Gyratory Shaker Research Articles

Standardization of optimum sieve size for maximizing seed quality in Amaranthus (Amaranthus tricolor L.)

Vegetables have a critical function in human health and nutritional security. Vegetables are considered an essential building block of any diet. Out of the leafy vegetables, Amaranthus is the most popular and salable vegetable consumed by people all over India. Seed processing experiment was undertaken in Amaranthus tricolor (CO 2) by using the sieves placed inside the mechanical seed shaker (Gyratory sieve shaker) to improve the quality of Amaranthus seeds. The seeds of Amaranthus were size graded with seed shaker attached with various sieve size of BSS 18 X18 (R), BSS 20 X 20 (R), BSS 22 X 22 (R) and BSP 22 X 22 (P). During processing, the machine could be adjusted for 2, 3, 4 and 5 minutes with an oscillating speed of 1440 rpm. The separated seeds were evaluated for seed quality characteristics such as seed germination percentage, shoot length, root length, dry matter production, 1000 seed weight and seed recovery percentage. The results revealed that the sieve size of BSS 22 X 22 (R=retained) mesh sieve had the better quality seeds with a maximum recovery of 67.41 g and when it is operated for a period of 5 minutes. The germination percentage was improved from 77 % to 95 % with 1000 seed weight of 73.21 mg, and the observed recovery was 56 per cent with the vigour index of 1145. Hence, BSS 22 X 22 retained mesh sieve with a duration of 5 minutes could be recommended as an optimum sieve size for grading Amaranthus seeds for improving the seed quality.

Cellular and physical microenvironments regulate the aggressiveness and sunitinib chemosensitivity of clear cell renal cell carcinoma.

Renal cell carcinoma (RCC) is the most predominant type of kidney cancer in adults and is responsible for approximately 85% of clinical cases. The tumor‐specific microenvironment includes both cellular and physical factors, and it regulates the homeostasis and function of cancer cells. Perirenal adipose tissue and tumor‐associated macrophages are the major cellular components of the RCC microenvironment. The RCC microvasculature network generates interstitial fluid flow, which is the movement of fluid through the extracellular compartments of tissues. This fluid flow is a specific physical characteristic of the microenvironment of RCC. We hypothesized that there may be an interaction between the cellular and physical microenvironments and that these two factors may play an important role in regulating the behavior of RCC. To elucidate the effects of adipose tissue, macrophages, and fluid flow stimulation on RCC and to investigate the relationships between these factors, we used a collagen gel culture method to generate cancer–stroma interactions and a gyratory shaker to create fluid flow stimulation. Adipose‐related cells, monocytes, and fluid flow influenced the proliferative potential and invasive capacity of RCC cells. Extracellular signal‐regulated kinase and p38 signaling were regulated either synergistically or independently by both fluid flow and cellular interactions between RCC and adipose tissue fragments or macrophages. Fluid flow stimulation synergistically enhanced the anti‐proliferative effect of sunitinib on RCC cells, but macrophages abolished the synergistic anti‐proliferative effect related to fluid flow stimulation. In conclusion, we established a reconstructed model to investigate the cellular and physical microenvironments of RCC in vitro. Our alternative culture model may provide a promising tool for further therapeutic investigations into many types of cancer. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Reproducibility of superhydrophobic and oleophobic polymeric micro surface topographies

Micro-structured surfaces can provide useful material properties, such as repellency to water, oil, or alcohols. Multiple polymers were investigated based on micro molding replication fidelity of repellent structures using both advancing and receding contact angle measurements. Five different polymers (Loctite 3525, ST-1060, TC-854, TC-8740, and Teflon AF) were chosen based on a range of durometers, for each of which, a recommended curing process was presented. These polymers were micro-structured via a one-step replica molding to create mushroom shaped fibers with overhanging caps. Teflon AF, a low surface energy polymer, produced innovative superhydrophobic as well as oleophobic Micro Surface Topographies (MSTs). Advancing contact angles (CAs) of these microstructures were 166 ± 4.2, 151 ± 2.9, and 119 ± 2.2 when in contact with a water, ethylene, and olive oil droplet respectively. The highest reproducibility was achieved by using a curing procedure of 100 °C for 2 h. However, even in these conditions, 35% of MSTs were not fully reproduced. ST-1060 was considered a good alternative to Teflon AF since, even after five uses of the same casting mold, the advancing CAs decreased less than 2% when in contact with any liquid tested. Polymeric MSTs resistance to an external force was also examined using a gyratory shaker suggesting that softer materials, such as ST-1060, were required to survive exposure to environmental conditions.

Utilization of Airlift Fermenters in the Mass Propagation of Pseudomonas and AureobasidiumSpecies for the Bioremediation of Crude Oil Polluted Aquatic Environments

This study involved the design and fabrication of airlift fermenters for the mass propagation of a consortium of three microorganisms (Pseudomonas aeruginosa, Pseudomonas fluorescence and Aureobasidium pullulans). The efficacy of the blend in the bioremediation of simulated crude oil polluted water systems environments was also tested with a view to generating more data on crude oil degrading microorganisms for bioremediation purposes. The organisms were separately grown on a gyratory shaker at 120 rpm and at 30 °C until notable growths were observed. Equal volume of the grown organisms (500 ml) of mixed culture was used as inoculum for the 4.5 L of medium in the 7 L airlift fermenter. Thereafter, this was transferred into the 45 litres medium in the 70 L fermenter until significant growth was observed at room temperature. Culture broths were withdrawn at intervals for the determination of biomass and residual hydrocarbon concentrations. The product from the 70 L fermenter was introduced into simulated polluted water systems from which samples were withdrawn weekly. Analyses for residual hydrocarbon contents were carried out using appropriate analytical techniques. The results showed that 78.5% hydrocarbon removal was observed in the 7 L fermenter after 72 h, while that of the 70 L fermenter was 97.8% after 180 h of fermentation. Polluted water sample treated with the microbes experienced 96.4% removal after 10 weeks of treatment. It was observed further that the application of NPK fertilizer biostimulant aided microbial activities in the removal of petroleum hydrocarbons than urea, cow dung and poultry droppings. The study has demonstrated that effective bioremediation of crude oil polluted water systems could be achieved through the application of biostimulants with mass propagated crude oil degrading organisms using airlift fermenter systems.
 Keywords: Pseudomonas aeruginosa; Pseudomonas fluorescence; Aureobasidium pullulans; Airlift fermenter; Bioremediation; Biostimulation; Bioaugmentation.

Mesenchymal cells and fluid flow stimulation synergistically regulate the kinetics of corneal epithelial cells at the air-liquid interface.

In vivo microenvironments are critical to tissue homeostasis and wound healing, and the cornea is regulated by a specific microenvironment complex that consists of cell-cell interactions, air-liquid interfaces, and fluid flow stimulation. In this study, we aimed to clarify the effects of and the correlations among these three component factors on the cell kinetics of corneal epithelial cells. Human corneal epithelial-transformed (HCE-T) cells were cocultured with either primary rat corneal fibroblasts or NIH 3T3 fibroblasts. We employed a double-dish culture method to create an air-liquid interface and a gyratory shaker to create fluid flow stimulation. Morphometric and protein expression analyses were performed for the HCE-T cells. Both the primary rat fibroblasts and the NIH 3T3 cells promoted HCE-T cell proliferation, and the presence of fluid flow synergistically enhanced this effect and inhibited the apoptosis of HCE-T cells. Moreover, fluid flow enhanced the emergence of myofibroblasts when cocultured with primary rat fibroblasts or NIH 3T3 cells. Extracellular signal-regulated kinase and p38 signaling were regulated either synergistically or independently by both fluid flow and cellular interaction between the HCE-T and NIH 3T3 cells. The cell-cell interaction and fluid flow stimulation in the air-liquid interface synergistically or independently regulated the behavior of HCE-T cells. Fluid flow accelerated the phenotypic change from corneal fibroblasts and NIH 3T3 cells to myofibroblasts. Elucidation of the multicomponent interplay in this microenvironment will be critical to the homeostasis and regeneration of the cornea and other ocular tissues.

Utilization of Airlift Fermenters in the Mass Propagation of Pseudomonas and AureobasidiumSpecies for the Bioremediation of Crude Oil Polluted Aquatic Environments

This study involved the design and fabrication of airlift fermenters for the mass propagation of a consortium of three microorganisms (Pseudomonas aeruginosa, Pseudomonas fluorescence and Aureobasidium pullulans). The efficacy of the blend in the bioremediation of simulated crude oil polluted water systems environments was also tested with a view to generating more data on crude oil degrading microorganisms for bioremediation purposes. The organisms were separately grown on a gyratory shaker at 120 rpm and at 30 °C until notable growths were observed. Equal volume of the grown organisms (500 ml) of mixed culture was used as inoculum for the 4.5 L of medium in the 7 L airlift fermenter. Thereafter, this was transferred into the 45 litres medium in the 70 L fermenter until significant growth was observed at room temperature. Culture broths were withdrawn at intervals for the determination of biomass and residual hydrocarbon concentrations. The product from the 70 L fermenter was introduced into simulated polluted water systems from which samples were withdrawn weekly. Analyses for residual hydrocarbon contents were carried out using appropriate analytical techniques. The results showed that 78.5% hydrocarbon removal was observed in the 7 L fermenter after 72 h, while that of the 70 L fermenter was 97.8% after 180 h of fermentation. Polluted water sample treated with the microbes experienced 96.4% removal after 10 weeks of treatment. It was observed further that the application of NPK fertilizer biostimulant aided microbial activities in the removal of petroleum hydrocarbons than urea, cow dung and poultry droppings. The study has demonstrated that effective bioremediation of crude oil polluted water systems could be achieved through the application of biostimulants with mass propagated crude oil degrading organisms using airlift fermenter systems. Keywords: Pseudomonas aeruginosa; Pseudomonas fluorescence; Aureobasidium pullulans; Airlift fermenter; Bioremediation; Biostimulation; Bioaugmentation.

Morpho-anatomical study of Stevia rebaudiana roots grown in vitro and in vivo

Stevia rebaudiana (Bertoni) Bertoni, Asteraceae, is used as a food additive because its leaves are a source of steviol glycosides. There are examples of tissue culture based on micropropagation and phytochemical production of S. rebaudiana leaves but there are few studies on adventitious root culture of S. rebaudiana. More than 90% of the plants used in industry are harvested indiscriminately. In order to overcome this situation, the development of methodologies that employ biotechnology, such as root culture, provides suitable alternatives for the sustainable use of plants. The aim of this study was to compare morpho-anatomical transverse sections of S. rebaudiana roots grown in vitro and in vivo. The in vitro system used to maintain root cultures consisted of a gyratory shaker under dark and light conditions and a roller bottle system. Transverse sections of S. rebaudiana roots grown in vitro were structurally and morphologically different when compared to the control plant; roots artificially maintained in culture media can have their development affected by the degree of media aeration, sugar concentration, and light. GC–MS and TLC confirmed that S. rebaudiana roots grown in vitro have the ability to produce metabolites, which can be similar to those produced by wild plants.

Hepatoprotective properties and biotransformation of berberine and berberrubine by cell suspension cultures of Dodonaea viscosa and Ocimum basilicum

IntroductionBiotransformations are having great potential to generate novel or known products more efficiently. Berberine (BB) represents one of the most studied among the naturally occurring protoberberine alkaloids due to its wide range of pharmacological activities. ObjectiveFirstly, to isolate BB from Berberis vulgaris roots, semisynthesis of berberrubine (M1). Secondly, to investigate the liability of BB and M1 for biotransformation by cell cultures of Ocimum basilicum and Dodonaea viscosa focusing on the production of more potent hepatoprotective metabolites. Finally, the hepatoprotective properties of both BB and M1 are studied against B. vulgaris alcoholic extract. MethodsFor the biotransformation, BB and M1 (10 mg/mL) in methanol, equal volume of methanol (control) and distilled water (blank) were added to Suspension cultures of O. basilicum and D. viscosa and were kept under light at 23°C on a gyratory shaker at 100rpm for 14days before the cells being harvested and the contents of each flask, lyophilized separately were dissolved in methanol and subjected to TLC and LC/MS analyses. For hepatoprotective properties TBARS was determined in liver homogenate, protein free supernatant according to the method described by Tappel and Zalkin (1959). The TBARS level was calculated against control according to the following equation:TBARSlevelnmol/mL=Absorbance/0.156. ResultsBB was identified in the root extract using HPLC. LC/MS analyses of lyophilized media from all cultures showed the absence of biotransformation. However, M1 was biotransformed by O. basilicum and D. viscosa cultures into BB (100%) and a new oxidized derivative of M1 (M1TD; 78.9%), respectively, while BB was transformed into palmatine (27.7%) by culture of D. viscosa only. For the hepatoprotective potential of M1and BB in comparison with B. vulgaris extract, all samples decreased Thiobarbituric acid reactive species (TBARS) level in a dose dependent manner. IC50 of BB, B. vulgaris ethanolic extract and M1 were 19.5, 16 and 9.8μg/mL, respectively. M1 with IC50 9.8μg/mL was the most potent inhibitor of lipid peroxidation induced by Fe2+ and H2O2 in liver homogenate at P<0.05. ConclusionThis study reports that the biocatalysis system of D. viscosa cultures showed appreciable capacity in oxidizing the phenolic group of M1 at C-9 and opening of the methylenedioxy ring of BB at non-extreme pH or temperature, followed by methylation to give palmatine. However, the biocatalysis system of O. basilicum culture showed a great capacity towards methylation of phenolic groups as evidenced by methylating OH of M1 at C-9 to give BB. In vitro biological tests indicated that M1 possess better hepatoprotective properties than BB and B. vulgaris methanolic extract.

بررسی بیان ژن های طویل ساز گونه Mortirella alpine در شرایط مناسب تولید آراشیدونیک اسید و روغن

بررسی بیان ژن های طویل ساز گونه Mortirella alpine در شرایط مناسب تولید آراشیدونیک اسید و روغن

21. Cryopreservation of Cleome rosea Vahl in vitro roots using the vitrification technique

Cleome rosea is a Brazilian native herbaceous species that occurs mainly in coastal sandy plains (restingas), ecosystems affected by human impact. Recent studies have reported its medicinal potential, such as anti-inflammatory, antigenotoxic, antiviral and antibacterial activities. Due to these aspects, the species has been investigated using tissue culture systems, including in vitro root cultures. These cultures are considered an efficient strategy to produce biomass, since they present a rapid growth and represent a stable form to produce secondary metabolites. Moreover, they represent a potential source of explants for mass propagation. Considering the rapid depletion of plant species found in the restingas, the development of conservation methods to study the potential presented by C . rosea becomes applicable. Whereas cryopreservation is the most reliable and economically effective method for long-term maintenance of germoplasms, the present study aimed to cryopreserve in vitro roots of C. rosea using the vitrification technique. Roots of micropropagated plants were excised (4–5 mm) and precultured for 4 days, in the dark, using liquid MS medium supplemented with naphthaleneacetic acid (NAA) at 1.34 μM and increasing concentrations of sucrose (0.2–0.4 M). The precultured root segments were transferred into 2 mL cryotubes (5 roots/cryotube) and treated or not with 1 mL of loading solution (0.4 M sucrose and 2.0 M glycerol in MS medium) for 20 min. After that, they were exposed for 15, 30, 45 or 60 min to 1 mL of PVS2 (30% glycerol, 15% Me 2 SO, 15% ethylene glycol, and 0.4 M sucrose in MS medium), PVS3 (50% glycerol and 50% sucrose in MS medium) or VSL (20% glycerol, 10% Me 2 SO, 30% ethylene glycol, 5% sucrose and 10 mM CaCl 2 in MS medium). The cryotubes were immersed directly into liquid nitrogen (−196 °C) and maintained in this condition for 1 h. The cryopreserved roots were thawed in a water bath at 38 °C for 2–3 min and washed for 30 min with liquid MS medium containing 1.2 M sucrose. For regrowth, the roots were maintained in liquid MS medium with decreasing concentrations of sucrose (0.4–0.2 M) for 4 days, in the dark. Then, the roots were transferred to liquid MS medium supplemented with NAA (1.34 μM). The culture flasks were maintained on a gyratory shaker (110 rpm). The recovery frequency (percentage of explants with lateral root formation) was recorded after 20 days and expressed as percentage. The highest values of recovery frequency were reached after treatment with the plant vitrification solutions PVS2 and PVS3. Roots treated with these solutions for 15 min achieved 77% and 100% of recovery frequency respectively. The use of loading before the exposure to the vitrification solutions did not influence the recovery process. On the other hand, root segments exposed to the VSL did not recover after freezing. The cryopreserved roots demonstrated an excellent ability to multiply, which remained stable for up to five subcultures. Furthermore, these roots also displayed capacity for shoot induction. These results demonstrated the establishment of an efficient cryopreservation protocol for in vitro roots of C. rosea , which achieved high recovery rates with the maintenance of their multiplication capacity.

Antimicrobial activity of nanosilver-coated socks fabrics against foot pathogens

Nanotechnology can be used in engineering-desired textile attributes, such as fabric softness and durability in fibres, yarns and fabrics. Nanocoating the surface of socks is one approach to the production of highly active surfaces with UV blocking, antimicrobial and self-cleaning properties. Synthesis of silver nanoparticles in this project was carried out chemically by wet reduction method (Ag-chem) and biologically by using neem (Azadirachta indica) leaves (Ag-neem). The formation of silver nanoparticles was monitored by UV–visible spectroscopy, which revealed the surface plasmon resonance peak at 420 nm for Ag-chem and 430 nm for Ag-neem, and transmission electron microscopy, which showed nanoparticles of various shapes and sizes (∼5–50 nm). Quantification of the prepared silver nanoparticles was done by atomic absorption spectroscopy, which revealed 0.044 M Ag+ and 0.042 M Ag+ ions in Ag-chem and Ag-neem, respectively. Coating of the socks fabrics (nylon and cotton) was carried out by exposing these fabrics to the prepared nanoparticle solutions on a gyratory shaker overnight. Antimicrobial activity of the Ag-chem and Ag-neem was carried out by performing minimum inhibitory concentration (MIC) and disc diffusion test against Sarcina lutea, an odour-producing organism, Klebsiella pnuemoniae, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus and Candida albicans, organisms causing foot infections. P. aeruginosa and S. lutea were found to be most sensitive to either of the above preparations. Ag-chem was found to be more effective than Ag-neem. Nylon and cotton socks fabrics were coated with each of the above preparations. The antibacterial efficacy of the nanosilver-finished fabrics was checked by zone inhibition test, antibacterial test and wash fastness test. In both cases, coated nylon fabrics showed better antimicrobial activity than coated cotton fabrics. S. lutea and K. pneumoniae showed greater zones of inhibition than the other test organisms. Nylon fabric coated with Ag-chem and Ag-neem gave maximum reduction in viable count of all test organisms as compared to cotton fabrics. Higher reduction in the viable count of all test organisms was observed with Ag-chem-coated nylon fabrics. Thus, coating of the nylon socks fabric with silver nanoparticles can be used as an effective way to combat foot-borne pathogens and thereby reducing discomforts like foot odour, perspiration, complications due to diabetes, athlete’s foot, etc.

DEVELOPMENT OF AN EMBRYOGENIC SUSPENSION CULTURE IN CUCUMIS MELO

Efficient somatic embryogenesis simplifies proliferation and gene transformation of many plants. A system for induction of direct somatic embryogenesis via suspension cultures in an Iranian melon cultivar (Khatooni) was optimized. Somatic embryos were induced from quiescent seed cotyledons on liquid MS basal medium supplemented with some osmotic materials. Effect of PEG 6000, PEG 4000, glucose and sucrose were evaluated on induction of somatic embryos. The excised cotyledons were cultured in liquid media and cultures were placed on a gyratory shaker (115 rpm) for two weeks and then crossed through 50 μM metal mesh. The caught embryos were eventually moved on a solid MS medium for further growth and maturation. The number of induced embryos was significantly different in various osmotic materials. The best result was obtained from medium supplemented with 60 g/L PEG 6000. A large number of cotyledonary somatic embryos (approximately 100 embryos from each explant) were produced in this treatment while the number of embryos in glucose and sucrose treatments was less than 30 for each explant. Using the PEG, suggested a quick and efficient way for direct induction of somatic embryogenesis in melon.

Binding and Orientation of Tricyclic Antidepressants within the Central Substrate Site of the Human Serotonin Transporter

Tricyclic antidepressants (TCAs) have been used for decades, but their orientation within and molecular interactions with their primary target is yet unsettled. The recent finding of a TCA binding site in the extracellular vestibule of the bacterial leucine transporter 11 A above the central site has prompted debate about whether this vestibular site in the bacterial transporter is applicable to binding of antidepressants to their relevant physiological target, the human serotonin transporter (hSERT). We present an experimentally validated structural model of imipramine and analogous TCAs in the central substrate binding site of hSERT. Two possible binding modes were observed from induced fit docking calculations. We experimentally validated a single binding mode by combining mutagenesis of hSERT with uptake inhibition studies of different TCA analogs according to the paired mutation ligand analog complementation paradigm. Using this experimental method, we identify a salt bridge between the tertiary aliphatic amine and Asp(98). Furthermore, the 7-position of the imipramine ring is found vicinal to Phe(335), and the pocket lined by Ala(173) and Thr(439) is utilized by 3-substituents. These protein-ligand contact points unambiguously orient the TCA within the central binding site and reveal differences between substrate binding and inhibitor binding, giving important clues to the inhibition mechanism. Consonant with the well established competitive inhibition of uptake by TCAs, the resulting binding site for TCAs in hSERT is fully overlapping with the serotonin binding site in hSERT and dissimilar to the low affinity noncompetitive TCA site reported in the leucine transporter (LeuT).

Decursin Production from Hairy Root Culture of Angelica gigas

Angelica gigas Nakai, commonly known as Korean Angelica, is a perennial herb belonging to the Umbelliferae family and one of the most important medicinal plants in Korea. For thousands of years, the roots of A. gigas has been used as traditional Oriental herbal medicine to treat anemia, abdominal pain, injuries, migraine, arthritis, and female afflictions, and the material has also been prescribed for health-promoting effects [Chi and Kim, 1970; Choi et al., 2003; Sarker and Nahar, 2004]. Decursin (Fig. 1) and decursinol angelate (a decursin isomer), the major secondary metabolites of A. gigas, have significant neuroprotective, anticancer, and anti-androgen-receptor signaling activities [Kang et al., 2005; Yim et al., 2005; Guo et al., 2007]. Various species of bacteria can transfer genes to higher plants, among which Agrobacterium rhizogenes, a Gramnegative soil bacterium, is one of the most widely studied. A. rhizogenes infects the plant cell and leads to the formation of hairy roots [Signs and Flores, 1990]. In many plants, hairy root cultures have proven to be an efficient production system for secondary metabolites. Such cultures have genetic and biochemical stabilities, rapid growth rate, and the ability to synthesize natural compounds at levels comparable to those of intact plants [Giri and Narasu, 2000; Guillon et al., 2006]. To the best of our knowledge, this is the first report on decursin production by hairy root culture of A. gigas. Here, we describe the production of decursin by hairy root cultures of A. gigas transformed with A. rhizogenes R1000. A. rhizogenes strain R1000 cultures were grown to the log phase at 28oC on a gyratory shaker (180 rev/min) in liquid Luria-Bertani medium. The bacterial cells were collected by centrifugation for 10 min at 2000 rpm, and resuspended at a cell density of A600 = 0.5 in MS [Murashige and Skoog, 1962] liquid inoculation medium. Young leaf and stem of A. gigas were taken from plants grown in vitro and were cut at the ends into sections of 0.7×0.7 cm2 and 0.7 cm. Excised leaves and stems were dipped into A. rhizogenes culture in the liquid inoculation medium for 10 min, blotted dry on a sterile filter paper, and incubated in the dark at 25oC on the agar-solidified MS medium. After 2 days of co-cultivation, the explant tissues were transferred to a hormone-free solid MS medium containing 200 mg/L Timentin. Within 4 weeks numerous hairy roots were observed emerging from the wound sites. The hairy roots were separated from the explant tissue and subcultured in the dark at 25oC on the agar-solidified MS medium. After repeated transfers to fresh media, rapidly growing hairy root cultures were obtained. Wild type root cultures were established by inoculating MS liquid medium with excised roots from A. gigas seedlings grown in vitro. Isolated wild type and hairy roots (200 mg) were transferred to 30 mL MS liquid medium, containing 30 g/ L sucrose, in 100 mL flasks. Wild type and hairy root cultures were maintained at 25oC on a gyratory shaker (100 rev/min) in a growth chamber. After 24 days of culture, hairy roots were harvested. Dry weight and decursin contents were determined. Three flasks were used for each culture condition, and the experiments were performed in duplicates. Hairy roots were dried at −80oC in brown paper bags for at least 48 h using a freeze-dryer. Dried samples were ground into a fine powder using a mortar and pestle. Samples (0.5 g) were extracted with 30 mL of 70:30 (% v/v) ethanol-water at 50oC in a water bath for 1 h. After *Corresponding author Phone: +82-42-821-5730; Fax: +82-42-822-2631 E-mail: supark@cnu.ac.kr

Reduction of Carboxylic Acids by Nocardia Aldehyde Oxidoreductase Requires a Phosphopantetheinylated Enzyme

Aldehyde oxidoreductase (carboxylic acid reductase (Car)) catalyzes the magnesium-, ATP-, and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes. Heterologous expression of the car gene in Escherichia coli afforded purified recombinant enzyme with a specific activity nearly 50-fold lower than that of purified native Nocardia sp. enzyme. The 5-fold increase in specific activity obtained by incubating purified recombinant Car with CoA and Nocardia cell-free extracts indicated that post-translational phosphopantetheinylation of Car is required for maximum enzyme activity. Nocardia phosphopantetheine transferase (PPTase) expressed in E. coli was isolated and characterized. When incubated with [(3)H]acetyl-CoA and Nocardia PPTase, the labeled acetylphosphopantetheine moiety was incorporated into recombinant Car. Coexpression of Nocardia Car and PPTase in E. coli gave a reductase with nearly 20-fold higher specific activity. Site-directed mutagenesis in which Ser(689) was replaced with Ala resulted in an inactive Car mutant. The results show that Car expressed in Escherichia coli is an apoenzyme that is converted to a holoenzyme by post-translational modification via phosphopantetheinylation. Doubly recombinant resting E. coli cells efficiently reduce vanillic acid to vanillin.

Latent Pathway Activation and Increased Pathway Capacity Enable Escherichia coli Adaptation to Loss of Key Metabolic Enzymes

The ability of biological systems to adapt to genetic and environmental perturbations is a fundamental but poorly understood process at the molecular level. By quantifying metabolic fluxes and global mRNA abundance, we investigated the genetic and metabolic mechanisms that underlie adaptive evolution of four metabolic gene deletion mutants of Escherichia coli (delta pgi, delta ppc, delta pta, and delta tpi) in parallel evolution experiments of each mutant. The initial response to the gene deletions was flux rerouting through local bypass reactions or normally latent pathways. The principal effect of evolution was improved capacity of already active pathways, whereas new flux distributions were not observed. Combinatorial changes in capacity and pathway activation, however, led to different intracellular flux states that enabled evolution in three of the four parallel cases tested. The molecular bases of the evolved phenotypes were then elucidated by global mRNA transcript analyses. Activation of latent pathways and flux changes in the tricarboxylic acid cycle were found to correlate well with molecular changes at the transcriptional level. Flux alterations in other central metabolic pathways, in contrast, were apparently not connected to changes in the transcriptional network. These results give new insight into the dynamics of the evolutionary process by demonstrating the flexibility of the metabolic network of E. coli to compensate for genetic perturbations and the utility of combining multiple high throughput data sets to differentiate between causal and noncausal mechanistic changes.

Calcium Binding by the Essential Virulence Factor BAD-1 of Blastomyces dermatitidis

BAD-1 (Blastomyces adhesin 1), a 120-kDa protein of Blastomyces dermatitidis, functions as an adhesin, immune modulator, and essential virulence factor. Structurally, BAD-1 is composed of a short N-terminal region, a core of 30 tandem repeats critical for virulence, and a C-terminal epidermal growth factor domain that binds the protein to yeast cell surface chitin. Each of the 30 acidic residue-rich tandem repeats contains a sequence that resembles the calcium-binding loop of the EF-hand domain found in many calcium-binding proteins. Here, we investigated the binding of calcium by BAD-1 and its biological significance. Yeast washed with double distilled H2O released surface-bound BAD-1, but EGTA washes were an order of magnitude more efficient, suggesting an interaction between BAD-1 and calcium. Immobilized BAD-1 was stained with ruthenium red dye, an indicator of calcium-binding proteins. In equilibrium dialysis, BAD-1 bound 45Ca2+ with an affinity of 0.41 x 10(-5) m and a capacity of 27 calcium/mol. Mass spectrometry confirmed this capacity. Elevated [Ca2+] diminished BAD-1 solubility. Upon deletion of its C-terminal epidermal growth factor-like domain, BAD-1 resisted aggregation by elevated [Ca2+] but retained its affinity and capacity for calcium. Removing 20 copies of the tandem repeat, however, sharply reduced the capacity of BAD-1 for calcium. Growth of the bad-1 null yeast was inhibited by 5 mm EGTA, and re-expression of BAD-1 in trans or the addition of exogenous purified BAD-1 restored growth. Thus, BAD-1 is a high capacity calcium-binding protein. This property contributes to the structure and function of BAD-1, as well as to B. dermatitidis acquisition of calcium from the environment.

Differential expression of a poplar copper chaperone gene in response to various abiotic stresses

Copper chaperone (CCH) is upregulated during Arabidopsis (Arabidopsis thaliana L. Columbia) leaf senescence, suggesting that it mobilizes certain metal ions in leaves and transports them to other growing parts of the plants. The CCHs are also involved in defense mechanisms against oxidative stress in Arabidopsis and tomato (Lycopersicon esculentum Mill. cv. 'Ailsa Craig'). To elucidate the functions of CCH in poplar, we cloned a CCH gene (PoCCH) from Populus (Populus alba x P. tremula var. glandulosa) suspension cells and tested its expression in response to various treatments including heavy metals, plant growth regulators and abiotic stresses. The PoCCH cDNA is 540 bp in length, including a 55-bp 5' noncoding domain, a 258-bp open reading frame (ORF), and 227-bp 3' termination region. The coding region of PoCCH represents a putative 85-amino-acid protein with a molecular weight of 8.9 kDa. The deduced amino acid sequence of the PoCCH gene product is 87 and 78% identical to those of tomato and Arabidopsis, respectively, with a high degree of conservation in both the metal-binding and lysine-rich regions. However, the PoCCH gene product lacks the C-terminal extension identified in Arabidopsis CCH. Southern blot analysis suggested that the PoCCH gene is present in low copy numbers in poplar. The expression of PoCCH increased under copper deprivation conditions. The expression of PoCCH was down-regulated by high concentrations of copper, whereas some metals, such as aluminum and zinc, markedly induced PoCCH, and others including cadmium, cobalt and lead had little effect on PoCCH expression. The plant growth regulator jasmonic acid caused an increase in PoCCH transcript whereas abscisic acid, salicylic acid and gibberellic acid did not. The gene was highly induced when cells were exposed to physical stress by high-speed agitation on a gyratory shaker. Other effective inducers of PoCCH expression in suspension culture were methyl viologen and NaCl. Thus, PoCCH does not respond to all stresses, but responds specifically to certain metals and abiotic stresses that induce oxidative damage. Our results suggest that JA is involved in regulating PoCCH expression in poplar cells.

Photochromicity of Anabaena Sensory Rhodopsin, an Atypical Microbial Receptor with a cis-Retinal Light-adapted Form

We characterize changes in isomeric states of the retinylidene chromophore during light-dark adaptation and photochemical reactions of Anabaena (Nostoc) sp. PCC7120 sensory rhodopsin (ASR). The results show that ASR represents a new type of microbial rhodopsin with a number of unusual characteristics. The three most striking are: (i) a primarily all-trans configuration of retinal in the dark-adapted state and (ii) a primarily 13-cis light-adapted state with a blue-shifted and lower extinction absorption spectrum, opposite of the case of bacteriorhodopsin; and (iii) efficient reversible light-induced interconversion between the 13-cis and all-trans unphotolyzed states of the pigment. The relative amount of ASR with cis and trans chromophore forms depends on the wavelength of illumination, providing a mechanism for single-pigment color sensing analogous to that of phytochrome pigments. In addition ASR exhibits unusually slow formation of L-like and M-like intermediates, with a dominant accumulation of M during the photocycle. Co-expression of ASR with its putative cytoplasmic transducer protein shifts the absorption maximum and strongly decreases the rate of dark adaptation of ASR, confirming interaction between the two proteins. Thus ASR, the first non-haloarchaeal sensory rhodopsin characterized, demonstrates the diversity of photochemistry of microbial rhodopsins. Its photochromic properties and the position of its two ground state absorption maxima suggest it as a candidate for controlling differential photosynthetic light-harvesting pigment synthesis (chromatic adaptation) or other color-sensitive physiological responses in Anabaena cells.

Cellular interaction between mouse pancreatic alpha-cell and beta-cell lines: possible contact-dependent inhibition of insulin secretion.

The endocrine cells in the pancreatic islet have cellular communication between the heterotypic cells as well as the homotypic cells. The present study was conducted to elucidate the cellular interaction between pancreatic alpha cells and beta cells utilizing differentiated mouse cell lines (i.e., alphaTC clone 6 and betaTC cells). Co-culture of these two cell lines on a gyratory shaker generated numerous cellular aggregates of homogenous size within 48 h. Immunohistochemical staining for insulin and glucagon demonstrated that betaTC cells were located in the central core of each aggregate, while alphaTC cells formed a mantle layer surrounding the betaTC cells. This segregation was observed regardless of the ratios of the two cell types employed. Although glucagon at concentrations of 10(-8) M or higher stimulated insulin secretion from betaTC cells in both monolayer and aggregates, an increase in the ratio of alphaTC/betaTC cells in aggregate cultures was accompanied by a decrease in secreted insulin and a rise in intracellular insulin content of the betaTC component. The inhibitory effect of alphaTC cells on betaTC insulin secretion was not limited to aggregate culture, since insulin secretion from betaTC cells was also suppressed, and intracellular insulin content increased, by co-culture of alphaTC with betaTC cells in monolayer. On the other hand, the secreted and intracellular insulin of betaTC cells was not affected by alphaTC cells in a Transwell co-culture system in which direct cell-to-cell contacts were prevented by a semipermeable membrane that permitted chemical communication via medium metabolites. These data suggest that the insulin secretion from betaTC cells may be inhibited possibly as a result of the contact with alphaTC cells.