Quantifying the Proteolytic Release of Extracellular Matrix-Sequestered VEGF with a Computational Model

Background

VEGF proteolysis by plasmin or matrix metalloproteinases (MMPs) is believed to play an important role in regulating vascular patterning in vivo by releasing VEGF from the extracellular matrix (ECM). However, a quantitative understanding of the kinetics of VEGF cleavage and the efficiency of cell-mediated VEGF release is currently lacking. To address these uncertainties, we develop a molecular-detailed quantitative model of VEGF proteolysis, used here in the context of an endothelial sprout.

Methodology and Findings

To study a cell''s ability to cleave VEGF, the model captures MMP secretion, VEGF-ECM binding, VEGF proteolysis from VEGF₁₆₅ to VEGF₁₁₄ (the expected MMP cleavage product of VEGF₁₆₅) and VEGF receptor-mediated recapture. Using experimental data, we estimated the effective bimolecular rate constant of VEGF₁₆₅ cleavage by plasmin to be 328 M⁻¹s⁻¹ at 25°C, which is relatively slow compared to typical MMP-ECM proteolysis reactions. While previous studies have implicated cellular proteolysis in growth factor processing, we show that single cells do not individually have the capacity to cleave VEGF to any appreciable extent (less than 0.1% conversion). In addition, we find that a tip cell''s receptor system will not efficiently recapture the cleaved VEGF due to an inability of cleaved VEGF to associate with Neuropilin-1.

Conclusions

Overall, VEGF₁₆₅ cleavage in vivo is likely to be mediated by the combined effect of numerous cells, instead of behaving in a single-cell-directed, autocrine manner. We show that heparan sulfate proteoglycans (HSPGs) potentiate VEGF cleavage by increasing the VEGF clearance time in tissues. In addition, we find that the VEGF-HSPG complex is more sensitive to proteases than is soluble VEGF, which may imply its potential relevance in receptor signaling. Finally, according to our calculations, experimentally measured soluble protease levels are approximately two orders of magnitude lower than that needed to reconcile levels of VEGF cleavage seen in pathological situations.     

Azithromycin Attenuates Fibroblast Growth Factors Induced Vascular Endothelial Growth Factor Via p38MAPK Signaling in Human Airway Smooth Muscle Cells

The airways in asthma and COPD are characterized by an increase in airway smooth muscle (ASM) mass and bronchial vascular changes associated with increased expression of pro-angiogenic growth factors, such as fibroblast growth factors (FGF-1 and FGF-2) and vascular endothelial growth factor (VEGF). We investigated the contribution of FGF-1/-2 in VEGF production in ASM cells and assessed the influence of azithromycin and dexamethasone and their underlying signaling mechanisms. Growth-synchronized human ASM cells were pre-treated with MAPK inhibitors, U0126 for ERK1/2^MAPK and SB239063 for p38^MAPK as well as with dexamethasone or azithromycin, 30 min before incubation with FGF-1 or FGF-2. Expression of VEGF (VEGF-A, VEGF₁₂₁, and VEGF₁₆₅) was assessed by quantitative PCR, VEGF release by ELISA and MAPK phosphorylation by Western blotting. Both FGF-1 and FGF-2 significantly induced mRNA levels of VEGF-A, VEGF₁₂₁, and VEGF₁₆₅. The VEGF protein release was increased 1.8-fold (FGF-1) and 5.5-fold (FGF-2) as compared to controls. Rapid transient increase in ERK1/2^MAPK and p38^MAPK phosphorylation and subsequent release of VEGF from FGF-1 or FGF-2-treated ASM cells were inhibited by respective blockers. Furthermore, azithromycin and dexamethasone significantly reduced both the VEGF release and the activation of p38^MAPK pathway in response to FGF-1 or FGF-2 treatment. Our Results demonstrate that FGF-1 and FGF-2 up-regulate VEGF production via ERK1/2^MAPK and p38^MAPK pathways. Both azithromycin and dexamethasone elicited their anti-angiogenic effects via p38^MAPK pathway in vitro, thereby suggesting a possible therapeutic approach to tackle VEGF-mediated vascular remodeling.     

Detection of antibodies against customized epitope: use of a coating antigen employing VEGF as fusion partner

Diagnosis of many infectious, autoimmune diseases and cancers depends on the detection of specific antibodies against peptide epitope by enzyme-linked immunosorbent assay (ELISA). However, small peptides are difficult to be coated on the plate surfaces. In this study, we selected GnRH as a model hapten to evaluate whether VEGF₁₂₁ would be suitable as an irrelevant hapten-carrier to develop a universal platform for specific antibodies detection. Firstly, GnRH was fused to the C terminus of VEGF₁₂₁ and the resultant fusion protein VEGF–GnRH expressed effectively as inclusion bodies in Escherichia coli. Thereafter, VEGF–GnRH was easily purified to near homogeneity with a yield of about 235 mg from 2.1 L induced culture. At last, VEGF–GnRH was used to perform ELISA and western blot, and our results suggested that VEGF–GnRH was capable of detecting anti-GnRH antibodies in sera both qualitatively and quantitatively. Indeed, previous studies of our laboratory had demonstrated that other fusion proteins such as VEGF–Aβ10, VEGF–GRP, VEGF–CETPC, and VEGF–βhCGCTP37 were able to detect their corresponding antibodies specifically. Therefore, VEGF₁₂₁ may be a suitable irrelevant fusion partner of important diagnostic peptide markers. Our works would shed some light on the development of a universal platform for detection of specific antibodies.     

Effect of oxygen supply on biomass and helvolic acid production in submerged fermentation of Cordyceps taii

The entomogenous fungus Cordyceps taii, a traditional Chinese medicinal mushroom, exhibits potent important pharmacological effects and it has great potential for health foods and medicine. In this work, the effects of oxygen supply on production of biomass and bioactive helvolic acid were studied in shake-flask fermentation of C. taii mycelia. The value of initial volumetric oxygen transfer coefficient (K_La) within 10.1–33.8 h⁻¹ affected the cell growth, helvolic acid production and expression levels of biosynthetic genes. The highest cell concentration of 17.2 g/L was obtained at 14.3 h⁻¹ of initial K_La. The highest helvolic acid production was 9.6 mg/L at 10.1 h⁻¹ of initial K_La. The expression levels of three genes encoding hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase and squalene synthase were down-regulated on day 2 and day 8 but up-regulated on day 14 at an initial K_La value of 10.1 h⁻¹ vs. 33.8 h⁻¹, which well corresponded to the helvolic acid biosynthesis in those conditions. The information obtained would be helpful for improving the biomass and helvolic acid production in large-scale fermentation of C. taii.     

Regulation of Vascular Endothelial Growth Factor (VEGF) Splicing from Pro-angiogenic to Anti-angiogenic Isoforms: A NOVEL THERAPEUTIC STRATEGY FOR ANGIOGENESIS*

Vascular endothelial growth factor (VEGF) is produced either as a pro-angiogenic or anti-angiogenic protein depending upon splice site choice in the terminal, eighth exon. Proximal splice site selection (PSS) in exon 8 generates pro-angiogenic isoforms such as VEGF₁₆₅, and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF₁₆₅b. Cellular decisions on splice site selection depend upon the activity of RNA-binding splice factors, such as ASF/SF2, which have previously been shown to regulate VEGF splice site choice. To determine the mechanism by which the pro-angiogenic splice site choice is mediated, we investigated the effect of inhibition of ASF/SF2 phosphorylation by SR protein kinases (SRPK1/2) on splice site choice in epithelial cells and in in vivo angiogenesis models. Epithelial cells treated with insulin-like growth factor-1 (IGF-1) increased PSS and produced more VEGF₁₆₅ and less VEGF₁₆₅b. This down-regulation of DSS and increased PSS was blocked by protein kinase C inhibition and SRPK1/2 inhibition. IGF-1 treatment resulted in nuclear localization of ASF/SF2, which was blocked by SPRK1/2 inhibition. Pull-down assay and RNA immunoprecipitation using VEGF mRNA sequences identified an 11-nucleotide sequence required for ASF/SF2 binding. Injection of an SRPK1/2 inhibitor reduced angiogenesis in a mouse model of retinal neovascularization, suggesting that regulation of alternative splicing could be a potential therapeutic strategy in angiogenic pathologies.     

Mechano-Regulation of Alternative Splicing

Alternative splicing contributes to the complexity of proteome by producing multiple mRNAs from a single gene. Affymetrix exon arrays and experiments in vivo or in vitro demonstrated that alternative splicing was regulated by mechanical stress. Expression of mechano-growth factor (MGF) which is the splicing isoform of insulin-like growth factor 1(IGF-1) and vascular endothelial growth factor (VEGF) splicing variants such as VEGF₁₂₁, VEGF_165, VEGF₂₀₆, VEGF₁₈₉, VEGF₁₆₅ and VEGF₁₄₅ are regulated by mechanical stress. However, the mechanism of this process is not yet clear. Increasing evidences showed that the possible mechanism is related to Ca²⁺ signal pathway and phosphorylation signal pathway. This review proposes possible mechanisms of mechanical splicing regulation. This will contribute to the biomechanical study of alternative splicing.     

Study on survival,growth, haematology and body composition of Cyprinus carpio under different acute and chronic salinity regimes

Pakistan’s most of the land is less productive or no productivity at all due to erosion and salinity of the soil, which can be utilized to develop fisheries. The project, “Survival, growth and body composition of Cyprinus carpio under different salinity regimes” was undertaken in two phases. In the first phase susceptibility of Cyprinus carpio at four salinity levels in triplicate within 0–10 g L⁻¹NaCl for 96 h in each aquarium was checked after one week acclamation at 0 g L⁻¹, 2 g L⁻¹ and 4 g L⁻¹ NaCl. LC₅₀ values varied from 7.67 to 10.65 g L⁻¹ after 96 h for C. carpio. Percentage mortality of the fish and important water quality parameters after every 12 h were observed for a period of 96-h. Probit analysis showed that 96-h LC₅₀ values ranged from 7.67 to 10.65 g L⁻¹. During experimental period aquaria water temperature ranged from 29.6 to 33.7 °C, pH values fluctuated between 7.8 and 9.7, Electrical conductivity values ranged from 2.40 to 20.13 dSm⁻¹ and Dissolved oxygen ranged between 2.23 and 10 mg L⁻¹. Sub-lethal salt concentration i.e. 0 g L⁻¹ to 3 g L⁻¹ NaCl upto 40 days showed that growth of C. carpio decreased with the increase of water salinity levels and ceased at 4 g L⁻¹ salinity and increase in salinity have negatively affected hematological parameters.     

Exogenous phosphatidic acid with saturated short-chain fatty acyl groups induces superoxide anion release from guinea pig peritoneal polymorphonuclear leukocytes by three different mechanisms

Treatment of suspensions of guinea pig peritoneal polymorphonuclear leukocytes (PMN) with four species of phosphatidate (PA) containing short-chain fatty acids induced sustained superoxide anion (O₂⁻) production after a lag time. The rank order of efficiency of these PAs in triggering O₂⁻ production was PA_8:0 [1,2-dioctanoyl-sn-glycerol-3-phosphate (GP)]>PA_10:0 (1,2-didecanoyl-GP)>PA_6:0 (1,2-dicaproyl-GP)≫PA_12:0 (1,2-dilauroyl-GP). The O₂⁻ release from PMN stimulated with PA_10:0 or PA_12:0, but not with PA_6:0 or PA_8:0, was lowered by the addition of 1 mM extracellular Ca²⁺. Studies with various inhibitors showed that the mechanism of multiphasic O₂⁻ production induced by PA_8:0 depended on its concentration: 1 and 3 μM PA_8:0 induced O₂⁻ production constantly after a lag time through a protein kinase-dependent mechanism that was inhibited by 100 nM staurosporine. With concentrations of PA of 10 μM or more, an additional mechanism that was independent of protein kinase became operative and predominant over the protein kinase-dependent one. This protein kinase-independent mechanism was inhibited selectively by 80 μM TMB-8. Concentrations of 30, 60 and 100 μM PA first elicited transient O₂⁻ production via another protein kinase-dependent mechanism that was more sensitive to H-7 than to staurosporine, and then sustained O₂⁻ production, mainly driven by the protein kinase-independent mechanism. Metabolism of exogenously added [¹⁴C]PA_8:0 in intact PMN was examined in the presence and absence of propranolol. Results suggest that PA itself is more important rather than its degradation products such as diacylglycerol, in inducing O₂⁻ production via three different mechanisms described above.     

The effect of nitrogen containing bisphosphonates,zoledronate and alendronate,on the production of pro-angiogenic factors by osteoblastic cells

Bisphosphonates (BPs) have been shown to influence angiogenesis. This may contribute to BP-associated side-effects such as osteonecrosis of the jaw (ONJ) or atypical femoral fractures (AFF). The effect of BPs on the production of angiogenic factors by osteoblasts is unclear. The aims were to investigate the effect of (1) alendronate on circulating angiogenic factors; vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANG-1) in vivo and (2) zoledronate and alendronate on the production of VEGF and ANG-1 by osteoblasts in vitro. We studied 18 post-menopausal women with T score ⩽ −2 randomized to calcium/vitamin D only (control arm, n = 8) or calcium/vitamin D and alendronate 70 mg weekly (treatment arm, n = 10). Circulating concentrations of VEGF and ANG-1 were measured at baseline, 3, 6 and 12 months. Two human osteoblastic cell lines (MG-63 and HCC1) and a murine osteocytic cell line (MLO-Y4) were treated with zoledronate or alendronate at concentrations of 10⁻¹²–10⁻⁶ M. VEGF and ANG-1 were measured in the cell culture supernatant. We observed a trend towards a decline in VEGF and ANG-1 at 6 and 12 months following treatment with alendronate (p = 0.08). Production of VEGF and ANG-1 by the MG-63 and HCC1 cells decreased significantly by 34–39% (p < 0.01) following treatment with zoledronate (10⁻⁹–10⁻⁶ M). Treatment of the MG-63 cells with alendronate (10⁻⁷ and 10⁻⁶) led to a smaller decrease (25–28%) in VEGF (p < 0.05). Zoledronate (10⁻¹⁰–10⁻⁶ M) suppressed the production of ANG-1 by MG-63 cells with a decrease of 43–49% (p < 0.01). Co-treatment with calcitriol (10⁻⁸ M) partially reversed this zoledronate-induced inhibition. BPs suppress osteoblastic production of angiogenic factors. This may explain, in part, the pathogenesis of the BP-associated side-effects.     

Effective biodegradation of 2,4,6-trinitrotoluene using a novel bacterial strain isolated from TNT-contaminated soil

In this environmental-sample based study, rapid microbial-mediated degradation of 2,4,6-trinitrotoluene (TNT) contaminated soils is demonstrated by a novel strain, Achromobacter spanius STE 11. Complete removal of 100 mg L⁻¹ TNT is achieved within only 20 h under aerobic conditions by the isolate. In this bio-conversion process, TNT is transformed to 2,4-dinitrotoluene (7 mg L⁻¹), 2,6-dinitrotoluene (3 mg L⁻¹), 4-aminodinitrotoluene (49 mg L⁻¹) and 2-aminodinitrotoluene (16 mg L⁻¹) as the key metabolites. A. spanius STE 11 has the ability to denitrate TNT in aerobic conditions as suggested by the dinitrotoluene and NO₃ productions during the growth period. Elemental analysis results indicate that 24.77 mg L⁻¹ nitrogen from TNT was accumulated in the cell biomass, showing that STE 11 can use TNT as its sole nitrogen source. TNT degradation was observed between pH 4.0–8.0 and 4–43 °C; however, the most efficient degradation was at pH 6.0–7.0 and 30 °C.     

Optimization of β-mannanase production by Bacillus subtilis US191 using economical agricultural substrates

The Bacillus subtilis US191 strain producing highly thermostable β-mannanase was previously selected as potential probiotic candidate for application as feed supplement in poultry industry. Initially, the level of extracellular β-mannanase production by this strain was 1.48 U ml⁻¹. To improve this enzyme titer, the present study was undertaken to optimize the fermentation conditions through experimental designs and valorization of agro-industrial byproducts. Using the Plackett–Burman design, in submerged fermentation, a set of 14 culture variables was evaluated in terms of their effects on β-mannanase production. Locust bean gum (LBG), soymeal, temperature, and inoculum size were subsequently optimized by response surface methodology using Box–Behnken design. Under optimized conditions (1 g L⁻¹ LBG, 8 g L⁻¹ soymeal, temperature of 30°C and inoculum size of 10¹⁰ CFU ml⁻¹), a 2.59-fold enhancement in β-mannanase titer was achieved. Next, to decrease the enzyme production cost, the effect of partial substitution of LBG (1 g L⁻¹) by agro-industrial byproducts was investigated, and a Taguchi design was applied. This allowed the attaining of a β-mannanase production level of 8.75 U ml⁻¹ in presence of 0.25 g L⁻¹ LBG, 5 g L⁻¹ of coffee residue powder, 5 g L⁻¹ of date seeds powder, and 5 g L⁻¹ of prickly pear seeds powder as mannans sources. Overall, a 5.91-fold improvement in β-mannanase production by B. subtilis US191 was achieved.     

Innovative development of membrane sparger for carbon dioxide supply in microalgae cultures

The present study was aimed to develop a membrane sparger (MS) integrated into a tubular photobioreactor to promote the increase of the carbon dioxide (CO₂) fixation by Spirulina sp. LEB 18 cultures. The use of MS for the CO₂ supply in Spirulina cultures resulted not only in the increase of DIC concentrations but also in the highest accumulated DIC concentration in the liquid medium (127.4 mg L⁻¹ d⁻¹). The highest values of biomass concentration (1.98 g L⁻¹), biomass productivity (131.8 mg L⁻¹ d⁻¹), carbon in biomass (47.9% w w⁻¹), CO₂ fixation rate (231.6 mg L⁻¹ d⁻¹), and CO₂ use efficiency (80.5% w w⁻¹) by Spirulina were verified with MS, compared to the culture with conventional sparger for CO₂ supply. Spirulina biomass in both culture conditions had high protein contents varying from 64.9 to 69% (w w⁻¹). MS can be considered an innovative system for the supply of carbon for the microalgae cultivation and biomass production. Moreover, the use of membrane system might contribute to increased process efficiency with a reduced cost of biomass production.     

Controlled Production of Carnosic Acid and Carnosol in Cell Suspensions of Lepechinia meyenii Treated with Different Elicitors and Biosynthetic Precursors

Lepechinia meyenii is a medicinal plant specialized in the biosynthesis of different types of antioxidants including the diterpenes carnosic (CA) acid and carnosol (CS). Herein we present the results of plant tissue culture approaches performed in this medicinal plant with particular emphasis on the generation and evaluation of a cell suspension system for CA and CS production. The effect of sucrose concentration, temperature, pH, and UV-light exposure was explored. In addition, diverse concentrations of microbial elicitors (salicylic acid, pyocyanin, Glucanex, and chitin), simulators of abiotic elicitors (polyethylene glycol and NaCl), and biosynthetic precursors (mevalonolactone, geranylgeraniol, and miltiradiene/abietatriene) were evaluated on batch cultures for 20 days. Miltiradiene/abietatriene obtainment was achieved through a metabolic engineering approach using a recombinant strain of Saccharomyces cerevisiae. Our results suggested that the maximum accumulation (Acc_max) of CA and CS was mainly conferred to stimuli associated with oxidative stress such as UV-light exposure (Acc_max, 6.2 mg L⁻¹) polyethylene glycol (Acc_max, 6.5 mg L⁻¹) NaCl (Acc_max, 5.9 mg L⁻¹) which simulated drought and saline stress, respectively. Nevertheless the bacterial elicitor pyocyanin was also effective to increase the production of both diterpenes (Acc_max, 6.4 mg L⁻¹). Outstandingly, the incorporation of upstream biosynthetic precursors such as geranylgeraniol and miltiradiene/abietatriene, generated the best results with Acc_max of 8.6 and 16.7 mg L⁻¹, respectively. Optimized batch cultures containing 100 mg L⁻¹ geranylgeraniol, 50 mg L⁻¹ miltiradiene/abietatriene (95 : 5 %) and 5 g L⁻¹ polyethylene glycol treated with 6 min UV light pulse during 30 days resulted in Acc_max of 26.7 mg L⁻¹ for CA and 17.3 mg L⁻¹ for CS on days 18–24. This strategy allowed to increase seven folds the amounts of CA and CS in comparison with batch cultures without elicitation (Acc_max, 4.3 mg L⁻¹).     

Fermentation and purification of microbial monomer 4-amminocinnamic acid to produce ultra-high performance bioplastics

Aromatic amines are base materials for generating super-engineering plastics such as polyamides and polyimides. Recombinant Escherichia coli ferments 4-aminocinnamic acid (4ACA) from glucose, and it can be derived to plastics of biomass origin with extreme thermal properties. Here, we scaled-up 4ACA production by optimizing microbial fermentation processes. The initial fermentation of 4-aminophenylalanine (4APhe) using E. coli generated the papABC genes of Pseudomonas fluorescens that produced 4APhe with a volumetric mass transfer coefficient (k_La) of 70 h⁻¹ in 115 L of culture broth, and 334 g of 4APhe at a final concentration of 2.9 g 4APhe L⁻¹. Crude 4APhe prepared from the fermentation broth was bioconverted to 4ACA by an E. coli strain producing phenylalanine ammonia lyase of the yeast Rhodotorula glutinis. The E. coli cells cultured under optimized conditions converted 4APhe to 4ACA at a rate of 0.65 g L⁻¹ 4ACA OD₆₀₀⁻¹. These processes resulted in the final derivation of 4.1 g L⁻¹ of 4ACA from glucose. The 4ACA was purified from the reaction as a hydrochloric acid salt and photodimerized to 4,4’-diaminotruxillic acid, which was polycondensed to produce bioaromatic polyimides. Large-scale 4ACA production will facilitate investigations of the physicochemical properties of biomass-derived aromatic polymers of 4ACA origin.     

Glycerol increases growth,protein production and alters the fatty acids profile of Spirulina (Arthrospira) sp LEB 18

Most of the crude glycerol produced globally is generated by biodiesel production, which makes this byproduct an environmental responsibility of the biofuel industries. Among the forms of this compound in use, microalgae cultivation is a promising alternative that may generate a reduction in crude glycerol treatment costs via using it as an organic, carbon-rich substrate in culture media. In this work, the influence of different concentrations of glycerol in the culture medium, the composition of fatty acids and proteins in Spirulina sp. LEB 18 biomass and their effect on its growth were investigated. The fatty acid profile of the biomass was altered, showing a 20% increase in the unsaturated concentration and a 60% reduction in the saturated concentration in the culture supplemented with 0.05 mol L⁻¹ of glycerol compared to those in the control. The addition of the substrate stimulated an increase in its cellular concentration (3.00 g L⁻¹, 0.05 mol L⁻¹), productivity (0.72 g L⁻¹ d⁻¹, 0.05 mol L⁻¹) and its protein production (69.78% w w⁻¹, 0.05 mol L⁻¹).     

Flow injection chemiluminescence determination of acetochlor and cartap-HCl in freshwater samples using an acidic KMnO4–rhodamine-B reaction system

A flow injection (FI) methodology using the acidic potassium permanganate (KMnO₄)–rhodamine-B (Rh-B) reaction with chemiluminescence (CL) detection was established to determine acetochlor and cartap-HCl pesticides in freshwater samples. Experimental parameters were optimized, and Chelex-100 cationic exchanger mini column and solid-phase extraction (SPE) were used as phase separation techniques. Linear calibration curves were observed for the standard solutions of acetochlor and cartap-HCl over the ranges 0.005–2.0 mg L⁻¹ [y = 1155.8x + 57.551, R² = 0.9999 (n = 8)] and 0.005–1.0 mg L⁻¹ [y = 979.76x + 14.491, R² = 0.9998 (n = 8)] with LODs and LOQs of 7.5 × 10⁻⁴ and 8.0 × 10⁻⁴ mg L⁻¹ (3σ blank) and 2.5 × 10⁻³ and 2.7 × 10⁻³ mg L⁻¹ (10σ blank), respectively, with an injection throughput of 140 h⁻¹. These methods were used to estimate acetochlor and cartap-HCl with or without the SPE procedure, respectively, in spiked freshwater samples. Results obtained were not significantly different at a 95% confidence level to those of other reported methods. Recoveries for acetochlor and cartap-HCl were obtained over the ranges 93–112% (RSD = 1.9–3.6%) and 98–109% (RSD = 1.7–3.8%), respectively. The most probable CL reaction mechanism was explored.     

Soil and tree chemistry reflected the cumulative impact of acid deposition in Pinus banksiana and Populus tremuloides stands in the Athabasca oil sands region in western Canada

The spatial variability of soil chemistry and Ca/Al ratios of soil solution and fine roots were investigated in jack pine (Pinus banksiana) and trembling aspen (Populus tremuloides, aspen) stands to assess the impact of chronic acid deposition on boreal forest ecosystems in the Athabasca oil sands region (AOSR) in Alberta, Canada. Available SO₄²⁻ (as the sum of soluble and adsorbed SO₄²⁻) accumulated in the soil near tree boles of both species, reflecting the influence of canopy intercepted SO₄²⁻. In jack pine stands, pH and soluble base cation concentrations decreased towards tree boles due to increased SO₄²⁻ leaching; the reverse was found in aspen stands due to deposition of base cations leached from the canopy. As a result, Ca/Al ratios in the soluble fraction in soils near jack pine boles were 5–20 times lower than that near aspen boles. The Ca/Al ratio did not reach the critical limits of 1.0 for soil solution (ranged from 1.0 to 4.1) or 0.5 for fine roots (0.7–7.9) in the studied watersheds. However, Alⁿ⁺ concentrations in the soil solution ranged from 0.2 to 4.1 mg L⁻¹ in NE7 and from 0.1 to 8.5 mg L⁻¹ in SM8 that can inhibit the growth of white spruce (Picea glauca) seedlings that commonly succeed aspen in upland sites in the AOSR. We suggest that the spatial variation caused by tree canopies/stems will affect forest regeneration and the effect of acid deposition on forest succession in the AOSR should be further studied.     

Characterization of recombinant L-ribose isomerase acquired from Cryobacterium sp. N21 with potential application in L-ribulose production

l-Ribose isomerase (lRI) is an enzyme that can catalyze the reversible isomerization between l-ribose and l-ribulose. It can also perform the conversion between many aldoses into their corresponding ketoses. l-RI was produced from Cryobacterium sp. N21 (CrL-RIse), and l-ribose was utilized as a substrate. The recombinant l-RI gene was cloned and overexpressed from Cryobacterium sp. N21. The purification of CrL-RIse was performed by metal-affinity chromatography. The enzyme displayed a corresponding band with an approximate size of 35 kDa on the SDS-PAGE analysis. The protein for this gene contains 266 amino acids with an expected molecular weight (M_w) of 29.6 kDa. The measured M_w of CrL-RIse calculated by HPLC was 125 kDa. CrL-RIse was extremely active in glycine buffer at 35 °C, pH 9.0, showing a specific activity of 54.96 U mg⁻¹. CrL-RIse displayed no major increase in activity with metal ions, excluding Mn²⁺. The estimated Km, K_cat, K_cat/Km and V_max values of CrL-RIse were 37.8 mM, 10,416 min⁻¹, 275.43 min⁻¹ mM⁻¹, and 250 U mg⁻¹, respectively. The rate of l-ribulose production was 31 % (6.24, 12.11, and 20.89 g L⁻¹) at equilibrium by utilizing 20, 40, and 70 g L⁻¹ of the substrate, respectively. The results indicated that CrL-RIse has the capability to manufacture l-ribulose from l-ribose.