Synthetic peptides from the carboxy-terminal globular domain of the A chain of laminin: their ability to promote cell adhesion and neurite outgrowth, and interact with heparin and the beta 1 integrin subunit

The large carboxy-terminal globular domain (G domain; residues 2,110-3,060) of the A chain of murine-derived laminin has been shown to promote heparin binding, cell adhesion, and neurite outgrowth. This study was conducted to define the potential sequence(s) originating from the G domain of laminin with any of these functional activities. A series of peptides were synthesized from the G domain, termed GD peptides, each approximately 20 amino acids long and containing multiple positively charged amino acids. In direct 3H-heparin binding assays, peptides GD-1 and GD-2 bound high levels of 3H-heparin, while peptides GD-3 and GD-4 bound lower levels of 3H-heparin, and GD-5 bound essentially no 3H-heparin. The binding of 3H-heparin to peptides GD-1 and GD-2 appeared to be of high affinity, since significant binding of 3H-heparin to these two peptides was still observed even when the NaCl concentration was raised to 1.0 M. Four of the peptides, GD-1, GD-2, GD-3, and GD-4, directly promoted the adhesion and spreading of HT-1080 human fibrosarcoma cells as well as the outgrowth of neurites from chick spinal cord and dorsal root ganglia neurons. In addition, solutions of these peptides or antibodies generated against these peptides inhibited laminin-mediated HT-1080 cell adhesion. Antibodies against the beta 1 integrin subunit inhibited HT-1080 cell adhesion and neurite outgrowth on surfaces adsorbed with peptides GD-3 and GD-4. Therefore, laminin appears to have multiple, independent sequences in the G domain that serve a similar cell adhesion promoting function for different cell types. Furthermore, these results suggest that the sequences comprising peptides GD-3 and GD-4 use an integrin as a receptor, of which the beta 1 integrin subunit is a component for these various cell types.     

Fed-batch culture of insect cells with exponential feeding of amino acid and yeastolate solution

Amino acids rather than sugars are the primary limiting substrates for the culture of insect cells in a Grace's medium. When cultures are supplemented with amino acids, the yeastolate components other than the amino acids become the secondary limiting substrates. For the fed-batch culture of insect cells, a solution containing concentrated amino acids and yeastolate was supplied using an exponential feed flow rate calculated from mass balance equations. During the batch period the specific growth rate was 0.02 h^у, whereas during the fed-batch period it was measured as 0.007 and 0.012 h^у on the basis of the cell numbers and the dry cell weight, respectively. This difference in the specific growth rates in the fed-batch period is caused by an increase in the cell size during this period. Furthermore, in fed-batch cultures, dissolved oxygen was found to be a limiting factor for high cell-density cultures.     

Influence of N- and/or C-terminal regions on activity, expression, characteristics and structure of lipase from Geobacillus sp. 95

GD-95 lipase from Geobacillus sp. strain 95 and its modified variants lacking N-terminal signal peptide and/or 10 or 20 C-terminal amino acids were successfully cloned, expressed and purified. To our knowledge, GD-95 lipase precursor (Pre-GD-95) is the first Geobacillus lipase possessing more than 80 % lipolytic activity at 5 °C. It has maximum activity at 55 °C and displays a broad pH activity range. GD-95 lipase was shown to hydrolyze p-NP dodecanoate, tricaprylin and canola oil better than other analyzed substrates. Structural and sequence alignments of bacterial lipases and GD-95 lipase revealed that the C-terminus forms an α helix, which is a conserved structure in lipases from Pseudomonas, Clostridium or Staphylococcus bacteria. This work demonstrates that 10 and 20 C-terminal amino acids of GD-95 lipase significantly affect stability and other physicochemical properties of this enzyme, which has never been reported before and can help create lipases with more specific properties for industrial application. GD-95 lipase and its modified variants GD-95-10 can be successfully applied to biofuel production, in leather and pulp industries, for the production of cosmetics or perfumes. These lipases are potential biocatalysts in processes, which require extreme conditions: low or high temperature, strongly acidic or alkaline environment and various organic solvents.     

Development and Quality Evaluation of Hypoallergenic Bakery Products Suitable for Patients with Wheat-associated Allergy

The influence of growth temperature (21–41°C) and light intensity on compositions of four carotenoids, and of fatty acids from carotenoid glucoside ester (carotenoid K-G-FA) and from the cellular lipids in Rhodococcus rhodochrous RNMS1 were quantitatively investigated. Lowering the temperature increased the total carotenoid content and the proportion of carotenoid K-G-FA. It increased the proportion of unsaturated fatty acids in both carotenoid K-G-FA and the cellular lipids, decreased that of saturated ones, and slightly decreased that of branched-chain ones. This bacterium adapted itself to low temperature by desaturating its fatty acids. The light intensity affected neither the content and the composition of carotenoids nor the composition of the fatty acids in carotenoid K-G-FA and the cellular lipids. This bacterium was a scotochromogenic strain.     

A Novel Recognition Site on Laminin for the α3β1 Integrin

Synthetic peptide GD-2 is a sequence of amino acids derived from the carboxy-terminal long arm of the A chain of laminin. Previous studies have shown that peptide GD-2 promotes the adhesion of human squamous cell carcinoma (SCC) cells as well as a variety of other cell lines. In this study, we attempted to identify the receptor that SCC cells use to adhere to peptide GD-2. Monoclonal antibodies (mAbs) against a human SCC cell line were generated. One of these mAbs, ASC-1, bound to the surface of SCC cells as determined by flow cytometry. This mAb inhibited SCC cell adhesion to peptide GD-2 and laminin, but not fibronectin or type IV collagen, suggesting that mAb ASC-1 binds to the SCC receptor for the peptide GD-2 sequence of laminin. MAb ASC-1 immunoprecipitated a complex composed of two components of 135 and 116 kDa. Immunoadsorption of ASC-1-binding material from the SCC cell extract by incubation with mAb ASC-1 resulted in the removal of the α3β1 integrin from the extract. Immunohistochemical staining of tissue from a normal human tongue and from a patient with SCC of the tongue revealed that mAb ASC-1 stained the surface of epithelial cells that were in contact with the basement membrane, as well as those cells located two to three layers above the basement membrane. This mAb also stained blood vessels in the squamous tissue. This staining pattern was identical to that observed when the same tissues were stained by a mAb against the α3 integrin subunit. In summary, by use of a new mAb, ASC-1, that recognizes the α3β1 integrin, we have determined that the α3β1 integrin mediates SCC cell adhesion to the peptide GD-2 sequence within laminin.     

Studies on the Cell Wall of Chlorella III. Incorporation of Photosynthetically Fixed Carbon into Cell Walls of Synchronously Growing Cells of Chlorella ellipsoidea

The carbon metabolism in cell walls of Chlorella ellipsoideawas studied by following ¹⁴C incorporation into cell wall constituentsin photosynthesizing, synchronously growing cells. The rateof incorporation was higher at an early growth phase of thecell cycle. The ¹⁴C was incorporated into both the major cellwall constituents, hemicellulose and ‘rigid wall’,and the radioactivity in the latter was distributed into itstwo components, glucosamine and amino acids. In pulse-chaseexperiments, the ¹⁴C fixed photosynthetically in the precedingcell cycle was rapidly transferred into the cell wall constituentsat the early growth phase of the ongoing cell cycle, and thereafterwas gradually released from the cell walls, although the totalamount of ¹⁴C in the cells remained constant. It was concludedthat the cell wall constituents are turned over during the growthphase of the algal cell cycle, and that the cell wall metabolismin the ongoing cell cycle is closely connected with the carbonmetabolism in the preceding cell cycle. (Received February 3, 1982; Accepted June 21, 1982)     

Exposure of Dunaliella salina to Low Temperature Mimics the High Light-Induced Accumulation of Carotenoids and the Carotenoid Binding Protein (Cbr)

We report that growth of Dunaliella salina at either 13°C/150µmol m^–2s^–1 or 30°C/2,500 µmol m^–2s^–1 results in the accumulation of comparable levels ofcarotenoids and the zeaxanthin-binding protein, Cbr. We concludethat carotenoid and Cbr abundance in this green alga respondto changes in PSII ‘excitation pressure’ ratherthan to high light per se. (Received September 19, 1996; Accepted November 20, 1996)     

Mechanism of Photoregulated Carotenogenesis in Rhodotorula minuta I. Photocontrol of Carotenoid Production

Rhodotorula minuta cells, which have only traces of carotenoidswhen grown in the dark, started carotenoid production with theonset of illumination and the amount increased almost linearlyuntil 70 hr then remained constant thereafter when incubationwas continued under illumination, with the number of cells continuingto increase. The rate of carotenoid production [Vc (µgg^–1 hr^–1)] depended on the intensity of light [I(ergcm^–2 sec^–1)], with the relationship of Vc=0.74 logI–1.46. The final carotenoid content [C(µg g^–1)]of cells incubated under continuous light was also controlledby the light intensity [I], with the relationship of C=52 logI–81. Control of carotenoid production by light occursas a two-phase process consisting of a temperatureindependentphotochemical reaction and light-independent biochemical reactions. (Received September 12, 1981; Accepted February 20, 1982)     

Growth and Osmoregulation of Chaetoceros muelleri in Relation to Salinity

The growth and osmoregulation of Chaetoceros muelleri Lemmermann(Bacillariophyceae) were investigated as a function of salinity.This centric diatom grew well over a wide range of salinityand required concentrations of NaCl above 10 mM for growth.Using gas chromatography- mass spectroscopy (GC-MS) analysisof cell extracts, we demonstrated that the alga contains anisomer of cyclohexanetetrol. The level of this isomer increasedwith increasing salinity. Levels of free amino acids also increasedwith increasing salinity, and quantitative determination withan amino acid analyzer revealed that the level of glutamic acidincreased the most with increases in salinity. Levels of intracellularK⁺ and Cl^– also increased significantly with increasesin salinity. Thus, in C. muelleri, not only organic solutessuch as the cyclohexanetetrol isomer and glutamic acid, butalso inorganic solutes such as K⁺ and Cl^– contribute toosmoregulation. (Received November 7, 1994; Accepted April 10, 1995)     

Further studies on the metabolism of glucose in the process of "glucose-bleaching" of Chlorella protothecoides

Previous studies have demonstrated that when cells of Chlorellaprotothecoides are incubated in a medium containing glucosebut no nitrogen source, they are profoundly bleached with degenerationof chloroplast structure and photosynthetic activity. When anitrogen source (urea) is added to the glucose medium, bleachingof algal cells is greatly suppressed. In this work the metabolismof glucose in the process of glucose-induced bleaching was studiedusing ¹⁴C-glucose as tracer. Changes in algal cell activityfor ¹⁴CO₂-evolution and ¹⁴C-incorporation into various cellularsubstances from ¹⁴C-glucose were followed. Most conspicuouswere increases in cellular activities for assimilating ¹⁴C-glucoseinto lipids (fatty acids) and glucose polymer. When urea wasadded to the glucose medium, the incorporation of ¹⁴C by algalcells into fatty acids was greatly reduced, while the assimilationof ¹⁴C into glucose polymer was increased. These and previous observations suggest that the formation oflarge amounts of lipids (fatty acids) probably is causally relatedto the induction of algal cell bleaching. (Received March 5, 1969; )     

Mechanism of Photoregulated Carotenogenesis in Rhodotorula minuta II. Aspects of Photoregulative Reaction

The photoregulation of carotenogenesis in Rhodotorula minutawas found to consist of tow phases, a temperature-independentphotochemical reaction (light process) and temperature-dependentbut light-independent biochemical reactions (dark process).These processes were separately examined by regulating the temperatureand were characterized as follows: 1) The quantity of carotenoid produced [C (µg g^–1)]and the rate of carotenoid production [Vc (µg g^–1hr^–1)] in the dark process were regulated by the lightdose [D (erg cm^–2)] to which cells were exposed in thelight process. These relationships were expressed by the equations:C=9.1 log D–62.0 and Vc=0.81 log D–5.60. This photoresponsefollowed the Roscoe-Bunsen reciprocity law. 2) The induced state toward carotenogenesis, once acquired inthe light process, was very stable, suggesting that the proposedphotochemical product is stable as an inducer of carotenogenesisand decreases only in conjunction with carotenoid biosynthesis. 3) The photochemical reaction was oxygen-independent, but subsequentdark reactions were completely dependent on oxygen. 4) Postulated compounds related to the photochemical reactionwere not metabolized in vivo. (Received September 12, 1981; Accepted February 20, 1982)     

The Effect of Different Levels of N Limitation on Sugars, Amino Acids, Growth and Biomass Partitioning in Broadbean (Vicia faba L.)

Broadbean plants (Vicia faba L.) were submitted to three differentlevels of steady state N limitation. Relative addition ratesof N were 0.06, 0.1 and 0.14d^-1. Plants were harvested at fiveevenly distributed times over a 2d period. Shoot growth correspondedwell with the imposed treatment. Root growth, relative to shootgrowth, was highest at the 0.06d^-1treatment. Diurnal patternsof soluble sugars, amino acids and starch were analysed. Onaverage, soluble sugar levels were highest in the plants ofthe 0.06d^-1treatment whereas average free amino acid levelswere highest in the 0.14d^-1treatment. Shoot growth increasedas the concentration of shoot amino acids increased. No suchcorrelation however could be found between root growth and freesugar levels in the root. Broadbean; Vicia faba L.; exponential addition; N limitation; free sugar; amino acids; diurnal cycle; functional equilibrium; starch     

Osmotic adjustment in marine yeast

The effect of environmental salinity on cell growth, and onthe composition and accumulation of compatible solutes, or osmotica,of five yeast strains (Aureobasidium pullulans, Candida sp.,Cryptococcus albidus var. albidus, Debaryomyces hansenii andRhodotorula rubra) was compared. All these yeast were isolatedfrom manne environments, but were able to grow in the absenceof salt and should therefore be considered as halotolerant strains.According to their specific cell growth rates at different saltconcentrations, these strains vary in their capacity to osmoticallyadjust to modifications in external salinity. Candida sp. appearsto be the most sensitive since the maximum salt concentrationat which it can grow is 1.54 mol 1^-1 NaCl; however, it showedthe highest specific cell growth in the range of 0 to 1.54 mol1^-1 NaCl. Aureobasidium pullulans, on the other hand, showedthe lowest specific growth rate, but the highest halotolerancerange from 0 to 5.13 mol 1^-1 NaCl. Debaryomyces hansenii, incontrast, showed higher specific growth at this salinity rangeCryptococcus albidus var. albidus and Rhodotorula rubra showedsimilar specific cell growth rate values and halotolerance between0 and 2.45 mol 1^-1 NaCl. The protein and carbohydrate contentof the biomass of the different yeast cells, as a result ofexternal salinity vanation, remained practically constant. Themost important effects of the increase in salt concentrationin the culture medium were the reduction of cell volume andthe accumulation of low-molecular-weight metabolites (LMWM).which appear to act as osmoregulators. Glycerol was found asthe major compatible solute in the different marine yeasts studiedherein with a total contribution of 64–96% of the internalcell osmolarity. Other LMWM, like carbohydrates and amino acids,contributed to a lesser extent to compensate for the rise inosmotic pressure promoted by the salinity of the external environment.     

Growth and Antithraquinone Biosynthesis by Galium mollugo L. Cells in Batch and Chemostat Culture

The kinetics of growth, nutrient uptake, and anthraquinone biosynthesisby suspension cultures of Galium mollugo L. cells were examinedin batch and continuous (chemostat) culture. In batch culture,although the initial growth rate was constant (minimum doublingtime = 35 h) characteristic changes in cell composition wereobserved during the growth cycle particularly cell dry weight(between 3.9 and 9.2 g/10⁹ cells), cell anthraquinone (22–80mg/10⁹ cells), and cell protein (0.7–1.6 g/10⁹ cells).Using a chemostat steady state growth was established at twodifferent specific growth rates with mean doubling times of40 h and 25 h. Phosphate was established as the growth-limitingnutrient in chemostat culture at a concentration of 11 µgP ml^–1. In steady state growth at a doubling time of 40h the cell composition remained constant although this was differentfrom any cells grown in batch culture. The cell anthraquinonelevel in steady state growth was between 7 and 30 times lowerthan in batch culture. This result raises the question of therelative importance of growth rate and the growth-limiting nutrientin determining accumulation of secondary products by culturedplant cells.     

EFFECT OF MANGANESE IONS ON THE IAA-INDUCED ELONGATION OF AVENA COLEOPTILE SECTIONS INHIBITED BY SOME ORGANIC ACIDS

1.Organic acids, such as citric, -ketoglutaric, succinic, fumaricand L-malic acids, inhibit the IAA-induced growth of Avena coleoptilesections. But pyruvic acid has no effect on the growth. 2.High concentrations of MnCl₂ (for example 10^–3 m) alleviatethe inhibition due to L-malic, -ketoglutaric, succinic and fumaricacids, but not that due to D-malic, tartaric and malonic acids. 3.A mechanism of the alleviating effect of Mn⁺⁺ on the inhibitiondue to the organic acids is discussed with the reference tothe activating effect of Mn⁺⁺ on "malic" enzyme. ¹Contribution No. 6 from the Botanical Gardens. Faculty of Science,University of Tokyo, Koishikawa, Tokyo.     

Differential mitogenic effects of methyl isobutyl xanthine and a tumor growth factor on G1-arrested 3T3 T proadipocytes at the predifferentiation GD state and the growth-factor deficiency GS state

Two growth-states exist in the G1 phase of the 3T3 T proadipocyte cell cycle. GD is the arrest state at which proadipocytes must growth-arrest prior to differentiation. GS is the arrest state at which proadipocytes growth-arrest following deprivation of serum or growth factors. In an attempt to further distinguish these arrest states, we have compared the relative ability of a variety of mitogens to induce GD- and GS-arrested cells to initiate DNA synthesis. The data show that GD-arrested cells at both high and low densities can be induced to proliferate by methyl-isobutyl-xanthine (MIX), whereas high and low density GS-arrested cells are not. The data also show that a tumor growth factor can stimulate the proliferation of both high and low density GD- and GS-arrested cells, whereas other agents are poor mitogens for high density GD-arrested cells. We conclude that MIX and a tumor growth factor (TUGF) can serve as probes to study the characteristics of the GD arrest state.     

Optimizations of carotenoid biosynthesis by controlling sucrose concentration

Summary The effects of sucrose onDaucus carota cell size and biosynthesis of carotenoid were investigated. At high concentration of sucrose, the growth rate and the final cell mass were low due to the substrate inhibition, and cell size was small compared to the result at low sucrose concentration. However, very high level of specific carotenoid content was obtained from small sized cells compared to the condition from large sized cells. The biosynthesis of carotenoid can be increased by optimizing the cell culture method as follow: (i) cultivate the cell initially at low sucrose concentration to increase the cell growth rate, and (ii) when cell mass reaches a certain level, increase the sucrose concentration to a high level to make the cell size small and to increase the biosynthesis of carotenoid.     

Specific Labeling of Cell Wall Polysaccharides with myo-[2-3H] Inositol during Germination and Growth of Phaseolus vulgaris L.

myo-[2-³H]Inositol was fed to bean seeds by imbibition and itsmetabolic fate was studied during germination and seedling growth.The largest amount of myo-inositol was taken up from a 500 HIMsupply (8 mg/seed) and the highest percentage was from 1 HIM(29%). myo-Inositol was incorporated to new cell wall polysaccharidesof hypocotyl and roots, mostly as uronic acid and pentose residues.In the 80% ethanolinsoluble cell walls of hypocotyls at 3, 4and 5 days after imbibition, 47 to 52% of ³H was detected asuronic acids, 20 to 24% as arabinose and 11 to 19% as xylose.Glucogenesis from myo-inositol was low: less than 6% was recoveredas hexoses. The ³H in uronic acid and arabinose residues decreasedwith increasing age (i.e. 0 to 6 cm from cotyledons) and increasedin older segments (further than 6 cm from cotyledons). In theoldest segment of 5-day-old hypocotyl (> 10 cm), ³H in thesugar residues was more than that in the youngest part (0–2cm). On the other hand, ³H in xylose residues increased steadilyin the older part, but did not exceed that in arabinose. The results show that the myo-inositol oxidation pathway functionsin growing hypocotyls and roots of bean seedlings to provideexclusively uronic acid and pentose units for cell wall synthesis.Results also show that incorporation of arabinose and uronicacids derived from myo-[2-³H]inositol to cell wall polysaccharidesis active in two regions of the hypocotyl; first, for the constructionof the primary walls in the young, growing region of the hypocotyl,and second, for thickening of the walls after completion ofelongation growth. ¹Supported by NSERC of Canada. (Received April 10, 1984; Accepted June 12, 1984)     

Involvement of Cell Wall-Bound Diferulic Acid in Light-Induced Decrease in Growth Rate and Cell Wall Extensibility of Oryza Coleoptiles

Irradiation of white fluorescent light (5 W m^–2) inhibitedthe growth of Oryza coleoptiles. Light irradiation increasedstress-relaxation parameters of coleoptile cell walls, minimumstressrelaxationtime and relaxation rate, and decreased cellwall extensibility (strain/load). Under light conditions, thecontents of ferulic and diferulic acids ester-linked to thehemicellulosic arabinose residue in cell walls increased andcorrelated with the modification of the cell wall mechanicalproperties. These results suggest that light irradiation enhancesthe formation of diferulic acid bridges in hemicelluloses, makingcell walls mechanically rigid and thus inhibits cell elongationin rice coleoptiles. Also, irrespective of coleoptile age orthe presence of light, the ratio of diferulic acid to ferulicacid was almost constant, suggesting that the rate limitingstep in the formation of diferulic acid bridges in Oryza cellwalls is in the step of feruloylation. (Received September 24, 1991; Accepted December 3, 1991)