Undergraduate research in a biotechnology teaching laboratory: expression and purification of a tyrosine kinase

The class consisted of senior molecular biology majors who had previously taken cell biology, microbiology, biochemistry and molecular biology lecture courses but who had little or no previous lab experience. These students were asked to design and create an expression vector and purify the expressed protein. This project provides the students with the opportunity to appreciate interconnections between experiments while learning the necessary techniques. Journal of Industrial Microbiology & Biotechnology (2000) 24, 359–363. Received 02 April 1999/ Accepted in revised form 10 November 1999     

Bioinformatics in the community college

Biotechnology is becoming an information-based field. In this article we describe some resources available to instructors, show how these resources are used in the biotechnology training program, and provide examples of activities used by non-science majors to increase their understanding of biology. We discuss some of the challenges we have encountered using these tools in the classroom. Journal of Industrial Microbiology & Biotechnology (2000) 24, 314–318. Received 08 April 1999/ Accepted in revised form 10 November 1999     

Integrated cell biology/biochemistry/molecular genetics laboratories: the cytoplasmic genome projects

This paper describes an integrated laboratory project for intermediate to advanced undergraduate students. The project spans an entire academic quarter (10 weeks) and involves a series of operations that give students experience with fundamental techniques in cell biology, molecular biology, biochemistry, genomics, and bioinformatics. In the process, the student learning community is strengthened, students gain increasing confidence in their abilities in the laboratory, and data are collected toward the eventual sequencing of a cytoplasmic genome. The culmination of the project is the preparation by students of a paper written in the format of a particularly accessible online journal. Journal of Industrial Microbiology & Biotechnology (2000) 24, 339–344. Received 02 April 1999/ Accepted in revised form 22 November 1999     

A series of laboratory exercises utilizing luxR gene of Vibrio fischeri and gfp gene of Aequoria victoria to teach the broad applications of polymerase chain reaction

Three experiments are described; directional cloning of the luxR gene from the bioluminescent marine bacterium, Vibrio fischeri, directional cloning of the gfpgene from the marine jelly fish, Aequoria victoria, and the construction of a LuxR-GFP fusion protein. Experiments are presented using lux and gfp in an undergraduate biology curriculum. Journal of Industrial Microbiology & Biotechnology (2000) 24, 345–352. Received 02 April 1999/ Accepted in revised form 19 November 1999     

Production process for recombinant human angiostatin in Pichia pastoris

A pilot-scale production method of recombinant human angiostatin, a 38-kD fragment of plasminogen which has been reported to have antiangiogenic activity, has been successfully established by expressing the protein in the methylotrophic yeast Pichia pastoris. The secreted protein inhibited cultured endothelial cell proliferation in vitro and Lewis lung carcinoma growth in mice. The fermentation process was carried out using an on-line methanol controller, administering methanol to the growing culture and keeping its concentration under 2 g L⁻¹. The fermentation lasted 90 h, of which 70 h were growth on methanol. During growth on methanol the culture volume increased 64%, from 7 L to 11.5 L, producing 200 mg angiostatin and 5 kg of biomass. Journal of Industrial Microbiology & Biotechnology (2000) 24, 31–35. Received 12 May 1999/ Accepted in revised form 06 September 1999     

Light-dependent transformation of anthranilate to indole by Rhodobacter sphaeroides OU5

Rhodobacter sphaeroides OU5 transformed anthranilate (2 mM) to an indole (0.7 mM) in a light-dependent process. Photobiotransformation was enhanced by tricarboxylic acid cycle intermediates and the indole formed was identified as 2,3 dihydroxy indole. Journal of Industrial Microbiology & Biotechnology (2000) 24, 219–221. Received 16 September 1999/ Accepted in revised form 20 December 1999     

Biotechnology training at California State University, Hayward: a model program

The Biotechnology Certificate Program (BCP) at California State University, Hayward was initiated in 1986 in response to industry demands for qualified employees in the molecular life sciences. This 9-month post-baccalaureate program includes laboratory courses in recombinant DNA techniques, protein chemistry, PCR, DNA sequencing, animal cell culture as well as two lecture courses in molecular biology. Rigorous selection at both entry and exit stages of the program ensures knowledgeable graduates with a greater than 90% employment placement. Corporate participation has been a cornerstone of the BCP and we anticipate continued cooperation in the future as the program evolves to meet the expanding needs of the biotechnology industry. Journal of Industrial Microbiology & Biotechnology (2000) 24, 364–366. Received 19 April 1999/ Accepted in revised form 09 November 1999     

Integration of laboratory exercises in developmental biology and neurobiology courses using the Xenopus oocyte expression system

This collaborative laboratory exercise integrates two upper division laboratory courses (Developmental Biology and Neurobiology) offered to biology majors at Wake Forest University. The laboratory exercise involves the use of the Xenopus oocyte expression system to study the function of specific membrane receptors and ligand-activated channels. cDNA or mRNA for receptor proteins is injected into Xenopus oocytes. The oocytes are assayed for expression of receptor proteins and two-electrode voltage clamping is done to determine whether the expressed proteins are functional in the oocyte system. This series of laboratory exercises is innovative in its interdisciplinary and collaborative approach to undergraduate teaching, and in its use of sophisticated molecular biological and physiological techniques in the undergraduate teaching laboratory. Students learn first-hand how these techniques have been used to achieve a new level of understanding of both development and neurobiology. Journal of Industrial Microbiology & Biotechnology (2000) 24, 353–358. Received 02 April 1999/ Accepted in revised form 10 November 1999     

Human trachea primary epithelial cells express both sialyl(α2-3)Gal receptor for human parainfluenza virus type 1 and avian influenza viruses, and sialyl(α2-6)Gal receptor for human influenza viruses

We reported previously that the dominant receptors of influenza A and B viruses, and human and murine respiroviruses, were sialylglycoproteins and gangliosides containing monosialo-lactosamine type I-and II-residues, such as sialic acid-α2-3(6)-Galβ1-3(4)-GlcNAcβ1-. In addition, the Siaα2-3Gal linkage was predominantly recognized by avian and horse influenza viruses, and human parainfluenza virus type 1 (hPIV-1), whereas the Siaα2-6Gal linkage was mainly recognized by human influenza viruses (Paulson JC in “The Receptors' [Conn M Ed] 2, 131–219 (1985); Suzuki Y, Prog Lipid Res 33, 429–57 (1994); Ito T, J Virol 73, 6743–51 (2000); Suzuki Y, J Virol 74, 11825–31 (2000); Suzuki T, J. Virol 75, 4604–4613 (2001); Suzuki Y, Biol. Pharm. Bull. 28, 399–408 (2005)). To clarify the distribution of influenza virus receptors on the human bronchial epithelium cell surface, we investigated a primary culture of normal human bronchial epithelial (NHBE) cells using two types of lectin (MAA and SNA), which recognize sialyl linkages (α2-3 and α2-6), using fluorescence-activated cell-sorting analysis. The results showed that both α2-3- and α2-6-linked Sias were expressed on the surface of primary human bronchial epithelial cells. The cells infected by hPIV-1 bound to MAA, confirming that cells targeted by hPIV-1 have α2-3-linked oligosaccharides. We also compared the ability of hPIV-1 and human influenza A virus to infect primary human bronchial epithelial cells pre-treated with Siaα2-3Gal-specific sialidase from Salmonella typhimurium. No difference was observed in the number of sialidase pre-treated and non-treated cells infected with human influenza A virus, which binds to Siaα2-6Gal-linked oligosaccharides. By contrast, the number of cells infected with hPIV-1 decreased significantly upon sialidase treatment. Thus, cultured NHBE cells showed both α2-3-linked Sias recognized by hPIV-1 and avian influenza virus receptors, and α2-6-linked Sias recognized by human influenza virus receptors.     

Process development for production of recombinant human insulin-like growth factor-I in Escherichia coli

Fed-batch cultures were carried out to overproduce human insulin-like growth factor I (IGF-I) in Escherichia coli. The effects of carbon sources (glucose or glycerol) and induction time on cell growth and IGF-I production were investigated in more detail. Glycerol was a better carbon source than glucose for IGF-I production in fed-batch culture. Induction at the mid-exponential phase with glycerol as a carbon source in the pH-stat fed-batch culture was optimal for IGF-I production. Under this condition, 2.8 g L⁻¹ of fusion IGF-I was produced as inclusion bodies. We have also developed downstream processing for preparative scale purification of IGF-I from the fusion protein produced by the fed-batch culture using glycerol as a carbon source. After the fusion protein expressed was solubilized in 8 M urea and cleaved with hydroxylamine, the released IGF-I was purified by cation exchange chromatography, refolding and preparative scale reverse phase HPLC (rp-HPLC) to give recombinant IGF-I of >98% purity. The biological activities of the purified IGF-I were measured and found to be identical to those of commercial IGF-I. Journal of Industrial Microbiology & Biotechnology (2000) 24, 94–99. Received 13 January 1999/ Accepted in revised form 02 October 1999     

Comparison of exopolysaccharide production by strains of Lactobacillus rhamnosus and Lactobacillus paracasei grown in chemically defined medium and milk

Exopolysaccharide (EPS) production was compared among three strains of lactobacilli. Lactobacillus rhamnosus strain 9595M can be classified among the highest EPS-producing strains of lactic acid bacteria reported to date with a maximum EPS production of 1275 mg L⁻¹. Under controlled pH, no significant differences in the quantity of EPS produced could be detected between carbon source (glucose or lactose) or fermentation temperature (32 or 37°C). In milk, strains ATCC 9595M and R produced more than 280 mg L⁻¹ EPS whereas strain Type V produced less than 80 mg L⁻¹ EPS. Journal of Industrial Microbiology & Biotechnology (2000) 24, 251–255. Received 10 September 1999/ Accepted in revised form 22 December 1999     

Production of carotenoids by Brevibacterium linens: variation among strains, kinetic aspects and HPLC profiles

This work describes carotenoid pigment production by the red bacterium Brevibacterium linens covering strain diversity, kinetic and analytical aspects. Pigment production of 23 B. linens strains ranged from 0.05 to 0.60 mg pigments L⁻¹ culture, with specific productivity from 0.2 to 0.6 mg pigments per g dry biomass. The pigment production time curve showed a sigmoid shape, that matched cell growth. HPLC analysis revealed three groups of peaks, possibly non-hydroxylated, mono- and di-hydroxylated carotenoids. Polar molecules were mainly represented. Journal of Industrial Microbiology & Biotechnology (2000) 24, 64–70. Received 19 April 1999/ Accepted in revised form 25 September 1999     

Simultaneous interpretation of Mössbauer, EPR and 57Fe ENDOR spectra of the [Fe4S4] cluster in the high-potential iron protein I Ectothiorhodospira halophila

4 S₄]^3 + and the reduced [Fe₄S₄]^2 + clusters in the high-potential iron protein I from Ectothiorhodospira halophila were measured in a temperature range from 5 K to 240 K. EPR measurements and ⁵⁷Fe electron-nuclear double resonance (ENDOR) experiments were carried out with the oxidized protein. In the oxidized state the cluster has a net spin S = 1/2 and is paramagnetic. As common in [Fe₄S₄]^3 + clusters, the M?ssbauer spectrum was simulated with two species contributing equally to the absorption area: two Fe^3 + atoms couple to the “ferric-ferric” pair, and one Fe^2 + and one Fe^3 + atom give the “ferric-ferrous pair”. For the simulation of the M?ssbauer spectrum, g-values were taken from EPR measurements. A-tensor components were determined by ⁵⁷Fe ENDOR experiments that turned out to be a necessary source of estimating parameters independently. In order to obtain a detailed agreement of M?ssbauer and ENDOR data, electronic relaxation has to be taken into account. Relaxing the symmetry condition in a way that the electric field gradient tensor does not coincide with g- and A-tensors yielded an even better agreement of experimental and theoretical M?ssbauer spectra. Spin-spin and spin-lattice relaxation times were estimated by pulsed EPR; the former turned out to be the dominating mechanism at T = 5 K. Relaxation times measured by pulsed EPR and obtained from the M?ssbauer fit were compared and yield nearly identical values. The reduced cluster has one additional electron and has a diamagnetic (S = 0) ground state. All the four irons are indistinguishable in the M?ssbauer spectrum, indicating a mixed-valence state of Fe^2.5 + for each. Received: 15 February 1999 / Accepted: 31 August 1999     

Formation of α-D-glucose-1-phosphate by thermophilic α-1,4-D-glucan phosphorylase

For the production of α-D-glucose-1-phosphate (G-1-P), α-1,4-D-glucan phosphorylase from Thermus caldophilus GK24 was partially purified to a specific activity of 13 U mg⁻¹ and an enzyme recovery of 15%. The amount of G-1-P reached maximum (18%) when soluble starch was used as substrate, and the smallest substrate for G-1-P formation was maltotriose. The structure of purified G-1-P was confirmed by comparison to ¹³C-NMR data for an authentic sample. In addition to G-1-P, glucose-6-phosphate (12%) was simultaneously produced when 10 mM maltoheptaose was used as substrate. Journal of Industrial Microbiology & Biotechnology (2000) 24, 89–93. Received 12 May 1999/ Accepted in revised form 29 August 1999     

Homology modeling and dynamics of the extracellular domain of rat and human neuronal nicotinic acetylcholine receptor subtypes α4β2 and α7

In recent years, it has become clear that the neuronal nicotinic acetylcholine receptor (nAChR) is a valid target in the treatment of a variety of diseases, including Alzheimer’s disease, anxiety, and nicotine addiction. As with most membrane proteins, information on the three-dimensional (3D) structure of nAChR is limited to data from electron microscopy, at a resolution that makes the application of structure-based design approaches to develop specific ligands difficult. Based on a high-resolution crystal structure of AChBP, homology models of the extracellular domain of the neuronal rat and human nAChR subtypes α4β2 and α7 (the subtypes most abundant in brain) were built, and their stability assessed with molecular dynamics (MD). All models built showed conformational stability over time, confirming the quality of the starting 3D model. Lipophilicity and electrostatic potential studies performed on the rat and human α4β2 and α7 nicotinic models were compared to AChBP, revealing the importance of the hydrophobic aromatic pocket and the critical role of the α-subunit Trp—the homolog of AChBP-Trp 143—for ligand binding. The models presented provide a valuable framework for the structure-based design of specific α4β2 nAChR subtype ligands aimed at improving therapeutic and diagnostic applications. Figure Electrostatic surface potential of the binding site cavity of the neuronal nicotinic acetylcholine receptor (nAChR). Nicotinic models performed with the MOLCAD program: a rat α7, b rat α4β2, c human α7, d human α4β2. All residues labeled are part of the α7 (a,c) or α4 (b,d) subunit with the exception of Phe 117, which belongs to subunit β2 (d). Violet Very negative, blue negative, yellow neutral, red very positive     

Increased astaxanthin production by a Phaffia rhodozyma mutant grown on date juice from Yucca fillifera

The wild strain and the astaxanthin-overproducing mutant strain 25–2 of Phaffia rhodozyma were analyzed in order to assess their ability to grow and synthesize astaxanthin in a minimal medium composed of g L⁻¹: KH₂PO₄ 2.0; MgSO₄ 0.5; CaCl₂ 0.1; urea 1.0 and supplemented with date juice of Yucca fillifera as a carbon source (yuca medium). The highest astaxanthin production (6170 μg L⁻¹) was obtained at 22.5 g L⁻¹ of reducing sugars. The addition of yeast extract to the yuca medium at concentrations of 0.5–3.0 g L⁻¹ inhibited astaxanthin synthesis. The yuca medium supported a higher production of astaxanthin, 2.5-fold more than that observed in the YM medium. Journal of Industrial Microbiology & Biotechnology (2000) 24, 187–190. Received 14 July 1999/ Accepted in revised form 02 December 1999     

Bacterial colonization of rigid gas permeable and hydrogel contact lenses by Staphylococcus aureus

Staphylococcus aureus ATCC 6538 and a clinical isolate of S. aureus from a bacterial keratitis patient were examined for their ability to adhere to etafilcon A, polymacon, silafocon, and pauflufocon A, B and C contact lenses. Both isolates adhered more to the rigid gas permeable (RGP) materials than to the hydrogel lenses tested (P < 0.05). S. aureus ATCC 6538 adhered to the etafilcon A material to a greater extent than did the clinical isolate (P < 0.05). there were no statistically significant differences in the recovery of staphylococci from unworn lens materials when surface area, composition and ionicity were evaluated for either the hydrogel or rgp lenses tested against lenses of a similar type. however, differences were observed when hydrogel lenses were evaluated against rgp lenses (P < 0.05). these differences may be related to water content. Journal of Industrial Microbiology & Biotechnology (2000) 24, 113–115. Received 12 August 1999/ Accepted in revised form 02 November 1999     

Saccharomyces cerevisiae strains with different degrees of ethanol tolerance exhibit different adaptive responses to produced ethanol

Two Saccharomyces cerevisiae strains with different degrees of ethanol tolerance adapted differently to produced ethanol. Adaptation in the less ethanol-tolerant strain was high and resulted in a reduced formation of ethanol-induced respiratory deficient mutants and an increased ergosterol content of the cells. Adaptation in the more ethanol-tolerant strain was less pronounced. Journal of Industrial Microbiology & Biotechnology (2000) 24, 75–78. Received 22 June 1999/ Accepted in revised form 06 October 1999     

Anomalies in the growth kinetics of Saccharomyces cerevisiae strains in aerobic chemostat cultures

Aerobic glucose-limited chemostat cultivations were conducted with Saccharomyces cerevisiae strains NRRL Y132, ATCC 4126 and CBS 8066, using a complex medium. At low dilution rates all three strains utilised glucose oxidatively with high biomass yield coefficients, no ethanol production and very low steady-state residual glucose concentrations in the culture. Above a threshold dilution rate, respiro-fermentative (oxido-reductive) metabolism commenced, with simultaneous respiration and fermentation occurring, which is typical of Crabtree-positive yeasts. However, at high dilution rates the three strains responded differently. At high dilution rates S. cerevisiae CBS 8066 produced 7–8 g ethanol L⁻¹ from 20 g glucose L⁻¹ with concomitant low levels of residual glucose, which increased markedly only close to the wash-out dilution rate. By contrast, in the respiro-fermentative region both S. cerevisiae ATCC 4126 and NRRL Y132 produced much lower levels of ethanol (3–4 g L⁻¹) than S. cerevisiae CBS 8066, concomitant with very high residual sugar concentrations, which was a significant deviation from Monod kinetics and appeared to be associated either with high growth rates or with a fermentative (or respiro-fermentative) metabolism. Supplementation of the cultures with inorganic or organic nutrients failed to improve ethanol production or glucose assimilation. Journal of Industrial Microbiology & Biotechnology (2000) 24, 231–236. Received 09 August 1999/ Accepted in revised form 18 December 1999     

Some considerations on relational equilibria

A previous study (Bull. Math. Biophysics,31, 417–427, 1969) on the definitions of stability of equilibria in organismic sets determined byQ relations is continued. An attempt is made to bring this definition into a form as similar as possible to that used in physical systems determined byF-relations. With examples taken from physics, biology and sociology, it is shown that a definition of equilibria forQ-relational systems similar to the definitions used in physics can be obtained, provided the concept of stable or unstable structures of a system determined byQ-relations is considered in a probabilistic manner. This offers an illustration of “fuzzy categories,” a notion introduced by I. Bąianu and M. Marinescu (Bull. Math. Biophysics,30, 625–635, 1968), in their paper on organismic supercategories, which is designed to provide a mathematical formalism for Rashevsky's theory of Organismic Sets (Bull. Math. Biophysics,29, 389–393, 1967;30, 163–174, 1968;31, 159–198, 1969). A suggestion is made for a method of mapping the abstract discrete space ofQ-relations on a continuum of variables ofF-relations. Problems of polymorphism and metamorphosis, both in biological and social organisms, are discussed in the light of the theory.