bluuebunny/crossref_metadata_2025 huggingface dataset:
Adenoviral vector–mediated gene transfer of IL‐13Rα2 chain followed by IL‐13 cytotoxin treatment offers potent targeted therapy for cytotoxin‐resistant cancers
Authors: Saito Makoto , Murata Takashi , Watanabe Ken , Kawakami Koji , Suzuki Motoyoshi , Koji Takehiko , Puri Raj K. , Kitazato Kaio , Kobayashi Nobuyuki
Published: March 2005

Previous studies demonstrated that IL‐13Rα2 chain–overexpressing cancer cells were highly sensitive to IL‐13 cytotoxin (IL13‐PE38QQR) and could be targeted by cytotoxin treatment. However, the majority of human tumors do not express high levels of IL‐13Rα2 chain. To expand the IL‐13 cytotoxin–mediated cancer targeting therapy, we combined cytotoxin treatment with gene transfer of IL‐13Rα2 chain. We constructed a recombinant adenoviral vector carrying the human IL‐13Rα2 gene (Ad‐IL‐13Rα2), which expresses high levels of IL‐13Rα2 chain on infected cells. Human cancer cell lines A549 and HOS, which originally show no IL‐13Rα2 expression and little sensitivity to IL‐13 cytotoxin, were effectively converted to become sensitive to this cytotoxin after Ad‐IL‐13Rα2 infection. The CC50 of IL‐13 cytotoxin for Ad‐IL‐13Rα2‐infected A549 cells was <10 ng/ml, whereas the CC50 for uninfected or control vector‐infected cells was >500 ng/ml. We also examined the antitumor activity of IL‐13 cytotoxin in an established xenograft model of cytotoxin‐resistant human lung tumor. Only a single i.t. injection of Ad‐IL‐13Rα2 markedly enhanced the sensitivity of established tumors to IL‐13 cytotoxin treatment; furthermore, this antitumor effect was significantly sustained for more than 1 month after the last treatment with IL‐13 cytotoxin. Taken together, these results suggest the combination of adenoviral vector–mediated IL‐13Rα2 gene transfer and IL‐13 cytotoxin administration can be an effective targeting approach for several types of IL‐13 cytotoxin–resistant cancers which show no or little expression of IL‐13Rα2 chain. © 2005 Wiley‐Liss, Inc.
Analytical performance evaluation of a new turbidimetric immunoassay for valproic acid on the ADVIA® 1650 analyzer: effect of gross hemolysis and high bilirubin
Authors: Datta Pradip , Dasgupta Amitava
Published: March 2005

Valproic acid is an anticonvulsant that requires careful therapeutic drug monitoring. Valproic acid is also used in psychiatric patients. Bayer Diagnostics (Tarrytown, NY) recently marketed a turbidimetric immunoassay for monitoring valproic acid concentrations in serum or plasma using the ADVIA® 1650 analyzer. We evaluated the performance of this new assay by comparing it with a widely used fluorescence polarization immunoassay (FPIA) on the AxSYM® analyzer (Abbott Laboratories, Abbott Park, IL). The total coefficient of variation (CV) for the low control of this new assay was 6.8% (mean=30.7, SD=2.1 µg/mL, n=44) while the corresponding CVs for the medium and high controls were 3.3% (mean=81.0, SD=2.7 µg/mL, n=44) and 5.9% (mean=142.9, SD=8.4 µg/mL, n=44), respectively. The assay is linear up to a serum valproic acid concentration of 170 µg/mL, and the detection limit is 4.4 µg/mL. We observed an excellent correlation between the FPIA of valproic acid and the turbidimetric assay using specimens from 52 different patients who were receiving valproic acid. Using the valproic acid concentrations obtained by the FPIA as the x‐axis, and the corresponding valproic acid concentrations obtained by the turbidimetric assay as the y‐axis, we developed the following regression equation: y=1.03 x+1.55 (r=0.98). With this new assay, high concentrations of bilirubin (unconjugated 30 mg/dL and conjugated 30 mg/dL) and gross hemolysis (4+, hemoglobin: 1,500 mg/dL) have no effect on measurements of valproic acid concentration. We conclude that the new turbidimetric assay for valproic acid can be used for routine therapeutic drug monitoring of valproic acid in clinical laboratories. J. Clin. Lab. Anal. 19:31–35, 2005. © 2005 Wiley‐Liss, Inc.
A study on the adsorption behavior of protein onto functional microspheres
Authors: Li Songjun , Hu Jie , Liu Bailing
Published: March 2005

Adsorption has been investigated using bovine serum albumin (BSA) as the model protein and sulfonated microspheres as the matrix. The adsorption appears to be sensitive to time, pH, and ionic strength, and the presence of sulfonate groups can play important roles in this process, increasing the adsorption rate and the amount of protein adsorbed, as well as influencing the interaction of BSA molecules. Fittings from Langmuir's and Freundlich's isotherms indicate that the actual adsorption becomes more complicated than the ideal process, due to the presence of intermolecular interactions of BSA. The determinations under different pH conditions (pH 2.2, 4.3, 7.4) show that near the isoelectric point of BSA (pI 4.7), the amount of protein adsorbed reached a maximum value, and a higher or lower pH resulted in a significant decrease in amount adsorbed. The effect of ionic strength is, however, closely associated with the operating conditions. Copyright © 2005 Society of Chemical Industry
Poor intercellular transport and absence of enhanced antiproliferative activity after non‐viral gene transfer of VP22‐P53 or P53‐VP22 fusions into p53 null cell lines in vitro or in vivo
Authors: Zavaglia David , Lin Erh‐Hsuan , Guidetti Mélanie , Pluquet Olivier , Hainaut Pierre , Favrot Marie‐Christine , Coll Jean‐luc
Published: March 2005

Background

The herpes simplex virus type 1 (HSV‐1) VP22 protein has the property to mediate intercellular trafficking of heterologous proteins fused to its C‐ or N‐terminus. We have previously shown improved delivery and enhanced therapeutic effect in vitro and in vivo with a P27‐VP22 fusion protein. In this report, we were interested in studying the spread and biological activity of VP22 fused to the P53 tumor suppressor.

Methods

Expression of the VP22‐P53 and P53‐VP22 fusion proteins was shown by Western blot and intercellular spreading was monitored by immunofluorescence on transiently transfected cells. In vitro antiproliferative activity of wild‐type (wt) P53 and P53‐VP22 was assessed by proliferation assays and transactivating ability was studied by a reporter gene test and a gel‐shift assay. Antitumor activity was also tested in vivo by intratumoral injections of naked DNA in a model of subcutaneous tumors implanted in nude mice.

Results

Our results show that the C‐terminal fusion or the N‐terminal P53‐VP22 fusion proteins are not able to spread as efficiently as VP22. Moreover, we demonstrate that VP22‐P53 does not possess any transactivating ability. P53‐VP22 has an antiproliferative activity, but this activity is not superior to the one of P53 alone, in vitro or in vivo.

Conclusions

Our study indicates that a gene transfer strategy using VP22 cannot be considered as a universal system to improve the delivery of any protein. Copyright © 2005 John Wiley & Sons, Ltd.
A QSAR‐modeling perspective on cationic transfection lipids. 1. Predicting efficiency and understanding mechanisms
Authors: Horobin Richard W. , Weissig Volkmar
Published: March 2005

Background

As gene therapy using viral vectors involves clinical risks, limited DNA‐carrying capacity, and manufacturing problems, non‐viral vectors, including cationic lipids, have been investigated. Unfortunately, these agents have significantly lower transfectional ability and, due to the complexity of the transfectional pathway, no general schemes exist for correlating cationic lipid chemistry with transfectional efficacy.

Methods

Quantitative structure–activity relationship (QSAR) analyses were carried out on sets of routinely used, experimental, and unsuccessful cationic lipid vectors taken from the literature. This approach described the amphipathic character, basicity, headgroup size, lipophilicity and shape of cationic lipids using numerical parameters. Compounds were plotted onto various parameter diagrams, and correlations were sought between numerical parameters and transfectional efficiency.

Results

Transfectionally effective cationic lipids fell into restricted zones in various parameter spaces, indicating that amphipathic character, lipid shape and lipophilicity were generally significant factors, whilst basicity and headgroup size were only important for certain compounds. The data supported the general significance of membrane mixing followed by induction of membrane curvature, and the more limited role of osmotic shock, as mechanisms of membrane disruption. QSAR descriptions of effective lipids permitted detailed chemical guidelines for optimizing cationic lipid structure to be given. Limitations of the approach and models are discussed.

Conclusions

QSAR modeling indicated that induction of membrane curvature and osmotic shock are important mechanisms for membrane disruption by cationic lipids. The models also allowed specification of chemically detailed guidelines for selection or design of optimal cationic lipids. Copyright © 2005 John Wiley & Sons, Ltd.
Rad51 siRNA delivered by HVJ envelope vector enhances the anti‐cancer effect of cisplatin
Authors: Ito Makoto , Yamamoto Seiji , Nimura Keisuke , Hiraoka Kazuya , Tamai Katsuto , Kaneda Yasufumi
Published: March 2005

Background

Every cancer therapy appears to be transiently effective for cancer regression, but cancers gradually transform to be resistant to the therapy. Cancers also develop machineries to resist chemotherapy. Short interfering RNA (siRNA) has been evaluated as an attractive and effective tool for suppressing a target protein by specifically digesting its mRNA. Suppression of the machineries using siRNA may enhance the sensitivity to chemotherapy in cancers when combined with an effective delivery system.

Methods

To enhance the anti‐cancer effect of chemotherapy, we transferred siRNA against Rad51 into various human cancer cells using the HVJ (hemagglutinating virus of Japan, Sendai virus) envelope vector in the presence or absence of cis‐diamminedichloroplatinum(II) (CDDP, cisplatin). The inhibition of cell growth was assessed by a modified MTT assay, counting cell number, or fluorescence‐activated cell sorting (FACS) analysis after Annexin V labeling. The synthetic Rad51 siRNA was also introduced into subcutaneous tumor masses of HeLa cells in SCID mice with or without intraperitoneal injection of CDDP, and tumor growth was monitored.

Results

When synthetic Rad51 siRNA was delivered into HeLa cells using the HVJ envelope vector, no Rad51 transcripts were detected on day 2, and Rad51 protein completely disappeared for 4 days after siRNA transfer. When HeLa cells were incubated with 0.02 µg/ml CDDP for 3 h after siRNA transfer, the number of colonies decreased to approximately 10% of that with scrambled siRNA. The sensitivity to CDDP was enhanced in various human cancer cells, but not in normal human fibroblasts. When Rad51 siRNA was delivered into tumors using the HVJ envelope vector, the Rad51 transcript level was reduced to approximately 25%. Rad51 siRNA combined with CDDP significantly inhibited tumor growth when compared to siRNA or CDDP alone.

Conclusions

Rad51 siRNA could enhance the sensitivity to CDDP in cancer cells both in vitro and in vivo. Our results suggest that the combination of CDDP and Rad51 siRNA will be an effective anti‐cancer protocol. Copyright © 2005 John Wiley & Sons, Ltd.
Replication‐dependent transgene expression from a conditionally replicating adenovirus via alternative splicing to a heterologous splice‐acceptor site
Authors: Carette Jan E. , Graat Harm C. A. , Schagen Frederik H. E. , Abou El Hassan Mohamed A. I. , Gerritsen Winald R. , van Beusechem Victor W.
Published: March 2005

Background

Oncolytic viruses are promising anticancer agents because they selectively kill cancer cells and multiply within a tumor. Their oncolytic potency might be improved by expressing a therapeutic gene from the virus genome. In this regard, proper kinetics and level of transgene expression are important. In addition, expression of cytotoxic transgene products should be confined to cancer cells. Here, we developed oncolytic adenoviruses that provide transgene expression dependent on viral replication.

Methods

We constructed an oncolytic adenovirus that expresses luciferase under regulation of the endogenous major late promoter (MLP) via alternative splicing to an inserted splice‐acceptor site analogous to that of the adenovirus serotype 40 long fiber gene. Splicing of the luciferase transcript was studied by RT‐PCR analysis. Expression was measured in the presence and absence of the flavonoid apigenin, an inhibitor of viral replication.

Results

The inserted splice‐acceptor site was properly recognized by the adenoviral splicing machinery. Luciferase expression levels were markedly higher than levels obtained with the cytomegalovirus (CMV) promoter, especially at late stages of infection. Inhibiting adenovirus replication reduced luciferase expression levels dramatically by 4 to 5 logs, whereas expression levels with the CMV‐luciferase adenovirus were only moderately affected (2 logs).

Conclusions

Transgene delivery using the endogenous late gene expression machinery resulted in an expression pattern distinct from expression driven by the conventional CMV promoter. The high expression levels and strict coupling of expression to viral replication should be useful for adequate monitoring of replication and might provide a platform for the design of armed conditionally replicating adenoviruses (CRAds) with enhanced oncolytic potency. Copyright © 2005 John Wiley & Sons, Ltd.
Decay of oxygen solid solution in plastically deformed silicon
Authors: Yarykin N. , Vdovin V. I.
Published: March 2005

Decay of the oxygen solid solution in silicon during annealing at 550–700 °C is studied by the IR absorption technique in the single crystalline samples subjected to the plastic deformation to a high dislocation density at 680 °C. The deformation is shown to significantly enhance the rate of the decay in the whole temperature range studied. Based on the simple model, which assumes the heterogeneous oxygen aggregation at dislocations, the effective oxygen diffusivity is calculated from the experimental data. The activation energy of oxygen diffusion in this temperature range is found to be about 1.6 eV, which is essentially lower than that for the isolated interstitial oxygen atom. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
A study of GaN regrowth on the micro‐facetted GaN template formed by in‐situ thermal etching
Authors: Moon Y. T. , Fu Y. , Yun F. , Dogan S. , Mikkelson M. , Johnstone D. , Morkoç H.
Published: March 2005

We report a study of GaN regrowth on micro‐facetted GaN templates formed by in‐situ thermal etching in a low‐pressure metalorganic chemical vapor deposition system. First, the 1.5 µm‐thick GaN epilayers were grown on c‐plane sapphire substrates. This was followed by an in‐situ thermal etching process under hydrogen and ammonia ambient with the etching time being a parameter. The thermally etched GaN templates showed hexagonal GaN pyramids, which were aligned along the growth direction on the c‐plane substrate. The 3 µm‐thick GaN regrowth was performed on these micro‐facetted GaN templates. The surface of the overgrown GaN was atomically smooth. The full width at half maximum of (102) peak in the X‐ray rocking curve profile decreased from 9.5 to 6.5 arcmin as the thermal etching time increased from 0 to 45 min. Etch pit density measurements revealed that the pit density of regrown GaN decreased by about one order of magnitude, compared to that of the control sample. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Influence of surface passivation on low‐frequency noise properties of AlGaN/GaN high electron mobility transistor structures
Authors: Vitusevich S. A. , Petrychuk M. V. , Danylyuk S. V. , Kurakin A. M. , Klein N. , Belyaev A. E.
Published: March 2005

We investigated low‐frequency noise in passivated and nonpassivated AlGaN/GaN high electron mobility transistor (HEMT) heterostructures grown on SiC substrate. The heterostructure layers are grown without intentionally doping the barrier material and two‐dimensional gas appears at the interface only due to polarization effects. Transmission line model structures with different lengths of the conducting channel were studied in a wide range of temperatures in a linear regime. The magnitude of the noise and Hooge parameter demonstrate different dependence on the distance between contacts in passivated and non‐passivated structures. Noise reduction in the structure for around one order of magnitude after passivation was revealed and different origins of the noise before and after passivation established. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Organic photodiodes on newspaper
Authors: Lamprecht B. , Thünauer R. , Ostermann M. , Jakopic G. , Leising G.
Published: March 2005

We report on the fabrication and characterization of organic photodiodes deposited on ordinary newspaper sheets. Parylene acts as water and chemical barrier layer, an ORMOCER layer serves as an excellent smoothing and base layer for the diodes, which correspond to classical Tang‐type bilayer cells. The photodiodes show excellent diode behaviour in current–voltage measurements of the devices under dark and illumination conditions. The spectral response covers the UV and visible spectral range.

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(© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
An empirical typology of narcissism and mental health in late adolescence
Authors: Lapsley Daniel K. , Aalsma Matthew C.
Published: March 2005

A two‐step cluster analytic strategy was used in two studies to identify an empirically derived typology of narcissism in late adolescence. In Study 1, late adolescents () responded to the profile of narcissistic dispositions and measures of grandiosity (“superiority”) and idealization (“goal instability”) inspired by Kohut's theory, along with several College Adjustment Scales and a measure of pathology of separation‐individuation. Cluster analysis revealed three clusters: covert narcissists (), moderate narcissists () and overt narcissists (). Moderate narcissists had significantly lower means scores on indices of anxiety, relationship problem, depression, esteem‐ and family problems and pathology of separation‐individuation. The overt and covert clusters showed comparable levels of dysfunction on most indices of adjustment. This general pattern was replicated in Study 2 (). Moderate narcissists showed a uniform profile of good adjustment, whereas covert and overt narcissist clusters showed a pervasive pattern of dysfunction. Results support the claim that narcissism has “two faces” and that a moderate degree of narcissism is associated with fewer adjustment problems or psychological symptoms. Directions for future research are discussed.
Fate of specific nucleolar perichromosomal proteins during mitosis: Cellular distribution and association with U3 snoRNA
Authors: Gautier Thierry , Fomproix Nathalie , Masson Claude , Azum‐Gélade Marie‐Claude , Gas Nicole , Hemandez‐Verdun Danièle
Published: March 2005

Summary— In mammalian cells, the nucleoli disintegrate during mitosis and some nucleolar proteins disperse at the periphery of allchromosomes forming a novel class of chromosomal passenger proteins. The nucleolar components which participate in the formation of this perichromosomal layer have been investigated to elucidate the role of these perichromosomal proteins in the assembly and disassembly of the nucleoli. i) Electron microscopy immunolabelling reveals that these proteins are predominantly located in the granular component of the nucleoli during interphase,. ii) Immunoprecipitation data suggest that they are distributed at the chromosome periphery in association with U3 small nucleolar RNA (snoRNA). In addition, the distribution of U3 snoRNA visualized by in situ hybridization, is similar to that observed for the perichromosomal proteins. iii) In cells which possess a nucleolar remnant during mitosis, U3 snoRNA and perichromosomal proteins were found both in the perichromosomal layer and in the nucleolar remnant. iv) Some of these proteins are conserved from yeast to man such as fibrillarin and a protein of 52 kDa. v) The location of these proteins observed in yeast by confocal microscopy shows that they are not dispersed during mitosis. Their partition between the two daughter cells is performed by scission of nucleolar structures forming a rod during the budding process. Therefore RNP complexes related to the processing steps of ribosome biogenesis in mammalian cells quit the nucleolus in late G2 and associate with the chromosome periphery until late telophase. They associate in the perichromosomal layer in human and PtK1 cells and both in the perichromosomal layer and the nucleolar remnant in CHO cells.
Effects of maitotoxin on calcium entry and phosphoinositidebreakdown in the rabbit ciliated tracheal epithelium
Authors: Venant Annick , Dazy Anne‐Catherine , Diogene Georges , Metezeau Philippe , Marano Francelyne
Published: March 2005

Summary— Maitotoxin induces a concentration‐dependent 45Ca uptake in primary cultures of rabbit tracheal epithelial cells. This response is insensitive to the calcium channel antagonists nifedipine, diltiazem and verapamil up to 20 μM. However, verapamil at 200 μM completely prevents 45Ca uptake. Measurements of indo‐1 fluorescence show that MTX induces a very sustained (≥ 2 h) [Ca]i rise, which is completely inhibited by 200 μM of verapamil. Genistein (110 μM) (an inhibitor of tyrosine kinases) also strongly inhibits it. The inhibitory effect of 50 μM miconazole (an inhibitor of cytochrome P450) is only partial. Okadaic acid (inhibitor of protein‐phosphatases) primarily delays the response to the toxin without decreasing its magnitude. MTX induces the formation of (1,4,5) inositol trisphosphate (IP3). The MTX response curve is biphasic. Stimulation is transient (5 10 min) and is not inhibited by chelation of intracellular Cai with BAPTA, nor by verapamil (200 μM) or U73122 (10 μM) (an inhibitor of activation of PLCPI through a trimeric G protein). Results suggest that MTX independently activates a calcium transport process (which might imply phosphorylation on tyrosine residues) and a PLC not linked to a trimeric G protein.
INFLUENCE OF SHADE ON STOMATAL DENSITY, LEAF SIZE AND OTHER LEAF CHARACTERISTICS IN THE MAJOR TROPICAL ROOT CROPS, TANNIA, SWEET POTATO, YAM, CASSAVA AND TARO
Authors: ONWUEME I. C. , JOHNSTON M.
Published: March 2005

Since shading often occurs under normal growing conditions in various cropping systems (for example, intercropping, alley cropping), field experiments were carried out to determine the effects of shading on stomatal density, leaf size, leaf dry matter, and leaf lamina thickness in the major tropical root and tuber crops, tannia (Xanthosoma sagittifolium), sweet potato (Ipomoea batatas), yam (Dioscorea esculenta), cassava (Manihot esculenta), and taro (Colocasia esculenta). Shading decreased stomatal density in the lower epidermis of tannia, sweet potato, yam and cassava, and in the upper epidermis also in tannia and sweet potato; the upper epidermis of yam and cassava were devoid of stomata. In contrast to the other species, taro under shade had an increased stomatal density in both the upper and lower epidermis, a finding which was confirmed in subsequent pot experiments. This response of taro was postulated as a possible manifestation of greater shade adaptation by the species.

For all the species, shading generally resulted in the production of larger (in terms of surface area) but thinner leaves, with a decreased dry matter concentration. For the two species (yam and cassava) that had stomata only on the lower epidermis, normal sun-grown plants had about twice as many stomata per unit area of lower epidermis than did corresponding plants of the other three species. The agronomic and physiological significance of the findings are discussed.
Poetic words, conservatism and the dating of Old English poetry
Authors: Cronan Dennis
Published: March 2005

Although the lexicon has frequently been used in discussions of the dating of Old English poetry, little attention has been paid to the evidence that poetic simplexes offer. One exception is an article by R. J. Menner, who noted that Beowulf and Genesis A share three poetic words, apart from compounds, that are not found elsewhere: freme ‘good, valiant’, gombe ‘tribute’, and secg ‘sword’. Menner used these words as part of an argument for an early date of Genesis A, an argument which hinged, in part, on lexical similarities between this poem and Beowulf, which he assumed was early. Although such an a priori assumption is no longer possible, evidence provided by the limited distribution of certain poetic simplexes is nonetheless useful for demonstrating the presence of a connection between two or more poems. Such a connection may be a matter of date or dialect, or it may indicate that the poems were the products of a single poetic school or subtradition. Unfortunately, we know little, if anything, about poetic subtraditions, and the poetic koiné makes the determination of the dialect of individual poems a complex and subtle matter that requires a much wider variety of evidence than poetic words can provide. However, the limited distribution of certain poetic simplexes can serve as an index of the poetic conservatism of the poems in which these words occur. This conservatism could be due to a number of factors: genre, content (that is, heroic legend vs biblical or hagiographical), style, or date of composition. As will emerge in the course of this discussion, the most straightforward explanation for this conservatism is that the poems which exhibit it were composed earlier than those which do not. Other explanations are, however, possible, and the evidence of poetic words is hardly sufficient by itself to determine the dating of Old English poems. But by focusing on patterns of distribution that centre upon Beowulf, we can examine what certain words may tell us about the conservatism of this poem and of those poems which are connected to it.
A Human Rights Approach to Statelessness in the Middle East
Authors: Doebbler Curtis F.
Published: March 2005

Despite repeated attempts to eliminate statelessness and to provide for the protection of stateless persons, international law has not been able to provide an adequate response to these problems. In the Middle East the problem has continued to grow as social and political change pushes people into becoming stateless and fails to provide those who are stateless with adequate protection. The treaties that have attempted to prevent this practice have failed. At the same time the lex specialis aimed at protecting people from the consequences of statelessness have also failed. The result has been a lacuna in the protection of stateless persons. This article suggests that a step towards filling this gap might be made by applying general international human rights law to protect stateless persons.
Geographical Hegelianism in Territorial Disputes Involving Non-European Land Relations: An Analysis of the Case Concerning Kasikili/Sedudu Island (Botswana/Namibia)
Authors: Gathii James Thuo
Published: March 2005

This article reviews the case concerning Kasikili/Sedudu Island with reference to acquisition of title to territory under international law. It traces the ICJ's use of evidence of African use and occupation to establish territorial sovereignty in a European state. The tests adopted by the Court are based on Eurocentric assumptions that only the consent of European states is necessary to adjudicate nineteenth century claims of title to territory. International law still carries forward within it the colonial notion that treaties between colonial powers in the nineteenth century extinguished pre-existing title to territory based on African use and occupation. The decision gives probative value to economic intentions of colonizing powers and geographical and scientific evidence in determining title to the disputed island while Africa use and occupation of territory is sidelined. Africa is treated as an unconscious geographical entity – a feature referred to as geographical Hegelianism in this article.
Differential Scanning Calorimetry Study of Phase Transitions Affecting the Quality of Dehydrated Materials
Authors: Roos Yrjö , Karel Marcus
Published: March 2005

Differential scanning calorimetry was used to determine the phase transitions of dried and rehumidified amorphous lactose, sucrose, and a mixture of sucrose and Amioca. Glass‐transition, crystallization, and melting temperatures decreased with increasing moisture content. The time to crystallization of amorphous lactose held isothermally above the glass‐transition temperature decreased as the temperature was increased. Isothermal crystallization time of lactose was a function of the temperature difference between the holding temperature and the glass‐transition temperature independently of moisture content. Amorphous biological materials are metastable showing temperature, moisture content, and time‐dependent phase transitions that affect their drying behavior, stickiness, storage stability, and quality.
Chemical and Biosynthetic Approaches to the Production of Novel Polypeptide Materials
Authors: Mcgrath Kevin P. , Tirrell David A. , Kawai Michio , Mason Thomas L. , Fournier Maurille J.
Published: March 2005

Three approaches to the synthesis of the repetitive copolypeptide [‐(GlyAla)3‐ GlyProGlu‐]n (1) are described. Direct chemical synthesis of 1 via classical solution methods required 18 steps and afforded a polydisperse product with an average molecular weight of less than 10 000. Two alternative genetic strategies were also explored. In the first, chemically synthesized DNA oligomers were self‐ligated to produce a population of multimers, which were fitted with translational start and stop signals and inserted into an expression plasmid containing the λ PL promoter and a synthetic ribosome binding site. Transformation of E. coli led to the isolation of a stable recombinant plasmid carrying an insert encoding 12 repeats of sequence 1. Attempts to identify polypeptide 1 after induction of transformed cultures were unsuccessful. A second strategy, generating a tripartite derivative of sequence 1 carrying short N‐and C‐terminal extensions, afforded excellent yields of product. The relative merits of chemical and genetic approaches to repetitive polypeptide materials are discussed.
Improved Bioreaction Kinetics for the Simulation of Continuous Ethanol Fermentation by Saccharomyces cerevisiae
Authors: Warren R. K. , Hill G. A. , Macdonald D. G.
Published: March 2005

A kinetic model for the production of ethanol by Saccharomyces cerevisiae has been developed from semiempirical analysis. The values for the parameters in this model were then determined by nonlinear multiple regression using the data of Bazua and Wilke ( 1977). The final equations were μ=0.427s(1‐(p/101.6)1.95)/(0.245+s), YX/p=0.291, and YX/s=0.152(1‐p/302.3). This model was then used to simulate a continuous stirred tank fermentor (CSTF) and compared to other models using the same experimental data but different kinetics. The equations required to use these kinetics in a CSTF with recycle were then developed. From this simulation, it was found that, for a CSTF with recycle, the best configuration to operate is an external recycle, with a low bleed and recycle ratio.
Growth and Fermentation Model for Alginate‐Entrapped Saccharomyces cerevisiae
Authors: Hannoun Betty J. M. , Stephanopoulos Gregory
Published: March 2005

A model of growth and fermentation for alginate‐entrapped Saccharomyces cerevisiae is presented. The model is based on Peringer's rate equations (Peringer, P.; et al. Biotechnol. Bioeng. Symp. 1973, 4, 27–42), developed for glucose‐repressed cultures of suspended cells at varying dissolved oxygen concentrations, and was fitted to estimates of growth and fermentation rates for immobilized cells obtained through a combination of experimental measurements and reaction‐diffusion analysis. Immobilized cell fermentation data with alginate beads in suspension compared well with simulations of reaction and diffusion in a similar system incorporating the proposed growth‐fermentation model. In view of these findings, the model was further applied to the simulation of transient and steady‐state operation of a packed‐bed immobilized cell biore‐actor. Results about the effect of operating parameters on bioreactor performance are presented, and implications regarding system design and operation for optimal overall performance are discussed.
Protective Effect of Methylcellulose and Other Polymers on Insect Cells Subjected to Laminar Shear Stress
Authors: Goldblum Seth , Bae Yong‐Kook , Hink W. Fred , Chalmers Jeff
Published: March 2005

The relative sensitivity of two insect cell lines to laminar shear stress was determined, and the protective effect of polymers added to the growth media of two insect cell lines, Trichoplusia ni (TN‐368) and Spodoptera frugiperda (SF‐9), was evaluated. TN‐368 and SF‐9 cells were found to be equally sensitive to laminar shear stress. Methylcellulose [0.5% (w/v) Dow E4M Methocel] and dextran [4.5% (w/v)] increased the resistance of suspended cells to lysis due to laminar shear stress by factors of up to 76 and 28, respectively, compared to cells in media without additives. It was observed that the protective effect of Pluronic F‐68 was concentration‐dependent: 0.2% and 0.3% (w/v) F‐68 increased the resistance of SF‐9 cells to shear stress by factors of 15 and 42, respectively. However, increasing the concentration to 0.5% did not significantly increase the cells' resistance compared to 0.3% (w/v). F‐68 at 0.2% only increased the resistance of TN‐368 cells by a factor of 6. It is believed that the protection is a result of the polymer adsorbing to the cell membrane. None of the polymer additives tested had a significant effect on SF‐9 or TN‐368 growth rate.
Effect of Dissolved Oxygen Control on Growth and Antibiotic Production in Streptomyces clavuligerus Fermentations
Authors: Yegneswaran P. K. , Gray M. R. , Thompson B. G.
Published: March 2005

A proportional‐integral control system was used to control dissolved oxygen in a fermentor at constant shear and mass transfer conditions. Growth and antibiotic production in Streptomyces clavuligerus were studied at different dissolved oxygen levels during the fermentation. Three protocols were employed: no‐oxygen control to provide a base case, oxygen controlled to a preset saturation level throughout the fermentation, and oxygen controlled at a high level only during the growth phase. The last protocol was aimed at optimizing the consumption of oxygen. Lower specific growth rates and cephamycin C yields were obtained when dissolved oxygen was controlled at 50% throughout the fermentation, compared to the base case. A 2.4‐fold increase in the final cephamycin yield was observed when dissolved oxygen was controlled at saturation levels during the growth phase, compared to the experiments without dissolved oxygen control. This enhancement in yield was independent of the dissolved oxygen (DO) level after exponential growth, in the range of 50–100% saturation. The most effective control strategy, therefore, was to control DO only during active growth when the biosynthetic enzymes were probably synthesized.
Removal of Hydrogen Sulfide by Chlorobium thiosulfatophilum in Immobilized‐Cell and Sulfur‐Settling Free‐Cell Recycle Reactors
Authors: Kim Byung Woo , Chang Ho Nam
Published: March 2005

Bioconversion of hydrogen sulfide to elementary sulfur by the photosynthetic bacterium Chlorobium thiosulfatophilum was studied in immobilized‐cell and sulfur‐settling free cell recycle reactors. The cells immobilized in strontium alginate beads excreted elementary sulfur and accumulated it as crystal in the bead matrices, which made it possible that the reactor broth remained clear and the light penetrated the reactor deeper than with the free cells. In comparison with the free cells, the immobilized cells required 30% less light energy at a H2S removal rate of 2 mM/(L‐h) and showed an activity of 2.4 times that of the free cells. However, in 40 h after the reaction the deterioration of the H2S removal efficiency became significant due to the accumulation of sulfur in the beads. The scanning electron micrograph (SEM) and energy‐dispersive X‐ray spectrometer (EDS) studies showed that the sulfur in the beads existed within a layer of 0.4 mm from the bead surface. In the sulfur‐settling free‐cell recycle reactor, about 80% of the sulfur excreted by the free cells could be removed in a settler. The 4‐L fed batch reactor with the settler improved the light transmission to result in a H2S removal rate of 3 μmol/ (mg of protein‐h), 50% higher than that without it. The settling recycle reactor was much better in the removal of H2S than the immobilized‐cell reactor because the former was a continuous system with the constant removal of sulfur particles by settling and of spent medium by supplying fresh medium at the same rate as the filtering rate of the reactor broth, while the latter was essentially a batch system where toxic metabolites and produced sulfur could not be removed.
Effect of Serum on the Plasma Membrane Fluidity of Hybridomas: An Insight into Its Shear Protective Mechanism
Authors: Ramirez Octavio T. , Mutharasan R.
Published: March 2005

We have previously shown that decreasing the concentration of fetal bovine serum (FBS) increased the fragility of a mouse hybridoma (HB‐32) during agitated batch cultivation and that increasing the plasma membrane fluidity (PMF) increased the shear sensitivity during exposure to laminar flow. In this study, the effect of FBS concentration on the PMF of HB‐32 was investigated. PMF was evaluated by steadystate fluorescence anisotropy (r8) of 1‐[4‐(trimethylamino) phenyl]‐6‐phenylhexa‐1,3,5‐triene. Increasing serum concentration increased the r8 of hybridomas, indicating a decrease in their PMF. The effect of cholesterol modulation on the PMF and shear sensitivity was also evaluated. Hybridomas were exposed to turbulent fluid shear after modification of PMF by cholesterol modulation. Direct cholesterol enrichment of the plasma membranes caused a decrease in the PMF and shear sensitivity, while cholesterol depletion caused an increase in PMF and shear sensitivity. Low‐and high‐density lipoprotein supplementation to cultures in serum‐free or complete medium decreased their shear sensitivity. Lipoprotein supplementation to serum‐free cultures decreased the PMF. Altogether, these results suggest that the protective mechanism of serum against hydrodynamic damage relies, at least partially, on its ability to decrease the PMF of hybridomas possibly through the transfer of cholesterol from the serum lipoproteins into the plasma membrane.
Kinetic Properties of Myrosinase in Hydrated Reverse Micelles
Authors: Hochkoeppler Alejandro , Palmieri Sandro
Published: March 2005

The enzymatic activity of myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) solubilized in reverse micelles, which were formed in isooctane by bis(2‐ethylhexyl) sodium sulfosuccinate (AOT), was studied using sinigrin (allyl glucosinolate) and glucotropeolin (benzyl glucosinolate) as substrates. The enzyme in reverse micelles follows Michaelis‐Menten kinetics for both substrates, showing similar kinetic values, whereas in water solution sinigrin seems to be the better substrate. The effect of the water concentration, W0 (W0=[H20]/[AOT]), on the steady‐state velocity of the myrosinase does not induce the bell‐shaped activity profile observed for many other enzymes. The optimal W0 for both substrates appears to be widespread between 10 and 40. The pH profile of myrosinase activity in reverse micelles shows an increase of activity from pH 4 to pH 6. Above this value and up to pH 9, the activity remains constant, establishing a broad plateau at its maximum. It was demonstrated that myrosinase activity in reverse micelles, as well as in water solution, strongly depends on the temperature, showing an extraordinary maximum of activity with both substrates around 60 °C. As in water, the enzyme appears to be strongly activated by ascorbate in reverse micelles with both sinigrin and glucotropeolin. All the above properties of the enzyme in reverse micelles, and in particular (i) the possibility of varying the water concentration and the pH within relatively wide ranges, (ii) the high thermal stability of the enzyme together with the exceptionally high optimum reaction temperature, and (iii) the activation of the enzyme by ascorbate more than in water solutions, strongly suggest the use of this enzyme solubilized in reverse micelles to good advantage for producing biologically interesting nitrogen‐and sulfur‐containing compounds from glucosinolate hydrolysis.
A Stable Long‐Term Hepatocyte Culture System for Studies of Physiologic Processes: Cytokine Stimulation of the Acute Phase Response in Rat and Human Hepatocytes
Authors: Bader Augustinus , Rinkes Inne H. Borel , Closs Ellen I. , Ryan Colleen M. , Toner Mehmet , Cunningham James M. , Tompkins Ronald G. , Yarmush Martin L.
Published: March 2005

Prior studies on the in vitro hepatic acute phase response have involved either hepatoma cell lines or conventional short‐term cultures of primary hepatocytes. No data are available on the response of primary hepatocytes in stable long‐term culture systems. In this study, the acute phase response of rat and human hepatocytes in a new long‐term culture system was examined in response to interleukin‐6 (IL‐6), interleukin‐1β (IL‐1β), and tumor necrosis factor α (TNF‐α). The cultured cells were sandwiched between two layers of collagen in a (double‐gel) configuration which has been shown to preserve both hepatocyte function and morphology over prolonged periods of time. The stability of this culture configuration enabled us to investigate, for the first time, the temporal aspects of the response in addition to the effects of the mediators on protein secretion. Exposure of rat hepatocytes to IL‐6 after culture for 16 days resulted in a 2‐fold reduction of albumin secretion and a 15‐fold increase in the secretion rates of fibrinogen and α2‐macroglobulin. In all instances, the peak response occurred at 48 h after IL‐6 exposure, and all protein secretion rates returned to pretreatment values within 5 days post‐treatment. Changes in the mRNA levels of these proteins in response to IL‐6 corresponded with those changes seen with the secreted products, indicating pretranslational regulation. Administration of IL‐1β to rat hepatocytes produced a similar decline of albumin secretion and a 5‐fold increase of fibrinogen secretion, whereas α2‐macroglobulin secretion remained undisturbed. In contrast, TNF‐α did not affect the secretion of any protein examined. Human hepatocytes in a double‐gel culture configuration reacted to IL‐6 and IL‐1β with an approximate 40‐fold increase in serum amyloid A secretion, peaking at day 3 posttreatment. Both the secretion pattern and temporal response of these cultured hepatocytes to cytokines appear to closely mimic the in vivo hepatocellular response. The double‐gel culture system is a stable, attractive tool for further investigation of the acute phase and other hepatocellular responses to physiologic stimuli.
Oxygen Mass Transfer Enhancement via Fermentor Headspace Pressurization
Authors: Yang Jeng‐Dar , Wang Nam Sun
Published: March 2005

Bioreactor headspace pressurization represents an excellent means of enhancing oxygen mass transfer to a culture. This method is particularly effective in situations where stirring or vigorous aeration is difficult. Because it in itself introduces no undesirable hydrodynamic force, the proposed method is also attractive for cells susceptible to agitation and sparging. Experiments were first conducted in an ideal fermentor by sparging air into a sulfite solution free from extraneous microbial effects. An increased oxygen mass transfer rate resulting from pressurization led to a superior cell growth rate and a higher maximum cell density in both of the microbial systems studied: a bacterial (Escherichia coli) culture up to 2.72 bar and a fragile algal (Ochromonas malhamensis) culture with pressure programming. Applying pressurization increased the maximum dry cell weight from 1.47 g/L to 1.77 g/L in the E. coli culture and increased the maximum viable cell density from 4 X107 cells/mL to 108 cells/mL in the algal culture. An additional advantage is that formation of undesirable products under oxygen limitation, e.g., acetic acid in the E. coli culture, can be suppressed. A significant (over 250%) improvement in the oxygen transfer rate can be achieved with existing fermentors with little modification as they are already designed to withstand reasonable pressure from autoclaving. This method is simple, clean, inexpensive, and easily implemented, and it can be applied alongside other existing methods of oxygen mass transfer enhancement.
Production and Purification of a Recombinant Elastomeric Polypeptide, G‐(VPGVG)19‐VPGV, from Escherichia coli
Authors: Mcpherson David T. , Morrow Casey , Minehan Daniel S. , Wu Jianguo , Hunter Eric , Urry Dan W.
Published: March 2005

An elastomeric polypeptide was produced, with the sequence G‐(VPGVG)19‐VPGV, as a fusion to glutathione S‐transferase using the vector pGEX‐3X. The fusion protein was expressed to high levels in Escherichia coli as indicated by SDS‐PAGE analysis of induced cells. The fusion protein was affinity purified and cleaved with protease factor Xa, and the elastomeric polypeptide was recovered to a high degree of purity as indicated by SDS—PAGE followed by staining with CuCl2. The physical characterizations of carbon‐13 and proton nuclear magnetic resonance and of the temperature profile for turbidity formation for the inverse temperature transition of hydrophobic folding and assembly attest to the successful microbial synthesis of the polypentapeptide of elastin. The results of these studies provide the initial progress toward achieving a more economical and practical means of producing material for elastic protein‐based polymer research and applications.
Fully‐Automatic Measurement of Mycelial Morphology by Image Analysis
Authors: Tucker Keith G. , Kelly Tom , Delgrazia Patricia , Thomas Colin R.
Published: March 2005

A fully‐automatic image analysis method is described for the comprehensive and quantitative analysis of the morphology of filamentous mycelia grown in submerged cultures. The method not only allows rapid measurement of important morphological parameters on freely dispersed mycelia but also provides a novel characterization of the aggregated (clumped) form. The latter can constitute more than 90% of the biomass in some fermentations and might therefore be expected to have a major influence on broth rheology, fermenter mixing, mass transfer, and hence fermentation productivity. Clumps are characterized not only in terms of the percentage of mycelia in this form, as in earlier work, but also in terms of clump area, perimeter, compactness, and roughness. The method has been tested on laboratory‐scale Streptomyces clavuligerus and Penicillium chrysogenum fermentations. It is not only more comprehensive than previous methods but also faster and thus will permit more extensive physiological and engineering studies on mycelial fermentations than has previously been possible.