeoxyglucose–positron emission tomography analysis revealed a significant (
P
<0.01) increase in myocardial viability in the infarct zone. There were no differences for any measured parameter between blood-derived or bone marrow–derived progenitor cells. No signs of an inflammatory response or malignant arrhythmias were observed.

Conclusions—

In patients with AMI, intracoronary infusion of autologous progenitor cells appears to be feasible and safe and may beneficially affect postinfarction remodeling processes.
Specific interactions and phase behavior of poly(2‐vinylpridine)/bisphenol blends
Authors: Li Xue‐Dong , Goh S. H. , Zheng J. W.
Published: December 2002

Hydrogen‐bonding interaction and miscibility between poly(2‐vinylpyrdine) (P2VPy) and five bisphenols are studied by Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and wide angle X‐ray diffraction (WAXD). It is found that the miscibility behavior strongly depends on the flexibility of the bisphenol molecules. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 87: 1137–1143, 2003
Optimization of a feed medium for fed‐batch culture of insect cells using a genetic algorithm
Authors: Marteijn R. C. L. , Jurrius O. , Dhont J. , de Gooijer C. D. , Tramper J. , Martens D. E.
Published: December 2002

Insect cells have been cultured for over 30 years, but their application is still hampered by low cell densities in batch fermentations and expensive culture media. With respect to the culture method, the fed‐batch culture mode is often found to give the best yields. However, optimization of the feed composition is usually a laborious task. In this report, the successful use of genetic algorithms (GAs) to optimize the growth of insect cells is described. A feed was developed from 11 different medium components, each used at a wide range of concentrations. The feed was optimized within four sets of 20 experiments. The optimized feed was tested in bioreactors and the addition scheme was further improved. The viable‐cell density of HzAm1 (Helicoverpa zea) insect cells improved 550% to 19.5 × 106 cells/mL compared to a control fermentation in an optimized commercial medium. No accumulation of waste products was found, and none of the amino acids was depleted. Glucose was depleted, which suggests that even further improvement is possible. We show that GAs are a successful method to optimize a complex fermentation in a relatively short time frame and without the need of detailed information concerning the cellular physiology or metabolism. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 269–278, 2003.
Bcl‐xL expression interferes with the effects of L‐glutamine supplementation on hybridoma cultures
Authors: Charbonneau Joel R. , Furtak Teresa , Lefebvre Jennie , Gauthier Eric R.
Published: December 2002

While feeding protocols and ectopic expression of anti‐apoptotic genes have been used to improve the viability of hybridoma cell lines, the effect of the expression levels of survival genes on the behavior of hybridomas following nutrient supplementation is unknown. In this study, we compared the behavior of the Sp2/0‐Ag14 hybridoma (Bcl‐xLlow) and the P3x63‐Ag8.653 myeloma (Bcl‐xLhigh) following culture supplementation with the amino acid L‐glutamine (L‐Gln). Our data revealed that L‐Gln addition substantially increased Sp2/0‐Ag14 cell viability and total cell density, concomitant with a decrease in the rate of cell death. This effect was not seen when other amino acids or D‐glucose (D‐Glc) replaced L‐Gln. The improvement in the culture behavior of Sp2/0‐Ag14 cells was attributed to a reduction in the rate of accumulation of apoptotic cells. On the other hand, L‐Gln supplementation had only a limited effect on the growth of the P3x63‐Ag8.653 cells. Interestingly, Sp2/0‐Ag14 cells over‐expressing Bcl‐xL showed a culture behavior upon L‐Gln complementation that was similar to the P3x63‐Ag8.653 myeloma. These results suggest that the anti‐apoptotic gene expression profile of hybridoma cells can markedly impact on the beneficial effects afforded by nutrient supplementation. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 279–290, 2003.
Overexpression of a heterologous protein, haloalkane dehalogenase, in a poly‐β‐hydroxybutyrate‐deficient strain of the facultative methylotroph Methylobacterium extorquens AM1
Authors: Fitzgerald Kelly A. , Lidstrom Mary E.
Published: December 2002

Using an expression vector containing pmxaF, a strong native promoter, expression of a model heterologous protein, haloalkane dehalogenase, from Xanthobacter autotrophicus GJ10 was achieved in the methylotrophic bacterium, Methylobacterium extorquens AM1. Although expression using the wild‐type strain was <5% of total cell protein, expression at a level of 10% of the total cell protein was achieved in a mutant unable to synthesize poly‐β‐hydroxybutyrate granules. Two other tested heterologous proteins, catechol dioxygenase and green fluorescent protein, were expressed at moderate levels in both wild‐type and the PHB‐negative strain. These results suggest that the M. extorquens PHB‐negative strain is a possible platform for overexpression of heterologous proteins with labeled or unlabeled methanol as a starting material. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 263–268, 2003.
Study of caspase inhibitors for limiting death in mammalian cell culture
Authors: Sauerwald Tina M. , Oyler George A. , Betenbaugh Michael J.
Published: December 2002

Apoptosis in mammalian cell culture is associated with decreased bioproduct yields and can be inhibited through altering the intracellular signaling pathways mediating programmed cell death. In this study, we evaluated the capacity to inhibit caspases to maintain high viable cell numbers in CHO and 293 cultures. Two genetic caspase inhibitors, XIAP and CrmA, were examined along with a mutant of each, XIAP‐BIR123NC, which contains three BIR domains but lacks the RING finger, and CrmA‐DQMD, which has CrmA's pseudosubstrate site replaced with that of another caspase inhibitor, p35. Stable CHO pooled and 293 clonal cell lines expressing each protein were exposed to apoptotic insults, including spent medium, Sindbis virus, and etoposide. For each insult the mutated protein resulted in higher viabilities than its wild‐type counterpart. However, the mutants provided different levels of protection, depending on the insult considered. CrmA‐DQMD was the preferred inhibitor for spent medium‐induced apoptosis, whereas XIAP‐BIR123NC conferred better protection for etoposide‐induced death. Addition of Z‐VAD.fmk to the genetically engineered cells enhanced viabilities in the presence of spent medium or etoposide; however, the largest increases in viability were experienced by the control cells, indicating an overlap in caspase inhibition between the genetic and chemical inhibitors. Finally, parental 293 cells were treated with caspase‐8 and ‐9 inhibitors, Z‐IETD.fmk and Z‐LEHD.fmk, in concert with spent medium or etoposide exposure. Spent medium‐induced death was delayed more readily with the caspase‐8 inhibitors, CrmA‐DQMD and Z‐IETD.fmk, and etoposide‐induced death was stalled more so with XIAP‐BIR123NC and Z‐LEHD.fmk. These results suggest that the apoptosis pathways induced and the level of protection afforded by a particular caspase inhibitor may vary with the insult considered. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 329–340, 2003.
Physicochemical characterization and antimicrobial properties of rhamnolipids produced byPseudomonas aeruginosa47T2 NCBIM 40044
Authors: Haba E. , Pinazo A. , Jauregui O. , Espuny M. J. , Infante M. R. , Manresa A.
Published: December 2002

Pseudomonas aeruginosa47T2, grown in submerged culture with waste frying oil as a carbon source, produced a mixture of rhamnolipids with surface activity. Up to 11 rhamnolipid homologs (Rha‐Rha‐C8‐C10; Rha‐C10‐C8/Rha‐C8‐C10;Rha‐Rha‐C8‐C12:1; Rha‐Rha‐C10‐C10; Rha‐Rha‐C10‐C12:1; Rha‐C10‐C10; Rha‐Rha‐C10‐C12/Rha‐Rha‐C12‐C10; Rha‐C10‐C12:1/Rha‐C12:1‐C10; Rha‐Rha‐C12:1‐C12; Rha‐Rha‐C10‐C14:1; Rha‐C10‐C12/Rha‐C12‐C10) were isolated from cultures ofP. aeruginosa47T2 from waste frying oil and identified by HPLC‐MS analysis. This article deals with the production, isolation, and chemical characterization of the rhamnolipid mixture RL47T2. The physicochemical and biological properties of RL47T2as a new product were also studied. Its surface tension decreased to 32.8 mN/m; and the interfacial tension against kerosene to 1 mN/m. The critical micellar concentration for RL47T2was 108.8 mg/mL. The product showed excellent antimicrobial properties. Antimicrobial activity was evaluated according to the minimum inhibitory concentration (MIC), the lowest concentration of an antimicrobial agent that inhibits development of visible microbial growth. Low MIC values were found for bacteriaSerratia marcescens(4 μg/mL),Enterobacter aerogenes(8 μg/mL),Klebsiella pneumoniae(0.5 μg/mL),Staphylococcus aureusandStaphylococcus epidermidis(32 μg/mL),Bacillus subtilis(16 μg/mL), and phytopathogenic fungal species:Chaetonium globosum(64 μg/mL),Penicillium funiculosum(16 μg/mL),Gliocadium virens(32 μg/mL) andFusarium solani(75 μg/mL). © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 316–322, 2003.
Water‐in‐oil macroemulsions sustain long‐term viability of microbial cells in organic solvents
Authors: Stefan Alessandra , Palazzo Gerardo , Ceglie Andrea , Panzavolta Eleonora , Hochkoeppler Alejandro
Published: December 2002

Extremely stable water‐in‐oil macroemulsions have been obtained by dispersing water in isooctane in the presence of lecithin. Either prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and Rhodotorula minuta) cells hosted in these water‐in‐oil macroemulsions are viable for weeks despite the consistent excess of organic solvent (ranging from 70 to 84%, v/v) in these ternary systems. Conjugation occurs upon mixing macroemulsions containing F+ or F− Escherichia coli strains, indicating consistent mass transfer between the water droplets. Populations of yeasts hosted in water‐in‐oil macroemulsion feature a higher frequency of cells aggregation when compared with the corresponding populations suspended in homogeneous aqueous media. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 323–328, 2003.
Pulsed feeding during fed‐batch fungal fermentation leads to reduced viscosity without detrimentally affecting protein expression
Authors: Bhargava Swapnil , Nandakumar M. P. , Roy Anindya , Wenger Kevin S. , Marten Mark R.
Published: December 2002

The goal in this study was to determine if pulsed addition of substrate could be used to alter filamentous fungal morphology during fermentation, to result in reduced broth viscosity. In all experiments, an industrially relevant strain of Aspergillus oryzae was grown in 20‐liter fermentors. As a control, cultures were fed limiting substrate (glucose) continuously. Tests were performed by altering the feeding strategy so that the same total amount of glucose was fed in repeated 300‐s cycles, with the feed pump on for either 30 or 150 s during each cycle. Variables indicative of cellular metabolic activity (biomass concentration, oxygen uptake rate, base consumed for pH control) showed no significant difference between continuous and pulse‐fed fermentations. In addition, there was no significant difference between total extracellular protein expression or the apparent distribution of these proteins. In contrast, fungal mycelia during the second half of pulse‐fed fermentations were approximately half the size (average projected area) of fungi during fermentations with continuous addition of glucose. As a result, broth viscosity during the second half of pulse‐fed fermentations was approximately half that during the second half of continuous fermentations. If these results prove to be applicable for other fungal strains and processes, then this method will represent a simple and inexpensive means to reduce viscosity during filamentous fungal fermentation. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 341–347, 2003.
Improved fuel cell and electrode designs for producing electricity from microbial degradation
Authors: Park Doo Hyun , Zeikus J. Gregory
Published: December 2002

A new one‐compartment fuel cell was composed of a rubber bunged bottle with a center‐inserted anode and a window‐mounted cathode containing an internal, proton‐permeable porcelain layer. This fuel cell design was less expensive and more practical than the conventional two‐compartment system, which requires aeration and a ferricyanide solution in the cathode compartment. Three new electrodes containing bound electron mediators including a Mn4+‐graphite anode, a neutral red (NR) covalently linked woven graphite anode, and an Fe3+‐graphite cathode were developed that greatly enhanced electrical energy production (i.e., microbial electron transfer) over conventional graphite electrodes. The potentials of these electrodes measured by cyclic voltametry at pH 7.0 were (in volts): +0.493 (Fe3+‐graphite); +0.15 (Mn4+‐graphite); and −0.53 (NR‐woven graphite). The maximal electrical productivities obtained with sewage sludge as the biocatalyst and using a Mn4+‐graphite anode and a Fe3+‐graphite cathode were 14 mA current, 0.45 V potential, 1,750 mA/m2 current density, and 788 mW/m2 of power density. With Escherichia coli as the biocatalyst and using a Mn4+‐graphite anode and a Fe3+‐graphite cathode, the maximal electrical productivities obtained were 2.6 mA current, 0.28 V potential, 325 mA/m2 current density, and 91 mW/m2 of power density. These results show that the amount of electrical energy produced by microbial fuel cells can be increased 1,000‐fold by incorporating electron mediators into graphite electrodes. These results also imply that sewage sludge may contain unique electrophilic microbes that transfer electrons more readily than E. coli and that microbial fuel cells using the new Mn4+‐graphite anode and Fe3+‐graphite cathode may have commercial utility for producing low amounts of electrical power needed in remote locations. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 348–355, 2003.
Kinetic locking‐on and auxiliary tactics for bioaffinity purification of NADP+‐dependent dehydrogenases using N6‐linked immobilized NADP+ derivatives: Studies with mammalian and microbial glutamate dehydrogenases
Authors: McMahon Mary , Tynan Julie , Mulcahy Patricia
Published: December 2002

This study is concerned with the development and application of kinetic locking‐on and auxiliary tactics for bioaffinity purification of NADP+‐dependent dehydrogenases, specifically (1) the synthesis and characterization of highly substituted N6‐linked immobilized NADP+ derivatives using a rapid solid‐phase modular approach; (2) the evaluation of the N6‐linked immobilized NADP+ derivatives for use with the kinetic locking‐on strategy for bioaffinity purification of NADP+‐dependent dehydrogenases: Model bioaffinity chromatographic studies with glutamate dehydrogenase from bovine liver (GDH with dual cofactor specificity, EC 1.4.1.3) and glutamate dehydrogenase from Candida utilis (GDH which is NADP+‐specific, EC 1.4.1.4); (3) the selection of an effective “stripping ligand” for NADP+‐dehydrogenase bioaffinity purifications using N6‐linked immobilized NADP+ derivatives in the locking‐on mode; and (4) the application of the developed bioaffinity chromatographic system to the purification of C. utilis GDH from a crude cellular extract.

Results confirm that the newly developed N6‐linked immobilized NADP+ derivatives are suitable for the one‐step bioaffinity purification of NADP+‐dependent GDH provided that they are used in the locking‐on mode, steps are taken to inhibit alkaline phosphatase activity in crude cellular extracts, and 2′,5′‐ADP is used as the stripping ligand during chromatography. The general principles described here are supported by a specific sample enzyme purification; the purification of C. utilis GDH to electrophoretic homogeneity in a single bioaffinity chromatographic step (specific activity, 9.12 μmol/min/mg; purification factor, 83.7; yield 88%). The potential for development of analogous bioaffinity systems for other NADP+‐dependent dehydrogenases is also discussed. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 356–369, 2003.
Testing for nandrolone metabolites in urine samples of professional athletes and sedentary subjects by GC/MS/MS analysis
Authors: Gambelunghe Cristiana , Sommavilla Marco , Rossi Ruggero
Published: December 2002

The concentrations of nandrolone metabolites, 19‐norandrosterone (19‐NA) and 19‐noretiocholanolone (19‐NE) were analysed in urine samples of professional athletes doing intense physical activity and sedentary subjects to verify if there was endogenous production of nandrolone and if there was any link between physical effort and the urinary metabolites of the steroid. We collected 18 urine samples from professional footballers age range 20–30 years, all from the same team, and 18 urine samples from males not doing any physical activity, age range 20–30 years. Neither group used nandrolone. Qualitative and quantitative analyses of urinary nandrolone metabolites were carried out by GC/MS followed by GC/MS/MS to confirm positive samples. This technique has been demostrated to be an excellent analytical approach for the determination of anabolic steroids at very low detection limits in complex matrices such as urine. In five urine samples from professional footballers traces of 19‐NA were detected. No trace of 19‐NA was found in the group of sedentary subjects and no trace of 19‐NE was found in any urine sample. The absence of nandrolone metabolites in sedentary subjects supports the hypothesis that the presence of 19‐NA and 19‐NE could be linked to physical effort even though the origin is not yet clear. Copyright © 2002 John Wiley & Sons, Ltd.
Screening for 18 diuretics and probenecid in doping analysis by liquid chromatography–tandem mass spectrometry
Authors: Deventer K. , Delbeke F. T. , Roels K. , Van Eenoo P.
Published: December 2002

A fast and selective liquid chromatography–tandem mass spectrometric (LC/MS/MS) method for the screening of 18 diuretics and probenecid in human urine is presented. Analyses were performed on a LCQ‐Deca® instrument equipped with ESI‐interface using scan by scan polarity changing. All diuretics and probenecid were separated in less than 20 min after liquid–liquid extraction with ethyl acetate. The LOD for all substances was 100 ng/mL or better. The method was applied to detect diuretics after the oral administration of several drugs including hydrochlorothiazide, bumetanide, spironolactone, furosemide, amiloride, triamterene, chlortalidone and epithizide. All diuretics could be detected for periods up to 96 h after the intake of therapeutic amounts. Copyright © 2002 John Wiley & Sons, Ltd.
Rat liver and kidney catechol‐O‐methyltransferase activity measured by high‐performance liquid chromatography with fluorescence detection
Authors: Tsunoda Makoto , Takezawa Kazuko , Masuda Mayumi , Imai Kazuhiro
Published: December 2002

We have previously reported a highly sensitive method for the measurement of catechol‐O‐methyltransferase (COMT) activities in rat erythrocytes with norepinephrine (NE), an endogenous native substrate, using high‐performance liquid chromatography (HPLC)‐fluorescence or peroxyoxalate chemiluminescence reaction detection. Applying this method to COMT activities in rat liver and kidney, known to have the highest activities of all organs, the optimum reaction conditions were investigated. Under the optimum conditions, soluble (S)‐COMT and membrane‐bound (MB)‐COMT activities in rat liver, with NE as a substrate, were 2.17 ± 0.33 and 0.16 ± 0.02 nmol/min/mg protein (n = 5), respectively. In rat kidney, S‐COMT and MB‐COMT activities were 1.81 ± 0.20 and 0.079 ± 0.009 nmol/min/mg protein (n = 5), respectively. Since liver and kidney play important roles in inactivating catecholamines, using the proposed method would yield critical information to delineate the role of metabolism of catecholamines in rat tissues. Copyright © 2002 John Wiley & Sons, Ltd.
A minimization principle for finite strain plasticity: incremental objectivity and immediate implementation
Authors: Lorentz Eric , Cano Valérie
Published: December 2002

A finite strain plasticity formulation is proposed which meets several requirements that often appear contradictory. On a physical ground, it is based on a multiplicative split of the deformation, hyperelasticity for the reversible part of the behaviour and the maximal dissipation principle to define the evolution laws. On a numerical ground, it is incrementally objective and the integration over a time increment can be expressed as a minimization problem, a proper framework to examine the questions of existence and uniqueness of the solutions. Last but not least, the implementation is immediate since it relies on the same equations for finite and infinitesimal transformations. Finally, the formulationis applied to von Mises plasticity with isotropic linear hardening and introduced in the finite element software Code_Aster®. The numerical computation of a cantilever beam shows that it leads to results in good agreement with those obtain with common approaches. Copyright © 2002 John Wiley & Sons, Ltd.