bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial dysfunction in autistic patients with 15q inverted duplication
Authors: Filipek Pauline A. , Juranek Jenifer , Smith Moyra , Mays Lee Z. , Ramos Erica R. , Bocian Maureen , Masser‐Frye Diane , Laulhere Tracy M. , Modahl Charlotte , Spence M. Anne , Gargus J. Jay
Published: May 2003

Two autistic children with a chromosome 15q11‐q13 inverted duplication are presented. Both had uneventful perinatal courses, normal electroencephalogram and magnetic resonance imaging scans, moderate motor delay, lethargy, severe hypotonia, and modest lactic acidosis. Both had muscle mitochondrial enzyme assays that showed a pronounced mitochondrial hyperproliferation and a partial respiratory chain block most parsimoniously placed at the level of complex III, suggesting candidate gene loci for autism within the critical region may affect pathways influencing mitochondrial function. Ann Neurol 2003;53:801–804
Regular exercise is beneficial to a mouse model of amyotrophic lateral sclerosis
Authors: Kirkinezos Ilias G. , Hernandez Dayami , Bradley Walter G. , Moraes Carlos T.
Published: May 2003

We tested whether a regular exercise regimen was associated with a change in the life span of G93A‐SOD1 transgenic mice, a model of familial ALS. Regular treadmill running for 10 weeks led to a significant increase in the life span of G93A‐SOD1 mice. The effect was stronger in male mice, whereas there was only a trend between exercised and sedentary female G93A‐SOD1 mice. The data suggest that regular exercise has a beneficial effect on the progression of ALS. Ann Neurol 2003;53:804–807
Apolipoprotein C‐II deficiency presenting as a lipid encephalopathy in infancy
Authors: Wilson Callum J. , Oliva Claudio Priore , Maggi Franco , Catapano Alberico L. , Calandra Sebastiano
Published: May 2003

An infant presented with massive hyperchylomicronemia and a severe encephalopathy. MRI showed marked lipid deposition throughout the brain. Despite the normalization of the biochemistry, there was little clinical improvement, and at 18 months of age she has severe developmental delay, a strikingly abnormal MRI. Apolipoprotein C‐II, the lipoprotein on chylomicrons responsible for the activation of lipoprotein lipase, was not detectable in blood. Analysis of the APO C‐II gene revealed a novel homozygous point mutation, 1118C→A. Subsequently, another sibling has been born with the same homozygous mutation and similar biochemistry but, perhaps because of early treatment, a normal neurological outcome. Ann Neurol 2003;53:807–810
Healthy‐side dominance of cortical neuromagnetic responses in sudden hearing loss
Authors: Po‐Hung Li Lieber , Shiao An‐Suey , Lin Yung‐Yang , Chen Li‐Fen , Niddam David M. , Chang Shyue‐Yih , Lien Chiang‐Feng , Chou Nan‐San , Ho Low‐Tone , Hsieh Jen‐Chuen
Published: May 2003

Previous brain imaging and mapping studies have reported findings indicating functional reorganization in the central auditory pathways of patients with profound unilateral hearing loss. This study reports for the first time to our knowledge, using a whole‐head neuromagnetometer with monaural stimulation of both intact and affected ears, a pattern of healthy‐side dominance for cortical neuromagnetic responses in adult patients in the early stage of idiopathic sudden sensorineural hearing loss, and a pattern of contralateral dominance is verified in controls. Ann Neurol 2003;53:810–815
Antiepileptic drugs as a possible neuroprotective strategy in brain ischemia
Authors: Calabresi Paolo , Cupini Letizia M. , Centonze Diego , Pisani Francesco , Bernardi Giorgio
Published: May 2003

Several new antiepileptic drugs (AEDs) have been introduced for clinical use recently. These new AEDs, as did the classic AEDs, target multiple cellular sites both pre‐ and postsynaptically. The major common goal of the pharmacological treatment using AEDs is to counteract abnormal brain excitability by either decreasing excitatory transmission or enhancing neuronal inhibition. Interestingly, an excessive release of excitatory amino acids and a reduced neuronal inhibition also occur in brain ischemia. Thus, recently, the use of AEDs as a possible neuroprotective strategy in brain ischemia is receiving increasing attention, and many AEDs have been tested in animal models of stroke, providing encouraging results. Experimental studies utilizing global or focal ischemia in rodents have provided insights into the possible neuroprotective action of the various AEDs. However, the implication of these studies in the treatment of acute stroke in humans is not always direct. In fact, various clinical studies with drugs targeting the same voltage‐ and ligand‐gated channels modulated by most of the AEDs failed to show neuroprotection. The differential mechanisms that underlie the development of focal ischemic injury in experimental animal models versus human stroke require further investigation to open a new therapeutic perspective for neuroprotection that might be applicable in the future. Ann Neurol 2003;53:693–702
N19S, a new SOD1 mutation in sporadic amyotrophic lateral sclerosis: No evidence for disease causation
Authors: Mayeux Véronique , Corcia Philippe , Besson Gérard , Jafari‐Schluep Hélène‐Farnase , Briolotti Valérie , Camu William ,   the French ALS Research Group
Published: May 2003

In a group of 331 sporadic amyotrophic lateral sclerosis (ALS) cases, we identified a new Cu/Zn superoxide dismutase single base substitution, N19S, in two patients. In the first case, seven healthy family members of 15 carried the substitution. Controls (n = 268) and familial ALS index cases (n = 180) were screened and one control subject with N19S was identified. Our data show that, despite a possible role of susceptibility factor for ALS, N19S alone cannot be considered as a direct cause for the disease. Ann Neurol 2003;53:815–818
Elevated osteopontin levels in active relapsing‐remitting multiple sclerosis
Authors: Vogt Mario H. J. , Lopatinskaya Luba , Smits Monique , Polman Chris H. , Nagelkerken Lex
Published: May 2003

In the search for proteins that might play a role in the pathogenesis of multiple sclerosis (MS), osteopontin (OPN) has been identified as the most prominent cytokine‐encoding gene expressed within MS lesions. Here, we report significantly increased OPN protein levels in plasma of relapsing‐remitting MS patients. In contrast, OPN protein levels in primary progressive and secondary progressive MS patients were similar to healthy control levels. Interestingly, active relapsing‐remitting patients had higher OPN protein levels than patients without relapses. Ann Neurol 2003;53:819–822
Polyamide‐reinforced ethylene–propylene–diene rubber compatibilized with chlorinated polyethylene
Authors: Liu Xin , Huang Hua , Zhang Yong , Zhang Yinxi
Published: May 2003

Vulcanizates of blends of ethylene–propylene–diene rubber and polyamide copolymers were prepared by reactive compatibilization. A reactive route was employed for compatibilizing these blends with the addition of chlorinated polyethylene (CPE). The influence of the compatibilizers, crosslinking agents, blend compositions, and addition modes of the compatibilizers on the mechanical properties of the blends was investigated. The morphologies of the blends were determined with scanning electron microscopy. The addition of CPE was found to reduce the particle size of the dispersed phase remarkably. The stability of the blends with compatibilizers was measured by high‐temperature thermal aging. The mechanical properties were examined by stress–strain measurements and dynamic mechanical thermal measurements; the addition of polyamide copolymers caused significant improvements in the tensile properties of these blends.© 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 1727–1736, 2003
Examination of selected synthesis and room‐temperature storage parameters for wood adhesive‐type urea–formaldehyde resins by 13C‐NMR spectroscopy. V
Authors: Kim Moon G. , Young No B. , Lee Sang M. , Nieh World L.
Published: May 2003

The effects of posttreatments of particleboard adhesive‐type urea–formaldehyde resins were studied. The resins were synthesized with formaldehyde/first urea (F/U1) mol ratios of 1.40, 1.60, 1.80, 2.10, and 2.40 and then the second urea was added to give a final formaldehyde/urea ratio of 1.15 in alkaline pH. The resins were posttreated at 60°C for up to 13.5 h and the 2.5‐h heat‐treated resin samples were stored at room temperature for up to 27 days. Resins sampled during the posttreatments were examined by 13C‐NMR and evaluated by bonding particleboards. In the posttreatments, hydroxymethyl groups on the polymeric resin components dissociated to formaldehyde and reacted with the second urea, and methylene and methylene–ether groups were formed from reactions involving the second urea. Methylene–diurea and urea groups bonded to UF polymers were identified. As a result, the viscosity of the resins initially decreased but later increased along with the cloudiness of the resins. Bond‐strength and formaldehyde‐emission values of particleboard varied with posttreatment variables as well as with the F/U1 mol ratios used in the resin syntheses. The results would be useful in optimizing resin synthesis and handling parameters. Various reaction mechanisms were considered. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 1896–1917, 2003
Highly filled blends of a vinylic copolymer with plasticized lignin: Thermal and mechanical properties
Authors: Feldman D. , Banu D. , Manley R. St. J. , Zhu H.
Published: May 2003

The objective of this study was the development of new vinyl flooring formulations with increased resistance to attack by fungi and microorganisms, formulated with plasticizers having chemical compositions different from that of common dioctyl phthalate (DOP). Alkyl phthalate plasticizers are considered to be toxicological and ecotoxicological hazards, although this is still under debate. It is suspected that during the service life of poly(vinyl chloride) (PVC) flooring, the attack of fungi and microorganisms leads to the degradation of DOP and the release of some volatile organic compounds. For this reason, in the new flooring formulations, the vinyl chloride/vinyl acetate copolymer (VC–VAc) was partially replaced with lignin, a natural polymer and a major component of wood and vascular plants. Besides its other functions in wood, lignin imparts resistance to microorganisms. An organosolv lignin from Alcell Technologies, Inc. (AL), was used as a partial replacement of PVC. The influence of the new plasticizers, as well as the influence of the partial replacement of VC–VAc with lignin, on the morphology and thermal and mechanical properties of the composites was investigated with scanning electron microscopy, differential scanning calorimetry, and tensile testing. Butyl benzyl phthalate and diethylene glycol dibenzoate were used as plasticizers; both were compatible with PVC and AL. The results indicated that diethylene glycol dibenzoate was the best plasticizer for these blend composites. In these formulations, AL could replace up to 20 parts of the copolymer. At this level of replacement, the key mechanical properties of the new composites compared very favorably with those of the DOP control formulations. The obtained formulation will be tested further for resistance to fungi and microorganisms. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 2000–2010, 2003
Synthesis of novel polyimides containing side‐chain azo‐2‐naphthol moieties
Authors: Mallakpour Shadpour , Hajipour Abdol‐Reza , Vahabi Ramin
Published: May 2003

An amine‐ester derivative of isoeugenol was prepared in three steps. This amine‐ester was converted to diazonium salt and subsequently was reacted with 2‐naphthol and a novel isoeugenol ester‐azo derivative as a new monomer was obtained in quantitative yield. This monomer was characterized by high‐field 1H‐NMR, IR, and elemental analysis and then was used for the preparation of model compound and polymerization reactions. 4‐Phenyl‐1,2,4‐triazoline‐3,5‐dione was allowed to react with this new monomer. The reaction was very fast and gave only one double adduct by Diels–Alder and ene pathways in excellent yield. The polymerization reactions of novel monomer with bistriazolinediones [bis(p‐3,5‐dioxo‐1,2,4‐triazolin‐4‐ylphenyl)methane and 1,6‐bis(3,5‐dioxo‐1,2,4‐triazolin‐4‐yl)hexane] were carried out in N,N‐dimethylacetamide at room temperature. The reactions were exothermic, fast, and gave novel heterocyclic polyimides by repetitive Diels–Alder‐ene polyaddition reactions. Some structural characterization and physical properties of these novel heterocyclic polyimides are reported. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 1942–1951, 2003
Developmental skeletal anomalies
Authors: Morcuende Jose A. , Weinstein Stuart L.
Published: May 2003

A genetic and molecular revolution is taking place in medicine today. Led by the Human Genome Project, genetic information and concepts are changing the way diseases are defined, diagnoses are made, and treatment strategies are developed. The profound implications of actually understanding the molecular abnormalities of many clinical problems are affecting virtually all medical and surgical disciplines. The ability to apply knowledge gleaned from the laboratory is our best hope for developing strategies to modify the pathologic effects of genes (by drug therapy), repair genes (gene therapy), and restore lost or affected tissues (tissue engineering). Instead of an empiric trial‐and‐error approach to therapy, it may become feasible to tailor treatment to the specific molecular malfunction. In this review we have chosen to emphasize a few selected musculoskeletal disorders, including skeletal dysplasias, spinal deformities, developmental dislocation of the hip, and idiopathic clubfoot. The logical extension of our understanding of the molecular players in many of these disorders is to establish precisely what the products of the affected genes do during skeletal development, and how mutations disturb these functions to produce the characteristic phenotype. Despite the many hypotheses generated from the work in human genetics, and the knowledge that has been gained from animal models, there remains a relatively poor understanding of how these genes interfere with skeletal development. Unraveling these mysteries and defining them in molecular and cellular terms will be the challenges for the near future. Birth Defects Research (Part C) 69:197–207, 2003. © 2003 Wiley‐Liss, Inc.
Physiology and pathophysiology of the growth plate
Authors: Ballock R. Tracy , O'Keefe Regis J.
Published: May 2003

Longitudinal growth of the skeleton is a result of endochondral ossification that occurs at the growth plate. Through a sequential process of cell proliferation, extracellular matrix synthesis, cellular hypertrophy, matrix mineralization, vascular invasion, and eventually apoptosis, the cartilage model is continually replaced by bone as length increases. The regulation of longitudinal growth at the growth plate occurs generally through the intimate interaction of circulating systemic hormones and locally produced peptide growth factors, the net result of which is to trigger changes in gene expression by growth plate chondrocytes. This review highlights recent advances in genetics and cell biology that are illuminating the important regulatory mechanisms governing the structure and biology of the growth plate, and provides selected examples of how studies of human mutations have yielded a wealth of new knowledge regarding the normal biology and pathophysiology of growth plate cartilage. Birth Defects Research (Part C) 69:123–143, 2003. © 2003 Wiley‐Liss, Inc.
Molecular ontogeny of the skeleton
Authors: Eames B. Frank , De La Fuente Luis , Helms Jill A.
Published: May 2003

From a traditional viewpoint, skeletal elements form by two distinct processes: endochondral ossification, during which a cartilage template is replaced by bone, and intramembranous ossification, whereby mesenchymal cells differentiate directly into osteoblasts. There are inherent difficulties with this historical classification scheme, not the least of which is that bones typically described as endochondral actually form bone through an intramembranous process, and that some membranous bones may have a transient chondrogenic phase. These innate contradictions can be circumvented if molecular and cellular, rather than histogenic, criteria are used to describe the process of skeletal tissue formation. Within the past decade, clinical examinations of human skeletal syndromes have led to the identification and subsequent characterization of regulatory molecules that direct chondrogenesis and osteogenesis in every skeletal element of the body. In this review, we survey these molecules and the tissue interactions that may regulate their expression. What emerges is a new paradigm, by which we can explain and understand the process of normal –and abnormal‐ skeletal development. Birth Defects Research (Part C) 69:93–101, 2003. © 2003 Wiley‐Liss, Inc.
Experimental investigation of the external nitrification biological nutrient removal activated sludge (ENBNRAS) system
Authors: Hu Zhi‐rong , Sötemann S. , Moodley R. , Wentzel M. C. , Ekama G. A.
Published: May 2003

A systematic lab‐scale experimental investigation is reported for the external nitrification (EN) biological nutrient removal (BNR) activated sludge (ENBNRAS) system, which is a combined fixed and suspended medium system. The ENBNRAS system was proposed to intensify the treatment capacity of BNR‐activated sludge (BNRAS) systems by addressing two difficulties often encountered in practice: (a) the long sludge age for nitrification requirement; and (b) sludge bulking. In the ENBNRAS system, nitrification is transferred from the aerobic reactor in the suspended medium activated sludge system to a fixed medium nitrification system. Thus, the sludge age of the suspended medium activated sludge system can be reduced from 20 to 25 days to 8 to 10 days, resulting in a decrease in reactor volume per ML wastewater treated of about 30%. Furthermore, the aerobic mass fraction can also be reduced from 50% to 60% to <30% and concommitantly the anoxic mass fraction can be increased from 25% to 35% to >55% (if the anaerobic mass fraction is 15%), and thus complete denitrification in the anoxic reactors becomes possible. Research indicates that both the short sludge age and complete denitrification could ameliorate anoxic aerobic (AA) or low food/microorganism (F/M) ratio filamentous bulking, and hence reduce the surface area of secondary settling tanks or increase the treatment capacity of existing systems. The lab‐scale experimental investigations indicate that the ENBNRAS system can obtain: (i) very good chemical oxygen demand (COD) removal, even with an aerobic mass fraction as low as 20%; (ii) high nitrogen removal, even for a wastewater with a high total kjeldahl nitrogen (TKN)/COD ratio, up to 0.14; (iii) adequate settling sludge (diluted sludge volume index [DSVI] <100 mL/g); and (iv) a significant reduction in oxygen demand. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 260–273, 2003.
Expression of a synthetic copy of the bovine chymosin gene in Aspergillus awamori from constitutive and pH‐regulated promoters and secretion using two different pre‐pro sequences
Authors: Cardoza R. E. , Gutiérrez S. , Ortega N. , Colina A. , Casqueiro J. , Martín J. F.
Published: May 2003

A copy of the bovine chymosin gene (chy) with a codon usage optimized for its expression in Aspergillus awamori was constructed starting from synthetic oligonucleotides. To study the ability of this filamentous fungus to secrete bovine prochymosin, two plasmids were constructed in which the transcriptional, translational, and secretory control regions of the A. nidulans gpdA gene and pepB genes were coupled to either preprochymosin or prochymosin genes. Secretion of a protein enzymatically and immunologically indistinguishable from bovine chymosin was achieved in A. awamori transformants with each of these constructions. In all cases, the primary translation product (40.5 kDa) was self‐processed to a mature chymosin polypeptide having a molecular weight of 35.6 kDa. Immunological assays indicated that most of the chymosin was secreted to the extracellular medium. Hybridization analysis of genomic DNA from chymosin transformants showed chromosomal integration of prochymosin sequences and, in some transformants, multiple copies of the expression cassettes were observed. Expression from the gpdA promoter was constitutive, whereas expression from the pepB promoter was strongly influenced by pH. A very high expression from the pepB promoter was observed during the growth phase. The A. awamori pepB gene terminator was more favorable for chymosin production than the S. cerevisiae CYC1 terminator. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 249–259, 2003.
Lipase‐catalyzed ethanolysis of fish oils: Multi‐response kinetics
Authors: Torres Carlos F. , Moeljadi Marlina , Hill Charles G.
Published: May 2003

The kinetics of the lipase‐catalyzed (Pseudomonas cepacia) ethanolysis of fish oil has been studied in a batch reactor using menhaden oil, tuna oil, and acylglycerol mixtures derived from menhaden oil. Multi‐response models derived from a generalized Michaelis–Menten mechanism were developed to describe the rates of formation of ethyl esters of the primary fatty acids present in the precursor oil. A first‐order model for deactivation of the lipase was fit simultaneously to one of the data sets. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 274–281, 2003.
Kinetic modeling of the light‐dependent photosynthetic activity of the green microalga Chlorella vulgaris
Authors: Yun Yeoung‐Sang , Park Jong Moon
Published: May 2003

Light‐dependent photosynthesis of Chlorella vulgaris was investigated by using a novel photosynthesis measurement system that could cover wide ranges of incident light and cell density and reproduce accurate readings. Various photosynthesis models, which have been reported elsewhere, were classified and/or reformulated based upon the underlying hypotheses of the light dependence of the algal photosynthesis. Four types of models were derived, which contained distinct light‐related variables such as the average or local photon flux density (APFD or LPFD) and the average or local photon absorption rate (APAR or LPAR). According to our experimental results, the LPFD and LPAR models could predict the experimental data more accurately although the APFD and APAR models have been widely used for the kinetic study of microalgal photosynthesis. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 303–311, 2003.
Inhibitory effect of carbon dioxide on the fed‐batch culture of Ralstonia eutropha: Evaluation by CO2 pulse injection and autogenous CO2 methods
Authors: Shang Longan , Jiang Min , Ryu Chul Hee , Chang Ho Nam , Cho Soon Haeng , Lee Jong Won
Published: May 2003

In order to see the effect of CO2 inhibition resulting from the use of pure oxygen, we carried out a comparative fed‐batch culture study of polyhydroxybutyric acid (PHB) production by Ralstonia eutropha using air and pure oxygen in 5‐L, 30‐L, and 300‐L fermentors. The final PHB concentrations obtained with pure O2 were 138.7 g/L in the 5‐L fermentor and 131.3 g/L in the 30‐L fermentor, which increased 2.9 and 6.2 times, respectively, as compared to those obtained with air. In the 300‐L fermentor, the fed‐batch culture with air yielded only 8.4 g/L PHB. However, the maximal CO2 concentrations in the 5‐L fermentor increased significantly from 4.1% (air) to 15.0% (pure O2), while it was only 1.6% in the 30‐L fermentor with air, but reached 14.2% in the case of pure O2. We used two different experimental methods for evaluating CO2 inhibition: CO2 pulse injection and autogenous CO2 methods. A 10 or 22% (v/v) CO2 pulse with a duration of 3 or 6 h was introduced in a pure‐oxygen culture of R. eutropha to investigate how CO2 affects the synthesis of biomass and PHB. CO2 inhibited the cell growth and PHB synthesis significantly. The inhibitory effect became stronger with the increase of the CO2 concentration and pulse duration. The new proposed autogenous CO2 method makes it possible to place microbial cells under different CO2 level environments by varying the gas flow rate. Introduction of O2 gas at a low flow rate of 0.42 vvm resulted in an increase of CO2 concentration to 30.2% in the exit gas. The final PHB of 97.2 g/L was obtained, which corresponded to 70% of the PHB production at 1.0 vvm O2 flow rate. This new method measures the inhibitory effect of CO2 produced autogenously by cells through the entire fermentation process and can avoid the overestimation of CO2 inhibition without introducing artificial CO2 into the fermentor. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 312–320, 2003.
Model‐based analysis of anaerobic acetate uptake by a mixed culture of polyphosphate‐accumulating and glycogen‐accumulating organisms
Authors: Zeng Raymond J. , Yuan Zhiguo , Keller Jürg
Published: May 2003

An increasing number of studies shows that the glycogen‐accumulating organisms (GAOs) can survive and may indeed proliferate under the alternating anaerobic/aerobic conditions found in EBPR systems, thus forming a strong competitor of the polyphosphate‐accumulating organisms (PAOs). Understanding their behaviors in a mixed PAO and GAO culture under various operational conditions is essential for developing operating strategies that disadvantage the growth of this group of unwanted organisms. A model‐based data analysis method is developed in this paper for the study of the anaerobic PAO and GAO activities in a mixed PAO and GAO culture. The method primarily makes use of the hydrogen ion production rate and the carbon dioxide transfer rate resulting from the acetate uptake processes by PAOs and GAOs, measured with a recently developed titration and off‐gas analysis (TOGA) sensor. The method is demonstrated using the data from a laboratory‐scale sequencing batch reactor (SBR) operated under alternating anaerobic and aerobic conditions. The data analysis using the proposed method strongly indicates a coexistence of PAOs and GAOs in the system, which was independently confirmed by fluorescent in situ hybridization (FISH) measurement. The model‐based analysis also allowed the identification of the respective acetate uptake rates by PAOs and GAOs, along with a number of kinetic and stoichiometric parameters involved in the PAO and GAO models. The excellent fit between the model predictions and the experimental data not involved in parameter identification shows that the parameter values found are reliable and accurate. It also demonstrates that the current anaerobic PAO and GAO models are able to accurately characterize the PAO/GAO mixed culture obtained in this study. This is of major importance as no pure culture of either PAOs or GAOs has been reported to date, and hence the current PAO and GAO models were developed for the interpretation of experimental results of mixed cultures. The proposed method is readily applicable for detailed investigations of the competition between PAOs and GAOs in enriched cultures. However, the fermentation of organic substrates carried out by ordinary heterotrophs needs to be accounted for when the method is applied to the study of PAO and GAO competition in full‐scale sludges. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 293–302, 2003.
Effects of buffering conditions and culture pH on production rates and glycosylation of clinical phase I anti‐melanoma mouse IgG3 monoclonal antibody R24
Authors: Müthing Johannes , Kemminer Sven E. , Conradt Harald S. , Šagi Dijana , Nimtz Manfred , Kärst Uwe , Peter‐Katalinić Jasna
Published: May 2003

R24, a mouse IgG3 monoclonal antibody (MAb) against ganglioside GD3 (Neu5Acα8Neu5Acα3Gal β4Glcβ1Cer), can block tumor growth as reported in a series of clinical trials in patients with metastatic melanoma. The IgG molecule basically contains an asparagine‐linked biantennary complex type oligosaccharide on the CH2 domain of each heavy chain, which is necessary for its in vivo effector function. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important MAb in CO2/HCO3− (pH 7.4, 7.2, and 6.9) and HEPES buffered serum‐free medium. Growth, metabolism, and IgG production of hybridoma cells (ATCC HB‐8445) were analyzed on a 2‐L bioreactor scale using fed‐batch mode. Specific growth rates (μ) and MAb production rates (qIgG) varied significantly with maximum product yields at pH 6.9 (qIgG = 42.9 μg 10−6 cells d−1, μ = 0.30 d−1) and lowest yields in pH 7.4 adjusted batches (qIgG = 10.8 μg 10−6 cells d−1, μ = 0.40 d−1). N‐glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD), matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF), and electrospray‐ionization quadrupole time‐of‐flight (ESI‐QTOF) mass spectrometry (MS). The highest relative amounts of agalacto and monogalacto biantennary complex type oligosaccharides were detected in the pH 7.2 (46% and 38%, respectively) and pH 6.9 (44% and 40%, respectively) cultivations and the uppermost quantities of digalacto (fully galactosylated) structures in the pH 7.4 (32%) and the HEPES (26%) buffered fermentation. In the experiments with HEPES buffering, antibodies with a molar Neu5Ac/Neu5Gc ratio of 3.067 were obtained. The fermentations at pH 7.2 and 6.9 resulted in almost equal molar Neu5Ac/Neu5Gc ratios of 1.008 and 0.985, respectively, while the alkaline shift caused a moderate overexpression of Neu5Ac deduced from the Neu5Ac/Neu5Gc quotient of 1.411. Different culture buffering gave rise to altered glycosylation pattern of the MAb R24. Consequently, a detailed molecular characterization of MAb glycosylation is generally recommended as a part of the development of MAbs for targeted in vivo immunotherapy to assure biochemical consistency of product lots and oligosaccharide‐dependent biological activity. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 321–334, 2003.
Properties of a novel magnetized alginate for magnetic resonance imaging
Authors: Shen Feng , Poncet‐Legrand Celine , Somers Sat , Slade Andrea , Yip Christopher , Duft Andy M. , Winnik Françoise M. , Chang Patricia L.
Published: May 2003

Implanting recombinant cells encapsulated in alginate microcapsules to secrete therapeutic proteins has been proven clinically effective in treating several murine models of human diseases. However, once implanted, these microcapsules cannot be assessed without invasive surgery. We now report the preparation and characterization of a novel ferrofluid to render these microcapsules visible with magnetic resonance imaging (MRI). The ferrofluid was prepared as a colloidal iron oxide stabilized in water by alginate. The presence of iron particles in the ferrofluid was verified with chemical titration, dynamic light scattering, and magnetization measurement. The microcapsules fabricated with various concentrations of the ferrofluid in the core, or on the surface of alginate microcapsules, or both, all produced microcapsules with smooth surfaces as shown with light and scanning electron microscopy. However, at the nanoscale level, as revealed with atomic force microscopy, the ferrofluid‐fabricated microcapsules demonstrated increased granularity, particularly when the ferrofluid was used to laminate the surface. From the force spectroscopy measurements, these modified microcapsules showed increasing surface rigidity in the following order: traditional alginate < ferrofluid in the core < ferrofluid on the surface. Although the mechanical stability of low‐concentration ferrofluid (0.1%) microcapsules was reduced, increasing concentrations, up to 20%, were able to improve stability. When these ferrofluid microcapsules were examined with MRI, their T2 relaxation time was reduced, thereby producing increased contrast readily detectable with MRI, whereas the traditional alginate microcapsules showed no difference when compared with water. In conclusion, such ferrofluid‐enhanced alginate is suitable for fabricating microcapsules that offer the potential for in vivo tracking of implanted microcapsules without invasive surgery. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 282–292, 2003.
Production of savinase and population viability of Bacillus clausii during high‐cell‐density fed‐batch cultivations
Authors: Christiansen Torben , Michaelsen Søren , Wümpelmann Mogens , Nielsen Jens
Published: May 2003

The growth and product formation of a Savinase‐producing Bacillus clausii were investigated in high‐cell‐density fed‐batch cultivations with both linear and exponential feed profiles. The highest specific productivity of Savinase was observed shortly after the end of the initial batch phase for all feed profiles applied and, in addition, there was a time‐dependent decrease in specific productivity. The specific glucose uptake rate increased with time for constant specific growth rate indicating that the maintenance requirements increased with time, possibly due to a decreasing K+ concentration. The physiological state of the cells was monitored during the cultivations using a flow cytometry assay based on the permeability of the cell membrane to propidium iodide. In the latter parts of the fed‐batch cultures with a linear feed profile, a large portion of the cell population was found to have a permeable membrane, indicating a large percentage of dead cells. By assuming that only cells with a nonpermeable membrane contributed to growth and product formation, the physiological properties of this subpopulation were calculated. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 344–352, 2003.
Multicriteria optimization of biochemical systems by linear programming: Application to production of ethanol by Saccharomyces cerevisiae
Authors: Vera Julio , De Atauri Pedro , Cascante Marta , Torres Néstor V.
Published: May 2003

In this study we present a method for simultaneous optimization of several metabolic responses of biochemical pathways. The method, based on the use of the power law formalism to obtain a linear system in logarithmic coordinates, is applied to ethanol production by Saccharomyces cerevisiae. Starting from an experimentally based kinetic model, we translated it to its power law equivalent. With this new model representation, we then applied the multiobjective optimization method. Our intent was to maximize ethanol production and minimize each of the internal metabolite concentrations. To ensure cell viability, all optimizations were carried out under imposed constraints. The different solutions obtained, which correspond to alternative patterns of enzyme overexpression, were implemented in the original model. We discovered few discrepancies between the S‐system‐optimized steady state and the corresponding optimized state in the original kinetic model, thus demonstrating the suitability of the S‐system representation as the basis for the optimization procedure. In all optimized solutions, the ATP level reached its maximum and any increase in its activity positively affected the optimization process. This work illustrates that in any optimization study no single criteria is of general application being the multiobjective and constrained task the proper way to address it. It is concluded that the proposed multiobjective method can serve to carry out, in a single study, the general pattern of behavior of a given metabolic system with regard to its control and optimization. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 335–343, 2003.
Continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus: Growth yields and morphological characterization
Authors: Robin J. , Lettier G. , McIntyre M. , Noorman H. , Nielsen J.
Published: May 2003

The growth stoichiometry of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus was determined in glucose‐limited chemostat cultivations using a chemically defined medium. This strain produces adipoyl‐7‐aminodeacetoxycephalosporanic acid (ad‐7‐ADCA) when it is fed with adipic acid. The biomass yield and maintenance coefficients for the strain were similar to those found for penicillin‐producing strains of Penicillium chrysogenum. The maximum specific growth rate in the chemostat was found to be 0.11 h−1. Metabolic degradation of adipate was found to take place in significant amounts only at dilution rates below 0.03 h−1. After three to five residence times, adipate degradation and ad‐7‐ADCA production disappeared, and this allowed determination of the biomass yield coefficient on adipate. The morphology was measured at different dilution rates and the mean total hyphal length and mean number of tips both increased with an increase in dilution rate from 0.015 to 0.065 h−1. Both variables decreased when the dilution rate was increased above 0.065 h−1. A correlation between mean total hyphal length and productivity of ad‐7‐ADCA was found. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 361–368, 2003.
Continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus: Kinetics of adipoyl‐7‐aminodeacetoxycephalosporanic acid and byproduct formations
Authors: Robin J. , Bruheim P. , Nielsen M. L. , Noorman H. , Nielsen J.
Published: May 2003

The production kinetics of a transformed strain of Penicillium chrysogenum expressing the expandase gene from Streptomyces clavuligerus was investigated in chemostat cultivations. The recombinant strain produces adipoyl‐7‐aminodeacetoxycephalosporanic acid (ad‐7‐ADCA) as the major product; however, during the cultivations, the appearance of a major unknown and poorly secreted product was observed. Investigations using high‐performance liquid chromatography (HPLC) and liquid chromatography–mass spectroscopy (LC‐MS) showed that this byproduct has a six‐membered dihydrothiazine ring, which is characteristic for cephalosporins. The byproduct may be formed via isopenicillin N by as‐yet unknown mechanisms, but involving expandase. It is likely that the unknown compound (UC) is deacetoxycephalosporin C (DAOC). Investigation of the instability of the various β‐lactams produced showed higher instability for compounds with a five‐membered thiazolidine ring than those with a six‐membered dihydrothiazine ring. Furthermore, secretion of products and byproducts was shown to be quite different. The productivity was studied as a function of the dilution rate in the range 0.015 to 0.090 h−1. The specific productivity of total β‐lactams was compared with that of the penicillin‐G‐producing host strain, and it was found to be lower at dilution rates of <0.06 h−1. Quantification of the fluxes through the pathway leading to ad‐7‐ADCA showed a decrease in flux toward ad‐7‐ADCA, and an increase in flux toward UC as the dilution rate increased. Northern analysis of the biosynthetic genes showed that expression of the enzymes involved in the ad‐7‐ADCA pathway decreased as the dilution rate increased. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 353–360, 2003.
Synthesis, Receptor Binding, Molecular Modeling, and Proliferative Assays of a Series of 17α‐Arylestradiols
Authors: Foy Nicolas , Stéphan Elie , Vessières Anne , Salomon Emmanuel , Heldt Jan‐Martin , Huché Michel , Jaouen Gérard
Published: June 2003

A series of new derivatives of estradiol substituted at position 17α by various aryls has been synthesized. This was made possible by efficient activation methods for the addition of aryllithiums to the carbonyl group at position 17 of estrone by using tetramethylethylenediamine (TMEDA) or BF3⋅OEt2. Their relative binding affinity (RBA) for the α and the β forms of the estrogen receptor (ER) have been measured. All except one of the compounds synthesized had an RBA value of around 10 % which indicates a level of tolerance towards the bulky substituent at position 17. The lipophilicity values measured for these compounds are higher than that found for estradiol (E2). A study of their proliferative/antiproliferative effects was carried out on hormone‐dependent (MCF7) and hormone‐independent (MDA‐MB231) breast cancer cell lines. It is interesting to note that all the compounds are estrogenic. The possibility of easily attaching an iodine at the end of a phenyl spacer opens up a route to new radiopharmaceuticals for use in radioimaging.
Simplified Synthetic TMC‐95A/B Analogues Retain the Potency of Proteasome Inhibitory Activity
Authors: Yang Zhi‐Qiang , Kwok Benjamin H. B. , Lin Songnian , Koldobskiy Michael A. , Crews Craig M. , Danishefsky Samuel J.
Published: June 2003

The proteasome regulates diverse intracellular processes, including cell‐cycle progression, antigen presentation, and inflammatory response. Selective inhibitors of the proteasome have great therapeutic potential for the treatment of cancer and inflammatory disorders. Natural cyclic peptides TMC‐95A and B represent a new class of noncovalent, selective proteasome inhibitors. To explore the structure–activity relationship of this class of proteasome inhibitors, a series of TMC‐95A/B analogues were prepared and analyzed. We found that the unique enamide functionality at the C8 position of TMC‐95s can be replaced with a simple allylamide. The asymmetric center at C36 that distinguishes TMC‐95A from TMC‐95B but which necessitates a complicated separation of the two compounds can be eliminated. Therefore, these findings could lead to the development of more accessible simple analogues as potential therapeutic agents.
Relative Stability of Major Types of β‐Turns as a Function of Amino Acid Composition: A Study Based on Ab Initio Energetic and Natural Abundance Data
Authors: Perczel András , Jákli Imre , McAllister Michael A. , Csizmadia Imre G.
Published: June 2003

Folding properties of small globular proteins are determined by their amino acid sequence (primary structure). This holds both for local (secondary structure) and for global conformational features of linear polypeptides and proteins composed from natural amino acid derivatives. It thus provides the rational basis of structure prediction algorithms. The shortest secondary structure element, the β‐turn, most typically adopts either a type I or a type II form, depending on the amino acid composition. Herein we investigate the sequence‐dependent folding stability of both major types of β‐turns using simple dipeptide models (‐Xxx‐Yyy‐). Gas‐phase ab initio properties of 16 carefully selected and suitably protected dipeptide models (for example Val‐Ser, Ala‐Gly, Ser‐Ser) were studied. For each backbone fold most probable side‐chain conformers were considered. Fully optimized 321G RHF molecular structures were employed in medium level [B3LYP/6‐311++G(d,p)//RHF/3‐21G] energy calculations to estimate relative populations of the different backbone conformers. Our results show that the preference for β‐turn forms as calculated by quantum mechanics and observed in Xray determined proteins correlates significantly.
Controlling Short‐ and Long‐Range Electron Transfer Processes in Molecular Dyads and Triads
Authors: Sánchez Luis , Pérez Ignacio , Martín Nazario , Guldi Dirk M.
Published: June 2003

Novel π‐extended tetrathiafulvalene (exTTF)‐based donor acceptor hybrids—dyads and triads—have been synthesized following a multistep synthetic procedure. Cyclic voltammetry and absorption spectroscopy, conducted in room temperature solutions, reveal features that are identical to the sum of the separate donor and acceptor moieties. Steady‐state and time‐resolved photolytic techniques confirm that upon photoexcitation of the fullerene chromophore, rapid (1.25×1010 s−1) and efficient (67 %) charge separation leads to long‐lived, charge‐separated radical pairs. Typical lifetimes for the dyad ensembles range between 54 and 460 ns, with the longer values found in more polar solvents. This indicates that the dynamics are located in the 'normal region' of the Marcus curve. In the triads, subsequent charge shifts transform the adjacent radical pair into the distant radical pair, for which we determined lifetimes of up to 111 μs in DMF—values never previously accomplished in molecular triads. In the final charge‐separated state, large donor‐acceptor separation (center‐to‐center distances: ≈30 Å) minimizes the coupling between reduced acceptor and oxidized donor. Analysis of the charge recombination kinetics shows that a stepwise mechanism accounts for the unusually long lifetimes.
Potassium Selective Calix[4]semitubes
Authors: Webber Philip R. A. , Cowley Andrew , Drew Michael G. B. , Beer Paul D.
Published: June 2003

A new class of ionophore consisting of two calix[4]arene units linked through the lower rim by two ethylene chains, in combination with propyl ether and phenolic functional groups, has been developed. These calix[4]semitube molecules exhibit remarkable selectivity and fast complexation kinetics for potassium over all Group 1 metal cations. Molecular modelling studies, using structural models derived from crystallographic data, suggest the potassium cation is complexed by a horizontal, side‐on route and not through the calix[4]arene annulus. The length of the bridging alkylene chain between the respective calix[4]arenes of the semitube structure dictates the strength and selectivity of alkali metal cation binding.
Facile Optical Resolution of tert‐Butanethiosulfinate by Molecular Complexation with (R)‐BINOL and Study of Chiral Discrimination of the Diastereomeric Complexes
Authors: Liao Jian , Sun Xiaoxia , Cui Xin , Yu Kaibei , Zhu Jin , Deng Jingen
Published: June 2003

An important synthon, tert‐butanethiosulfinate (2), has been effectively resolved by forming molecular complexes with (R)‐2,2′‐dihydroxy‐1,1′‐binaphthyl (BINOL, 3) in high enantioselectivity (>99 % ee). The present procedure represents the first example of the resolution of thiosulfinate. The mechanism of chiral discrimination is discussed in terms of molecular recognition based on IR and Xray analyses of the diastereomeric complexes during the resolution. In the less‐soluble complex, (R)‐3 and (R)‐2 self‐assembled as a linear supramolecule; however, in the more‐soluble complex, (R)‐3 and (S)‐2 formed a simple bimolecular complex by one stronger hydrogen bond. Hydrogen bonding is the major driving force for effective resolution.
Polymer‐Supported Triazenes as Smart Reagents for the Alkylation of Carboxylic Acids
Authors: Erb Bernhard , Kucma Jean‐Philippe , Mourey Sandrine , Struber Fritz
Published: June 2003

Starting from polystyrene, a simple four‐step synthesis of polymer‐supported alkyltriazenes (alkyl=Me, Et, benzyl) is described. With this synthesis, a loading capacity of 2.2 mmol g−1 can be reached. The most prominent application of these polymer‐supported reagents is the rapid, highly selective and high‐yielding esterification of carboxylic acids, which involves a simple “mix and filter off” procedure at room temperature. If stored in a refrigerator, these reagents are stable for many months and they can be recycled several times.
Phospholipase A2 Hydrolysis of Mixed Phospholipid Vesicles Formed on Polyelectrolyte Hollow Capsules
Authors: Ge Liqin , Möhwald Helmuth , Li Junbai
Published: June 2003

Mixtures of the phospholipids L‐α‐dimyristoylphosphatidic acid (DMPA) and L‐α‐dipalmitoylphosphatidylcholine (DPPC) have been successfully adsorbed onto the charged surface of multilayer polyelectrolyte capsules to form a novel vesicle. Leaving such vesicles in phospholipase A2 solution, we observed the hydrolysis reaction on the surface of the lipid/polymer vesicles and a permeability change before and after the reaction by confocal‐laser scanning microscopy (CLSM). A capsule with adjustable permeability was constructed. This method may provide new features for drug‐release vesicles.