bluuebunny/crossref_metadata_2025 huggingface dataset:
Communal nesting in the usually solitary marsupial, Phascogale tapoatafa
Authors: Rhind Susan G.
Published: December 2003

The marsupial brushtailed phascogale Phascogale tapoatafa is a solitary, hollow‐dependent, arboreal insectivore that occurs at low densities in open forests and woodlands of Australia. Data gathered from nest box surveys (4 years), and from radio‐tracking phascogales to nest sites (3 years) in south‐western Australia confirm solitary nesting after dispersal. However, in the winter of a single year, nest box surveys in one study area showed that 56% of individuals were nest sharing. On a neighbouring site, 81 group nests were also recorded among 18 radio‐collared individuals. In both areas, groups comprised two to four individuals of any age/sex combination. The same phascogales tended to nest together and in a number of different sites. Nest sharing between females was restricted to territory boundaries and continued after the annual die‐off of the males. Nest sharing coincided with prolonged drought conditions and in this year mature phascogales were significantly smaller than normal, i.e. males 25% less in weight, females 12% less. Communal nesting seemed to be a response to thermoregulatory difficulties posed by the three interrelated factors of low body mass, declining temperatures and declining food availability. Nest sharing in this species appeared to be a behavioural indicator of an energetics crisis, there was a population decline during the drought period and a population crash in the following year.
Host fidelity of a symbiotic porcellanid crab: the importance of host characteristics
Authors: Thiel Martin , Zander Anke , Valdivia Nelson , Baeza Juan A. , Rueffler Claus
Published: December 2003

The social behaviour of symbiotic organisms is influenced by the density and distribution pattern of hosts. Herein we examined the host‐use behaviour of the anemone‐dwelling crab Allopetrolisthes spinifrons (Porcellanidae) in which adults usually live as solitary individuals on their hosts. Adults of this crab use two different sea anemone species, one intermediate‐sized species that can be found at relatively high densities in the intertidal zone and another large one that occurs at significantly lower densities in the shallow subtidal zone along exposed rocky shores of the south‐east Pacific. Mark–recapture experiments demonstrated that crabs in subtidal waters (low abundance of hosts) remained for long time periods on the same hosts while crabs in the intertidal environment (high abundance of hosts) frequently changed hosts. There were no differences in host fidelity between male and female crabs. In an immigration experiment in the intertidal zone, significantly more juveniles immigrated than had been present originally, indicating that host‐use behaviour is age dependent. The sex ratio between resident and immigrated individuals did not change. In an additional experiment hosts were planted at two different densities (high and low) both in the shallow subtidal and the intertidal zone to test whether host fidelity of crabs depends on distance between hosts. At the subtidal site, about half the crabs remained on their sea anemones for 14 days while at the intertidal site most crabs disappeared within 1 day in both density treatments. At both sites crabs were seen changing hosts in high density treatments, but no such events could be witnessed at low densities of hosts. Although the results are not fully conclusive, they suggest that host movements are affected by host densities. This relationship may be mediated by host (anemone species, size, distance) and site‐specific (predation pressure, exposure time) factors. In general, the present study indicates that host characteristics influence host fidelity of symbiotic organisms and thereby, their social behaviour.
The functional morphology of Bentholyonsia teramachii (Bivalvia: Lyonsiellidae): clues to the origin of predation in the deep water Anomalodesmata
Authors: Morton Brian
Published: December 2003

Bentholyonsia teramachii has hitherto been described from Japan, but a conspecific specimen is herein recorded from Western Australian shelf waters at a depth of ∼100 m. Hitherto placed in the Lyonsiidae, B. teramachii is herein relocated in the Lyonsiellidae, alongside a second genus –Lyonsiella. Features of the anatomy of Bentholyonsiateramachii, including highly sensory siphons, reduced ctenidia unable to collect and transport particulate food, non‐sorting labial palps, and a simplified Type II stomach and muscular gut all suggest that the species is a predator like Lyonsiella formosa. Other features of the anatomy, such as pallial taenioid muscles, radial mantle glands, and the structure of the ctenidia all point to a link between B. teramachii and the Lyonsiellidae and thus with the Verticordiidae and Parilimyidae and, therefore, with some of the oldest anomalodesmatans, the Pholadomyidae. In the possession by B. teramachii of a unique pair of antero‐dorsal suspensory muscles, the link with fossil pholadomyoideans is reinforced. Bentholyonsia thus represents another genus of predatory bivalve, not only expanding our view of the prevalence of this feeding mode in the deep sea by this class but also helping us show how it evolved, in anatomical terms, from a suspension‐ (or deposit‐) feeding ancestor.
Sperm competition in Odonata (Insecta): the evolution of female sperm storage and rivals' sperm displacement
Authors: Córdoba‐Aguilar A. , Uhía E. , Rivera A. Cordero
Published: December 2003

Odonates (dragonflies) are well known for the ability of the males to displace sperm stored in the female's sperm‐storage organs during copulation. By this means, copulating males are able to increase their fertilization success. This ability has been used as an example to illustrate a conflict of interests between the sexes in which males have evolved sperm‐displacement mechanisms whilst females have presumably evolved means to avoid sperm displacement. The present review has four aims: (1) to describe the copulatory mechanisms used during sperm displacement; (2) to analyse the causes of sperm usage patterns; (3) to discuss this information using current hypotheses on conflict between the sexes; and (4) to illuminate topics for further research. Four copulatory mechanisms are described: sperm removal (physical withdrawal of stored sperm), sperm repositioning (‘pushing’ of rival sperm to sites where its use will be least likely), female sensory stimulation to induce sperm ejection, and sperm flushing (displacement of sperm using the copulating male's sperm). Sperm‐precedence studies in Odonata are scarce and their values vary considerably between species. In those species in which sperm displacement is incomplete, the last copulating male obtains a high but variable short‐term fertilization success which decreases with time. Some male and female factors affecting sperm precedence patterns are mentioned: (1) male variation in genital morphology; (2) duration of copulation influenced by the male (the longer the copulation, the more stored sperm displaced); (3) adaptations of the sperm‐storage organs that allow the female to manipulate the sperm she has received (i.e. avoiding sperm displacement, re‐distributing sperm masses, favouring sperm located in certain sites and ejecting sperm after copulation). We suggest that male and female odonates have co‐evolved at the level of genital function with the control of stored sperm as the focus of the conflict. The benefits for males in this co‐evolution lie in maximizing their fertilization success. However, it is not clear what females obtain from storing sperm and making it unreachable during sperm displacement. Two hypothetical benefits that females may obtain for which some evidence has been gathered are genetic diversity and viability genes. It is finally suggested that odonates can become excellent subjects of study for testing current ideas related to sexual conflict and speciation processes through sexual selection.
A New HPLC‐ELSD Method To Quantify Indican in Polygonum tinctorium L. and To Evaluate β‐Glucosidase Hydrolysis of Indican for Indigo Production
Authors: Angelini Luciana G. , Campeol Elisabetta , Tozzi Sabrina , Gilbert Kerry G. , Cooke David T. , John Philip
Published: December 2003

A method to quantify the indigo precursor indican (indoxyl‐β‐d‐glucoside) in Polygonum tinctoriumL. has been developed. Plant material was extracted in deionized water, and indican was identified and quantified using high performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD). Results confirmed that with this method it is possible to measure indican content in a short time, obtaining reliable and reproducible data. Using this method, leaf indican content was quantified every 15 days during the growing season (from May to October) in P. tinctorium crops grown in a field experiment in Central Italy. Results showed that indican increased along the growing season until flowering and was positively affected by photosynthetic active radiation (PAR). Indican is naturally hydrolyzed by native β‐glucosidase to indoxyl and glucose, the indoxyl yielding indigo. The activity of two enzymes, sweet almond β‐glucosidase and Novarom G preparation, were compared with P. tinctorium native β‐glucosidase to evaluate indigo production. Results showed that the ability to promote indigo formation increased as follows: almond β‐glucosidase ≤ Novarom G.
Application of Magnetic Agarose Support in Liquid Magnetically Stabilized Fluidized Bed for Protein Adsorption
Authors: Tong Xiao‐Dong , Sun Yan
Published: December 2003

A novel magnetic agarose support (MAS) was fabricated for application in a liquid magnetically stabilized fluidized bed (MSFB). It was produced by water‐in‐oil emulsification method using a mixture of agarose solution and nanometer‐sized superparamagnetic Fe3O4 particles as the aqueous phase. The MAS showed good superparamagnetic responsiveness in a magnetic field. A reactive triazine dye, Cibacron blue 3GA (CB), was coupled to the gel to prepare a CB‐modified magnetic agarose support (CB‐MAS) for protein adsorption. Lysozyme was used as a model protein to test the adsorption equilibrium and kinetic behavior of the CB‐MAS. The dependence of bed expansion in the MSFB with a transverse magnetic field on liquid velocity and magnetic field intensity was investigated. Liquid‐phase dispersion behavior in the MSFB was examined by measurements of residence time distributions and compared with that obtained in packed and expanded beds. Dynamic lysozyme adsorption in the MSFB was also compared with those in packed and expanded beds. The dynamic binding capacity at 10% breakthrough was estimated at 55.8 mg/mL in the MSFB, higher than that in the expanded bed (31.1 mg/mL) at a liquid velocity of 45 cm/h. The results indicate that the CB‐MAS is promising for use in liquid MSFB for protein adsorption.
Monte Carlo Simulation of LNCaP Human Prostate Cancer Cell Aggregation in Liquid‐Overlay Culture
Authors: Song Hong , O/Connor Kim C. , Lacks Daniel J. , Enmon Richard M. , Jain Shamik K.
Published: December 2003

Neoplastic cells self‐assemble in liquid‐overlay cultures into multicellular spheroids that resemble micrometastases and avascular regions of larger tumors. A Monte Carlo simulation based on Meakinapos;s cluster‐cluster aggregation model resolved the physical mechanisms by which LNCaP human prostate cancer cells aggregate in this environment. The best‐fit solution suggests that LNCaP cells aggregate with an adhesion probability of 0.5% when they migrate within a radius of influence between cell centers of 180 μm, 10 times the cell diameter. The sweeping radius of influence is indicative of cell tethering and/or chemotaxis and results in an intrinsic rate of self‐aggregation that increases from k11 = 1.5 h‐1 for single cells to k1010 = 17.5 h‐1 for 10‐mers. Similar rates are predicted by Smoluchowskiapos;s collision theory (1), suggesting that they are inherent properties of LNCaP liquid‐overlay culture. Aggregates form more compact structures in culture than during simulation as measured by the fractal dimension: DF = 1.74 ± 0.04 for 10‐mers in culture vs DF = 1.25 ± 0.10 for simulated 10‐mers. Additional restructuring would further extend the radius of influence and diminish adhesion. Applications of this work include the production of highly viable spheroids for drug testing and basic oncological research.
Evaluation of Ion Exchange Resins for Removal of Inhibitory Compounds from Corn Stover Hydrolyzate for Xylitol Fermentation
Authors: de Mancilha Ismael Maciel , Karim M. Nazmul
Published: December 2003

The use of dilute acids to catalyze the hydrolysis of hemicellulose to its sugar constituents is well‐known and effective. However, a major problem associated with this pretreatment is the poor fermentability of the produced hydrolyzate as a result of the presence of the microorganismapos;s inhibitory compounds. In the present work, seven ion‐exchange resins were tested in order to detoxify corn stover hydrolyzate. Regarding xylose recovery, it was observed that more than 92% recovery was feasible. Furfural removal varied from 53.% to 99.%, and hydroxymethylfurfural (HMF) removal was effective between 37% and 100%. Acetic acid was totally removed by Purolite A 103 S resin. Corn stover hydrolyzate (CSH) treated with Purolite A 103 S, and Finex CS 14 GC resins, was tested as substrate for xylitol production using a yeast, Candida mogii. Product yields, Yp/s, of 0.41 and 0.37 g/g and cellular yields, Yx/s, of 0.24 and 0.13 g/g, respectively, were obtained using the two types of resin‐treated hydrolyzates.
Geranium Leaf Assisted Biosynthesis of Silver Nanoparticles
Authors: Shankar S. Shiv , Ahmad Absar , Sastry Murali
Published: December 2003

Development of biologically inspired experimental processes for the synthesis of nanoparticles is evolving into an important branch of nanotechnology. In this paper, we report on the use of Geranium (Pelargonium graveolens) leaf extract in the extracellular synthesis of silver nanoparticles. On treating aqueous silver nitrate solution with geranium leaf extract, rapid reduction of the silver ions is observed leading to the formation of highly stable, crystalline silver nanoparticles in solution. Transmission electron microscopy analysis of the silver particles indicated that they ranged in size from 16 to 40 nm and were assembled in solution into quasilinear superstructures. The rate of reduction of the silver ions by the geranium leaf extract is faster than that observed by us in an earlier study using a fungus, Fusarium oxysporum, thus highlighting the possibility that nanoparticle biosynthesis methodologies will achieve rates of synthesis comparable to those of chemical methods. This study also represents an important advance in the use of plants over microorganisms in the biosynthesis of metal nanoparticles.
Development of a Segmented Model for a Continuous Electrophoretic Moving Bed Enantiomer Separation
Authors: Thome Brian M. , Ivory Cornelius F.
Published: December 2003

With the recent demonstration of a continuous electrophoretic “moving bed” enantiomer separation at mg/h throughputs, interest has now turned to scaling up the process for use as a benchtop pharmaceutical production tool. To scale the method, a steady‐state mathematical model was developed that predicts the process response to changes in input feed rate and counterflow or “moving bed” velocities. The vortex‐stabilized apparatus used for the separation was modeled using four regions based on the different hydrodynamic flows in each section. Concentration profiles were then derived on the basis of the properties of the Piperoxan‐sulfated β‐cyclodextrin system being studied. The effects of different regional flow rates on the concentration profiles were evaluated and used to predict the maximum processing rate and the hydrodynamic profiles required for a separation. Although the model was able to qualitatively predict the shapes of the concentration profiles and show where the theoretical limits of operation existed, it was not able to quantitatively match the data from actual enantiomer separations to better than 50% accuracy. This is believed to be due to the simplifying assumptions involved, namely, the neglect of electric field variations and the lack of a competitive binding isotherm in the analysis. Although the model cannot accurately predict concentrations from a separation, it provides a good theoretical framework for analyzing how the process responds to changes in counterflow rate, feed rate, and the properties of the molecules being separated.
Continuous d‐Tagatose Production by Immobilized Thermostable l‐Arabinose Isomerase in a Packed‐Bed Bioreactor
Authors: Ryu Se‐Ah , Kim Chang Sup , Kim Hye‐Jung , Baek Dae Heoun , Oh Deok‐Kun
Published: December 2003

d‐Tagatose was continuously produced using thermostable l‐arabinose isomerase immobilized in alginate with d‐galactose solution in a packed‐bed bioreactor. Bead size, L/D (length/diameter) of reactor, dilution rate, total loaded enzyme amount, and substrate concentration were found to be optimal at 0.8 mm, 520/7 mm, 0.375 h‐1, 5.65 units, and 300 g/L, respectively. Under these conditions, the bioreactor produced about 145 g/L tagatose with an average productivity of 54 g tagatose/L·h and an average conversion yield of 48% (w/w). Operational stability of the immobilized enzyme was demonstrated, with a tagatose production half‐life of 24 days.
Enzymatic Synthesis of 3′‐Hydroxyacetaminophen Catalyzed by Tyrosinase
Authors: Valero Edelmira , Lozano M. Isabel , Varón Ramón , García‐Carmona Francisco
Published: December 2003

3′‐Hydroxyacetaminophen, a catechol metabolite of N‐acetyl‐p‐aminophenol (acetaminophen) and N‐acetyl‐m‐aminophenol (a structural analogue of acetaminophen and considered as a possible alternative because it is not hepatotoxic), is enzymatically synthesized for the first time using mushroom tyrosinase. Although reported to be weakly hepatotoxic in vivo, this catechol derivative of acetaminophen is not commercially available. This compound was obtained from its monophenolic precursor, acetaminophen, using the enzyme tyrosinase in the presence of an excess of ascorbic acid, thus reducing back the o‐quinone product of catalytic activity to the catechol acetaminophen derivative. A mathematical model of the system is proposed, which is also applicable to the tyrosinase‐mediated synthesis of any o‐diphenolic compound from its corresponding monophenol. This synthesis procedure is continuous, easy to perform and control, and adaptable to a bioreactor with the immobilized enzyme for industrial purposes in a nonpolluting way.
Electrophysiological and Morphological Characterization of Rat Embryonic Motoneurons in a Defined System
Authors: Das Mainak , Molnar Peter , Devaraj Halagowder , Poeta Matthew , Hickman James J.
Published: December 2003

In an attempt to integrate biological components with silicon‐based devices and systems, artificial silane surfaces have been successfully used to grow motoneurons in a defined environment. In this study we characterized the morphology and electrophysiology of purified rat embryonic (E14) motoneurons grown on a self‐assembled monolayer (SAM) of N‐1[3‐(trimethoxysilyl)propyl]diethylenetriamine (DETA) versus that on ornithine/laminin surfaces in serum‐free media. On DETA motoneurons were flat and grew more processes, whereas on ornithine/laminin they tended to aggregate. The membrane time constant, a characteristic associated with electrotonic compactness, was significantly longer for motoneurons grown on DETA. Other electrophysiological parameters were similar for the motoneurons on the different surfaces. This is the first study where purified ventral horn motoneurons were cultured in a completely defined (nonbiological surface, serum‐free) environment.
Improving the Industrial Production of 6‐APA: Enzymatic Hydrolysis of Penicillin G in the Presence of Organic Solvents
Authors: Abian Olga , Mateo César , Fernández‐Lorente Gloria , Guisán José M. , Fernández‐Lafuente Roberto
Published: December 2003

The hydrolysis of penicillin G in the presence of an organic solvent, used with the purpose of extracting it from the culture medium, may greatly simplify the industrial preparation of 6‐APA. However, under these conditions, PGA immobilized onto Eupergit displays very low stability (half‐life of 5 h in butanone‐saturated water) and a significant degree of inhibition by the organic solvent (30%). The negative effect of the organic solvent strongly depended on the type of solvent utilized: water saturated with butanone (around 28% v/v) had a much more pronounced negative effect than that of methylisobutyl ketone (MIBK) (solubility in water was only 2%). These problems were sorted out by using a new penicillin G acylase derivative designed to work in the presence of organic solvents (with each enzyme molecule surrounded by an hydrophilic artificial environment) and a suitable organic solvent (MIBK). Using such solvent, this derivative kept its activity unaltered for 1 week at 32 °C. Moreover, the enzyme activity was hardly inhibited by the presence of the organic solvent. In this way, the new enzyme derivative thus prepared enables simplification of the industrial hydrolysis of penicillin G.
Reduced Formation of Byproduct Component C in Acarbose Fermentation by Actinoplanessp. CKD485–16
Authors: Choi Byoung Taek , Shin Chul Soo
Published: December 2003

Acarbose fermentation was conducted by cultivation of Actinoplanessp. CKD485–16. Approximately 2,300 mg/L of acarbose was produced at the end of cultivation along with 600 mg/L of the acarbose byproduct component C. Maltose, a known moiety of acarbose, should be maintained at high concentration levels in culture broths for efficient acarbose production. The acarbose yield increased with an increasing osmolality of the culture medium, with a maximum value of 3,200 mg/L obtained at 500 mOsm/kg. Component C was also produced in proportion to the osmolality. Conversion of acarbose to component C was accomplished with resting whole cells. Inhibitors of the conversion of acarbose to component C were sought since component C is probably derived from acarbose. Valienamine was found to be a potent inhibitor, resulting in a more than 90% reduction in component C formation at a 10 μM concentration. Effects were similar in a 1,500‐L pilot fermentor with acarbose and component C yields of 3,490 and 43 mg/L at 500 mOsm/kg, respectively.
Oxidation‐Reduction Potential as a Control Variable for the Anaerobic Stage during Anaerobic‐Aerobic p‐Nitrophenol Degradation
Authors: Buitrón Germán , Betancur Manuel J. , Moreno Gloria , Moreno Jaime A.
Published: December 2003

Combined anaerobic‐aerobic processes are a viable alternative for the treatment of xenobiotic compounds that are difficult to treat by traditional processes. The variable nature of the sequencing batch reactors, SBR, systems allows manipulation of the selective pressure on the microorganisms. Then, the activity of the community can be dynamically adjusted to meet changing effluents conditions. To improve the response of the SBR to changing influent conditions, several efforts have been made to automate and control the duration of the sequential phases of the SBR. The objective of this work is to present and discuss the feasibility of the use of the oxidation‐reduction potential, ORP, as a control variable for the determination of the anaerobic phase length in an anaerobic‐aerobic SBR used to degrade p‐nitrophenol, PNP. The control of the anaerobic phase of the anaerobic‐aerobic reactor was achieved with software developed at the Institute of Engineering‐UNAM. During the anaerobic stage, the PNP is reduced to p‐aminophenol, PAP. As a consequence of the compound transformation, there is a change in the oxidation‐reduction potential of the culture medium. This change was used to indicate the minimal concentration of PNP and, as a consequence, the maximal PAP concentration. The feasibility of the algorithm for using the variations in the ORP to determine on‐line the length of the anaerobic stage in an anaerobic‐aerobic process was demonstrated in our laboratory.
Using PC12 Cells To Evaluate Poly(caprolactone) and Collagenous Microcarriers for Applications in Nerve Guide Fabrication
Authors: Waddell Rebecca L. , Marra Kacey G. , Collins Kelly L. , Leung Jade T. , Doctor John S.
Published: December 2003

Tissue‐engineered nerve guides can provide mechanical support as well as chemical stimulation for nerve regeneration. PC12 cells were used to test the novel combination of poly(caprolactone) (PCL) and macroporous collagen‐based microcarriers (CultiSphers) as an initial phase in the fabrication of multichanneled nerve guides. Laminin‐coated PCL was an effective matrix for the attachment, proliferation, and viability of PC12 cells, relative to uncoated PCL. PC12 cells attached to laminin‐coated PCL and extended neurites when cultured in the presence of nerve growth factor (NGF). PC12 cells attached and proliferated on CultiSphers and extended neurites in response to NGF. A novel PCL/CultiSpher composite material also supported PC12 attachment and proliferation and provides a potentially useful material for the fabrication of synthetic nerve guides.
Assessment of In‐Line Near‐Infrared Spectroscopy for Continuous Monitoring of Fermentation Processes
Authors: Tosi Simona , Rossi Maddalena , Tamburini Elena , Vaccari Giuseppe , Amaretti Alberto , Matteuzzi Diego
Published: December 2003

The application of NIR in‐line to monitor and control fermentation processes was investigated. Determination of biomass, glucose, and lactic and acetic acids during fermentations of Staphylococcus xylosus ES13 was performed by an interactance fiber optic probe immersed into the culture broth and connected to a NIR instrument. Partial least squares regression (PLSR) calibration models of second derivative NIR spectra in the 700–1800 nm region gave satisfactory predictive models for all parameters of interest: biomass, glucose, and lactic and acetic acids. Batch, repeated batch, and continuous fermentations were monitored and automatically controlled by interfacing the NIR to the bioreactor control unit. The high frequency of data collection permitted an accurate study of the kinetics, supplying lots of data that describe the cultural broth composition and strengthen statistical analysis. Comparison of spectra collected throughout fermentation runs of S. xylosus ES13, Lactobacillus fermentum ES15, and Streptococcus thermophylus ES17 demonstrated the successful extension of a unique calibration model, developed for S. xylosus ES13, to other strains that were differently shaped but growing in the same medium and fermentation conditions. NIR in‐line was so versatile as to measure several biochemical parameters of different bacteria by means of slightly adapted models, avoiding a separate calibration for each strain.
Optimal NS0 Cell Growth in a Hollow Fiber Bioreactor Requires Increased Serum Concentration or a Cholesterol Supplement on the Cell Side of the Fiber
Authors: Gramer Michael J. , Maas Jodi
Published: December 2003

An NSO/GS cell line secreting a humanized antibody was routinely propagated in a T‐flask using 2% serum. For scale‐up of antibody production, this cell line was inoculated into a hollow fiber system using the same serum concentration. The metabolic activity increased for a few days in the hollow fiber system, but invariably the activity dropped dramatically as the cells died by day 7. A hollow fiber micro‐bioreactor was used as a screening tool to examine possible reasons for cell death in the large‐scale system. As seen in the hollow fiber system, cells died when 2% serum was used either on the cell side only or on both sides of the fiber in the micro‐bioreactor. In contrast, the use of 20% serum on the cell side of the fiber and basal medium on the non‐cell side resulted in good cell expansion at high viability. Regardless of the cell side serum concentration, no further growth enhancements were seen when up to 20% serum was placed on the non‐cell side of the fiber. These results suggest that a serum component that does not readily cross the fiber is limiting cell growth in the hollow fiber bioreactors. The addition of a cholesterol‐rich lipid supplement resulted in better cell growth in the micro‐bioreactor, while the addition of other non‐cholesterol lipid supplements resulted in no growth enhancement. The growth‐enhancing properties of the cholesterol supplement were more pronounced at lower serum concentrations, suggesting that poor growth at low serum concentration was due to suboptimal cholesterol levels. When the cell side serum concentration was increased to 20% in the hollow fiber system, cells grew and filled the bioreactor, allowing a 39‐day production run. These results demonstrate that this NSO cell line requires an increased cell side serum concentration for optimal growth and that this requirement is likely due to the inherent cholesterol dependency of this cell line.
Retroviral Delivery of the Ecdysone‐Regulatable Gene Expression System
Authors: Vaillancourt Peter , Felts Katherine A.
Published: December 2003

We describe a set of Moloney Murine Leukemia Virus (MoMLV)‐based replication‐defective retroviral vectors for delivery of the ecdysone‐inducible system into mammalian cells. The vector pFB‐ERV contains a tricistronic CMV expression cassette from which the ecdysone receptor proteins RXR and VgEcR are expressed, with the neo‐resistance marker expressed as the third open reading frame (ORF). The inducible vector pCFB‐EGSH contains an ecdysone‐inducible expression cassette inserted between the viral LTRs in the antisense orientation relative to that for the viral promoter. Potential interference from the proviral 5′ LTR is obviated due to a SIN deletion in the 3′ LTR. When used together, induction ratios of over 1000‐fold were achieved in NIH3T3 cells using firefly luciferase as a reporter.
Neural‐Network‐Based Identification of Tissue‐Type Plasminogen Activator Protein Production and Glycosylation in CHO Cell Culture under Shear Environment
Authors: Senger Ryan S. , Karim M. Nazmul
Published: December 2003

An artificial neural network (ANN) modeling scheme has been constructed for the identification of both recombinant tissue‐type plasminogen activator (r‐tPA) protein production and glycosylation from Chinese hamster ovary (CHO) cell culture, cultivated in a stirred bioreactor. A series of hybrid feed‐forward backpropagation neural networks were constructed to function as a software sensor. This enabled predictions of viable cell density, r‐tPA content, and r‐tPA glycosylation. The sensor was based on an initial input vector space consisting of simple metabolite concentrations, batch cultivation time, and a description of shear stress applied to the culture. Metabolite concentrations of the culture supernatant, included in the input vector space, were obtained from a single isocratic HPLC measurement. The shear stress component of the input space enabled accurate culture state prediction over a wide range of agitation rates. Coefficient of determination (r2) values between ANN predicted and experimental measurements of 0.945, 0.943, 0.956, and 0.990 were calculated to validate individual ANN prediction accuracy for total ammonia, apparent viable cell density, total r‐tPA, and Type II glycoform concentrations, respectively.
An Optimized Method for Aspergillusniger Spore Production on Natural Carrier Substrates
Authors: Bapat Prashant M. , Kundu Sucharita , Wangikar Pramod P.
Published: December 2003

Aspergillus niger spores have wide ranging applications in the fermentation industry as well as in wastewater treatment. We present an optimized method for production of A. niger spores on natural substrates such as rice, split pea, and millet. The specific productivity (number of spores per gram of dry substrate) was 31‐fold greater and volumetric productivity was 750‐fold greater compared to agar slopes. The important process variables were incubation temperature, moisture content, and inoculum quantity. We find that the optimal condition for total spore count is different from the viable spore count for millet. The optimum lies in a narrow region defined by the process parameters. Of the three substrates tested split pea gave the highest specific spore productivity of 3.1 × 1010 spores per gram of dry substrate. This is the first report of systematic study on the effect of process parameters on spore viability. The method of A. niger spore production on natural substrate appears advantageous as compared to the currently practiced method in terms of scale‐up, cost, and ease of operation.
Determination of Size Distribution and Encapsulation Efficiency of Liposome‐Encapsulated Hemoglobin Blood Substitutes Using Asymmetric Flow Field‐Flow Fractionation Coupled with Multi‐Angle Static Light Scattering
Authors: Arifin Dian R. , Palmer Andre F.
Published: December 2003

In this study, we investigated the size distribution, encapsulation efficiency, and oxygen affinity of liposome‐encapsulated tetrameric hemoglobin (LEHb) dispersions and correlated the data with the variation in extruder membrane pore size, ionic strength of the extrusion buffer, and hemoglobin (Hb) concentration. Asymmetric flow field‐flow fractionation (AFFF) in series with multi‐angle static light scattering (MASLS) was used to study the LEHb size distribution. We also introduced a novel method to measure the encapsulation efficiency using a differential interferometric refractive index (DIR) detector coupled to the AFFF‐MASLS system. This technique was nondestructive toward the sample and easy to implement. LEHbs were prepared by extrusion using a lipid combination of dimyristoyl‐phosphatidylcholine, cholesterol, and dimyristoyl‐phosphatidylglycerol in a 10:9:1 molar ratio. Five initial Hb concentrations (50, 100, 150, 200, and 300 mg Hb per mL of buffer) extruded through five different membrane pore diameters (400, 200, 100, 80, and 50 nm) were studied. Phosphate buffered saline (PBS) and phosphate buffer (PB) both at pH 7.3 were used as extrusion buffers. Despite the variation, extrusion through 400‐nm pore diameter membranes produced LEHbs smaller than the pore size, extrusion through 200‐nm membranes produced LEHbs with diameters close to the pore diameter, and extrusion through 100‐, 80‐, and 50‐nm membranes produced LEHbs larger than the pore sizes. We found that the choice of extrusion buffer had the greatest effect on the LEHb size distribution compared to either Hb concentration or extruder membrane pore size. Extrusion in PBS produced larger LEHbs and more monodisperse LEHb dispersions. However, LEHbs extruded in PB generally had higher Hb encapsulation efficiencies and lower methemoglobin (metHb) levels. The choice of extrusion buffer also affected how the encapsulation efficiency correlated with Hb concentration, extruder pore size, and the metHb level. The most optimum encapsulation efficiency and amount of Hb entrapped were achieved at the highest Hb concentration and the largest pore size for both extrusion buffers (62.38% and 187.14 mg Hb/mL of LEHb dispersion extruded in PBS, and 69.98% and 209.94 mg Hb/mL of LEHb dispersion extruded in PB). All LEHbs displayed good oxygen‐carrying properties as indicated by their P50 and cooperativity coefficients. LEHbs extruded in PB had an average P50 of 23.04 mmHg and an average Hill number of 2.29, and those extruded in PBS had average values of 27.25 mmHg and 2.49. These oxygen‐binding properties indicate that LEHbs possess strong potential as artificial blood substitutes. In addition, the metHb levels in PB‐LEHb dispersions are significantly low even in the absence of antioxidants such as N‐acetyl‐l‐cysteine.
Highly Efficient Conversion of Lactate to Pyruvate Using Whole Cells of Acinetobacter sp.
Authors: Ma Cui Qing , Xu Ping , Dou Yi Meng , Qu Yin Bo
Published: December 2003

On an industrial scale, the production of pyruvate at a high concentration from the cheaper lactate substrate is a valuable process. To produce pyruvate from lactate by whole cells, various lactate‐utilizing microorganisms were isolated from soil samples. Among them, strain WLIS, identified as Acinetobacter sp., was screened as a pyruvate producer. For the pyruvate preparation from lactate, the preparative conditions were optimized with whole cells of the strain. The cells cultivated in the medium containing 100 mM of l‐lactate showed the highest biotransformation efficiency from lactate to pyruvate. The optimized dry‐cell concentration, pH, and temperature of reaction were 6 g/L, pH 7.0–7.5, and 30 °C, respectively. The influences of ethylenediaminetetraacetic acid (EDTA) and aeration on a biotransformation reaction were carried out under the test conditions. Under the optimized reaction conditions, l‐lactate at concentrations of 200 and 500 mM were almost totally stoichiometrically converted into pyruvate in 8 and 12 h, respectively. About 60% of 800 mM of l‐lactate was transformed into pyruvate in 24 h. This reduced conversion rate is probably due to the high substrate inhibition in biotransformation.
Effects of Epidermal Growth Factor on Fibroblast Migration through Biomimetic Hydrogels
Authors: Gobin Andrea S. , West Jennifer L.
Published: December 2003

We have previously reported on the development and use of synthetic hydrogel extracellular matrix (ECM) analogues that can be used to study the mechanisms of migration. These biomimetic hydrogels consist of bioinert poly(ethylene glycol) diacrylate derivatives with proteolytically degradable peptide sequences included in the backbone of the polymer and adhesion peptide sequences grafted into the network. Cells adhere to the hydrogel via interaction between the grafted adhesion ligands and receptors on the cell surface. The cells migrate through the three‐dimensional system by secreting the appropriate proteolytic enzymes, which are involved in cell migration and are targeted to the peptide sequences incorporated in the backbone of the polymer. It was observed that cell migration has a biphasic dependence on adhesion ligand concentration, with optimal migration at intermediate ligand levels. In this study, we demonstrate that we can covalently attach epidermal growth factor (EGF) to PEG and graft them into the hydrogels. It was observed that EGF when tethered maintained mitogenic activity. It was also observed that fibroblast migration significantly increased in the presence of the grafted EGF through the collagenase‐sensitive hydrogels. In addition, the increase in migration was found to be independent from the proliferative response of the cells. These synthetic ECM analogues allow one to systematically control identities and concentrations of biomolecules and are useful tools to study mechanisms of cell migration.
Field‐Flow Fractionation as Analytical Technique for the Characterization of Dry Yeast: Correlation with Wine Fermentation Activity
Authors: Sanz Ramsés , Galceran M Teresa , Puignou Lluís
Published: December 2003

Important oenological properties of wine depend on the winemaking yeast used in the fermentation process. There is considerable controversy about the quality of yeast, and a simple and cheap analytical methodology for quality control of yeast is needed. Gravitational field flow fractionation (GFFF) was used to characterize several commercial active dry wine yeasts from Saccharomyces cerevisiae and Saccharomyces bayanus and to assess the quality of the raw material before use. Laboratory‐scale fermentations were performed using two different S. cerevisiae strains as inocula, and GFFF was used to follow the behavior of yeast cells during alcoholic fermentation. The viable/nonviable cell ratio was obtained by flow cytometry (FC) using propidium iodide as fluorescent dye. In each experiment, the amount of dry wine yeast to be used was calculated in order to provide the same quantity of viable cells. Kinetic studies of the fermentation process were performed controlling the density of the must, from 1.071 to 0.989 (20/20 density), and the total residual sugars, from 170 to 3 g/L. During the wine fermentation process, differences in the peak profiles obtained by GFFF between the two types of commercial yeasts that can be related with the unlike cell growth were observed. Moreover, the strains showed different fermentation kinetic profiles that could be correlated with the corresponding fractograms monitored by GFFF. These results allow optimism that sedimentation FFF techniques could be successfully used for quality assessment of the raw material and to predict yeast behavior during yeast‐based bioprocesses such as wine production.
Purification and Concentration of Alkaline Phosphatase by Selective Permeabilization of Escherichia coli Using Reverse Micellar Solutions
Authors: Bansal‐Mutalik Ritu , Gaikar Vilas G.
Published: December 2003

Recovery of alkaline phosphatase (AP) from the periplasm of Escherichia coli using reverse micellar solutions (RMSs) of sodium dioctyl sulfosuccinate (AOT) in aliphatic hydrocarbons has been attempted. A variety of surface‐active agents, solvents, and reverse micellar conditions were screened, and an excellent recovery of the enzyme in a concentrated form, with a high purification factor, was obtained in a single‐step process. The permeabilization process strongly depended on the water content of the RMS as well as on the amount of water coating the microbial cell surface. The product was almost free from nucleic acids. In addition, because of the low affinity of AOT and the organic solvent for the aqueous phase, contamination by the permeabilizing agents would also be negligible.
Generation of a Telomere‐Based Episomal Vector
Authors: d'Aiuto Leonardo , Heras Jose I. de las , Ross Andrew , Shen Ming Hong , Cooke Howard
Published: December 2003

We have developed a telomere‐based episome by large‐scale amplification in Escherichia colicells. This episome consists of a PAC vector in which a 6 Kb sequence, containing an array of telomeric repeats spaced by a synthetic sequence, is tandemly repeated by large‐scale multimerization in E. coli. After transfection in human HT1080 cells, the construct, called clone 106, was able to persist in episomal form or integrated into some endogenous chromosomes. Integrations occurred exclusively at the telomeres. Episomes were still present in HT1080 cells after more than 100 days in the absence of selection. Integrations of clone 106 into the telomeric regions were retained only under selective conditions, and when the selection was removed the construct was progressively eliminated from the chromosome. The long‐term maintenance of clone 106 into human cells as an episome and its ability to integrate transiently into the telomeres of the host chromosomes suggest that this PAC‐based episome is potentially a good candidate vector for gene therapy applications.
Scale‐Up of a Liquid Static Culture Process for Hyperproduction of Ganoderic Acid by the Medicinal Mushroom Ganodermalucidum
Authors: Tang Ya‐Jie , Zhong Jian‐Jiang
Published: December 2003

Scale‐up of a liquid static culture process was studied for hyperproduction of ganoderic acid (GA) by a famous Chinese traditional medicinal mushroom, Ganoderma lucidum. Initial volumetric oxygen transfer coefficient (KLa) and area of liquid surface per liquid volume (As) were identified as key factors affecting cell growth and GA accumulation in liquid static cultures of G. lucidum, on the basis of which a multilayer static bioreactor was designed. At a low initial KLa level of 2.1 h‐1, a thick layer of white mycelia was formed on the liquid surface, and an optimal production of total GA (i.e., GA production in the liquid and on the liquid surface) was obtained. Both the formation of white mycelia and production of GA on the liquid surface were enhanced with an increase of As within the range as investigated (0.24–1.53 cm2/mL). At an As value of 0.90 cm2/mL, the total GA production reached maximum. A successful scale‐up from a 20‐mL static T‐flask to a 7.5‐L three‐layer static bioreactor was achieved based on initial KLa. The maximum biomass (20.8 ± 0.1 g DW/L), GA content (4.96 ± 0.13 mg/100 mg DW), and total GA production (976 ± 35 mg/L) were attained in static bioreactors. Not only GA content but also its production obtained in this work were the highest ever reported.
Mechanisms Controlling the Temperature‐Dependent Binding of Proteins to Poly(N‐isopropylacrylamide) Microgels
Authors: Grabstain Violet , Bianco‐Peled Havazelet
Published: December 2003

Poly(N‐isopropylacrylamide) (PNIPA) microgels may offer several advantages over PNIPA‐modified surfaces when used as sorbents in temperature‐sensitive chromatography. Yet, a full exploitation of these advantages requires a better understanding of the mechanisms controlling the separation process. As a model system, we have studied the binding of three proteins (bovine serum albumin (BSA), ovalbumin, and lysozyme) to PNIPA microgels. Binding experiments were conducted both below (25 °C) and above (37 °C) the volume phase transition temperature of the gel, Tc. The analysis of the binding isotherms has shown that although an average gel particle contained a larger amount of protein below the phase transition temperature, the concentration of the protein within the particle was higher above this temperature. These findings were attributed to changes in the binding loci due to temperature swings around Tc: whereas a sorption mechanism is dominant below this temperature, surface‐adsorption was more important above it. A comparison between the three studied proteins has shown that below Tc the binding increases with a decrease in the molecular weight. On the other hand, no significant difference in the bound protein amounts was observed above the phase transition temperature. Our results imply that, despite the increase in the gelapos;s hydrophobicity above the phase transition temperature, the resolution in bioseparations based on PNIPA gels is not necessarily better above Tc.
Detection of Benzene, Toluene, Ethyl Benzene, and Xylenes (BTEX) Using Toluene Dioxygenase‐Peroxidase Coupling Reactions
Authors: Xu Zhaohui , Mulchandani Ashok , Chen Wilfred
Published: December 2003

We have developed a simple, whole‐cell bioassay for the detection of bioavailable benzene, toluene, ethyl benzene, and xylenes (BTEX) and similar compounds. A genetically engineered E. coli strain expressing toluene dioxygenase (TDO) and toluene dihydrodiol dehydrogenase (TodD) was constructed, enabling the conversion of BTEX into their respective catechols, which were quickly converted into colored products by a horseradish peroxidase (HRP)‐coupled reaction. The intensity of the color formation was correlated to concentrations of the BTEX compounds. Under the optimized conditions, a detection limit (defined as three times the standard deviation of the response obtained from the blank) of 10, 10, 20, and 50 μM was observed for benzene, toluene, ethyl benzene, and xylene, respectively. The bioassay was selective toward BTEX‐related compounds with no interference observed with commonly used pesticides, herbicides, and organic solvent. The bioassay was very stable with little change in response over a 10‐week period. The excellent stability suggests that the reported bioassay may be suitable for field monitoring of BTEX to identify and track contaminated water and follow the bioremediation progress.