Expression of <Emphasis Type="Italic">Vitreoscilla</Emphasis> hemoglobin in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> improve the cell density and insecticidal crystal proteins yield

The Vitreoscilla hemoglobin (VHb) gene (vgb) was integrated into the chromosome of Bacillus thuringiensis BMB171 using integrative vector pEG491. The production of VHb was confirmed by CO-difference spectra analysis. Fermentation experiments results showed that with the production of VHb, the critical oxygen concentration (COC) of the host strain was reduced from 18 to 12%. The maximum viable cell counts of the VHb⁺ strain in high, middle, and low aeration/agitation fermentations were 0.94-, 1.23-, and 1.59-fold of those of the VHb⁻ strain, respectively. Under the same conditions, the yields of insecticidal crystal proteins (ICP) by VHb⁺ strain were 1.22-, 1.63-, and 3.13-fold of those of the VHb⁻ strain. The production of VHb also accelerated the formation of ICP and spores. These results indicated that the production of VHb could improve the cell density and ICP yield of B. thuringiensis, especially under low aeration/agitation condition.     

Production and characterization of homopolymer poly(3-hydroxyvalerate) (PHV) accumulated by wild type and recombinant Aeromonas hydrophila strain 4AK4

Aeromonas hydrophila 4AK4 normally produces copolyesters (PHBHHx) consisting of 3-hydroxybutyrate (C4) and 3-hydroxyhexanoate (C6). Wild type and recombinant A. hydrophila 4AK4 (pSXW02) expressing vgb and fadD genes encoding Vitreoscilla haemoglobin and Escherichia coli acyl-CoA synthase respectively, were found able to produce homopolyester poly(3-hydroxyvalerate) (PHV) (C5) on undecanoic acid as a single carbon source. The recombinant grew to 5.59 g/L cell dry weight (CDW) containing 47.74 wt% PHV in shake flasks when growth was conducted in LB medium and PHV production in undecanoic acid. The cells grew to 47.12 g/L CDW containing 60.08 wt% PHV in a 6 L fermentor study. Physical characterization of PHV produced by recombinant A. hydrophila 4AK4 (pSXW02) in fermentor showed a weight average molecular weight (M_w) of 230,000 Da, a polydispersity of 3.52, a melting temperature of 103 °C and a glass transition temperature of −15.8 °C. The degradation temperature at 5% weight loss of the PHV was around 258 °C.     

Hypoxia-induced growth rate reduction in two juvenile estuary-dependent fishes

As eutrophication of coastal waters increases, water quality issues such as hypoxia have come to the forefront of environmental concerns for many estuarine systems. Chronic hypoxia during the summer has become a common occurrence in numerous estuaries, degrading nursery habitat and increasing the potential for exposure of juvenile fish to low levels of dissolved oxygen (DO).We conducted a laboratory study to investigate how hypoxic conditions and temperature affect growth rates of two juvenile estuary-dependent fish: the Atlantic menhaden (Brevoortia tyrannus) and spot (Leiostomus xanthurus). For a 2-week period, we exposed the fish to one of four constant DO levels (6.0, 4.0, 2.0 or 1.5 mg O₂ l⁻¹), at one of two temperatures (25 or 30 °C). A fifth DO treatment, included for spot at 30 °C, allowed DO to fluctuate from 10.0 mg O₂ l⁻¹ during the day, to 2.0 mg O₂ l⁻¹ at night. This diel fluctuation approximated the natural DO cycle in tidal estuarine creeks. Size measurements were recorded at the beginning, middle and end of experiments.Growth rates were generally unaffected by low DO until concentrations dropped to 1.5 mg O₂ l⁻¹, resulting in 31-89% growth reductions. Our results suggest that DO levels must be severely depressed, and in fact, approaching lethal limits, to negatively impact growth of juvenile spot and Atlantic menhaden.     

High expression of Trigonopsis variabilis -amino acid oxidase in Pichia pastoris

To explore a new approach of high expression of -amino acid oxidase (DAAO) in Pichia pastoris, a gene encoding DAAO from Trigonopsis variabilis (TvDAAO gene) deleted intron was prepared by PCR amplification and cloned into the intracellular expression vector pPIC3.5K. The expression plasmid pPIC3.5K-DAAO linearized by SalI was transformed into Pichia pastoris strain GS115 (his⁻mut⁺). By means of MM and MD plates and PCR, the recombinant P. pastoris strains (his⁺mut⁺) were obtained. Activity assay and SDS-PAGE demonstrated that DAAO was intracellularly expressed in P. pastoris with the induction of methanol. The recombinant strain PD27 with the highest expression of DAAO was screened through activity assay and its high-density fermentation was carried out in a 1-l fermentor. Activity assay and SDS-PAGE demonstrated that DAAO was intracellularly expressed in P. pastoris with the induction of methanol. The recombinant cells with high expression of DAAO were screened and the high-density fermentation was carried out in a 1-l fermentor. Interestingly, the DAAO expression level reached up to 473 U/g dry cell weight in fermentation yield. Finally, 1-hexanol was used to break recombinant cells and the specific activity of DAAO was 1.46 U/mg protein in crude extraction.     

Microbial production of L -glutamate and L -glutamine by recombinant Corynebacterium glutamicum harboring Vitreoscilla hemoglobin gene vgb

Vitreoscilla hemoglobin (VHb) gene vgb equipped with a native promoter Pvgb or a tac promoter Ptac was introduced into Corynebacterium glutamicum ATCC14067, respectively. Ptac was proven to be more suitable for expressing VHb protein in higher concentration in both Escherichia coli and C. glutamicum strains compared with the native vgb promoter Pvgb. VHb-expressing C. glutamicum exhibited higher oxygen uptake rate and enhanced cell growth. Recombinant C. glutamicum harboring vgb gene equipped with Ptac promoter produced 23% more l-glutamate in shake-flask culture and grew to 30% more cell density and formed 22% more l-glutamate in fermentor studies compared with the wild-type strain. When a site-directed mutagenesis in which Tyr405 was replaced by a phenylalanine residue (Y405F) was performed on glutamine synthesis gene, recombinant C. glutamicum overexpressing the mutated gene glnA′ was able to produce l-glutamine effectively. Co-expression of vgb and glnA′ genes in C. glutamicum produced 17 g/l l-glutamine in shake flask culture, approximately 30% more than that produced by the recombinant harboring only glnA′ gene. In fermentor cultivation, the recombinant yielded 25% more cells and produced 40.5 g/l l-glutamine. In this study, it was clearly demonstrated that VHb significantly enhanced cell growth, l-glutamate, and l-glutamine production by recombinant C. glutamicum.     

Effect of nutrient limitation and two-stage continuous fermentor design on productivities during "Clostridium ragsdalei" syngas fermentation

The effect of three limiting nutrients, calcium pantothenate, vitamin B₁₂ and cobalt chloride (CoCl₂), on syngas fermentation using “Clostridium ragsdalei” was determined using serum bottle fermentation studies. Significant results from the bottle studies were translated into single- and two-stage continuous fermentor designs. Studies indicated that three-way interactions between the three limiting nutrients, and two-way interactions between vitamin B₁₂ and CoCl₂ had a significant positive effect on ethanol and acetic acid formation. In general, ethanol and acetic acid production ceased at the end of 9 days corresponding to the production of 2.01 and 1.95 g L⁻¹ for the above interactions. Reactor studies indicated the three-way nutrient limitation in two-stage fermentor showed improved acetic acid (17.51 g g⁻¹ cells) and ethanol (14.74 g g⁻¹ cells) yield compared to treatments in single-stage fermentors. These results further support the hypothesis that it is possible to modulate the product formation by limiting key nutrients during C. ragsdalei syngas fermentation.     

Impact of hypoxia on the survival, egg production and population dynamics of Acartia tonsa Dana

Concern for the increasing occurrence of coastal zone hypoxia has generally focused on the direct, short-term impact of reduced dissolved oxygen (DO) concentrations on the survival of commercially important species such as fish and crabs. Copepods, especially the naupliar stages, are important pelagic food web components, yet only a few studies have considered the effect of reduced DO concentrations on their survival and population dynamics. This study considers the impact of both lethal and sublethal DO concentrations on copepods. Acartia tonsa were reared at 25 °C at saturating DO (normoxic control) and reduced (hypoxic) DO concentrations of 1.5 or 0.7 ml l⁻¹. Oxygen concentrations were maintained in replicate flasks, by bubbling seawater with air (control), or mixtures of nitrogen and oxygen. Egg production, but not survival, was significantly higher in the controls compared to the 1.5 ml l⁻¹ DO treatment. Survival and egg production were significantly lower at 0.7 ml l⁻¹ DO compared to the control. The results suggest that the sublethal as well as the lethal effects of hypoxia may have important repercussions on population and community dynamics in coastal systems.     

Expression of Vitreoscilla Hemoglobin Enhances Cell Growth and Dihydroxyacetone Production in Gluconobacter oxydans

Dihydroxyacetone (DHA) is an important ketose sugar, which is extensively used in the cosmetic, chemical, and pharmaceutical industries. DHA has been industrially produced by Gluconobacter oxydans with a high demand of oxygen. To improve the production of DHA, the gene vgb encoding Vitreoscilla hemoglobin (VHb) was successfully introduced into G. oxydans, where it was stably maintained, and expressed at about 76.0 nmol/g dry cell weight. Results indicated that the constitutively expressed VHb improved cell growth and DHA production in G. oxydans under different aeration conditions. Especially at low aeration rates, the VHb-expressing strain (VHb⁺) displayed 23.13% more biomass and 37.36% more DHA production than those of VHb-free strain (VHb⁻) after 32 h fermentation in bioreactors. In addition, oxygen uptake rate (OUR) was also increased in VHb⁺ strain relative to the control strain during fermentation processes.     

Short term effects of hypoxia and bioturbation on solute fluxes, denitrification and buffering capacity in a shallow dystrophic pond

The effects of short term hypoxia on bioturbation activity and inherent solute fluxes are scarcely investigated even if increasing number of coastal areas are subjected to transient oxygen deficits. In this work dark fluxes of oxygen (O₂), dissolved inorganic carbon (TCO₂) and nutrients across the sediment-water interface, as well as rates of denitrification (isotope pairing), were measured in intact sediment cores collected from the dystrophic pond of Sali e Pauli (Sardinia, Italy). Sediments were incubated at 100, 70, 40 and 10% of O₂ saturation in the overlying water, with both natural benthic communities, dominated by the polychaete Polydora ciliata (11.100 ± 2.500  ind. m^− 2), and after the addition of individuals of the deep-burrower polychaete Hediste diversicolor. Below an uppermost oxic layer of ~ 1 mm, sediments were highly reduced, with up to 6 mM of S²⁻ in the 5 mm layer. Flux of S²⁻ and O₂ calculated from pore water gradients were 8.61 ± 1.12 and − 2.27 ± 0.56 mmol m^− 2 h^− 1, respectively. However, sediment oxygen demand (SOD) calculated from core incubation was − 10.52 ± 0.33 mmol m^− 2 h^− 1, suggesting a major contribution of P. ciliata to O₂-mediated sulphide oxidation. P. ciliata also strongly stimulated NH₄⁺ and PO₄³⁻ fluxes, with rates ~ 15 and ~ 30 folds higher, respectively, than those estimated from pore water gradients. P. ciliata activity was significantly reduced at 10% O₂ saturation, coupled to decreased rates of solutes transfer. The addition of H. diversicolor further stimulated SOD, NH₄⁺ efflux and SiO₂ mobilisation. Similarly to P. ciliata, the degree of stimulation of SOD and NH₄⁺ flux by H. diversicolor depended on the level of oxygen saturation. TCO₂ regeneration, respiratory quotients, PO₄³⁻ fluxes and denitrification of added ¹⁵NO₃⁻ were not affected by the addition of H. diversicolor, but depended upon the O₂ levels in the water column. Denitrification rates supported by water column ¹⁴NO₃⁻ and sedimentary nitrification were both negligible (< 0.5 µmol m^− 2 h^− 1). They were not significantly affected by oxygen saturation nor by bioturbation, probably due to the limited availability of NO₃⁻ in the water column (< 3 µM) and O₂ in the sediments. This study demonstrates for the first time the integrated short term effect of transient hypoxia and bioturbation on solute fluxes across the sediment-water interface within a simplified lagoonal benthic community.     

Recombinant glucose oxidase from Penicillium amagasakiense for efficient bioelectrochemical applications in physiological conditions

GOX is the most widely used enzyme for the development of electrochemical glucose biosensors and biofuel cell in physiological conditions. The present work describes the production of a recombinant glucose oxidase from Penicillium amagasakiense (yGOXpenag) displaying a more efficient glucose catalysis (k_cat/K_M(glucose) = 93 μM⁻¹ s⁻¹) than the native GOX from Aspergillus niger (nGOXaspng), which is the most industrially used (k_cat/K_M(glucose) = 27 μM⁻¹ s⁻¹). Expression in Pichia pastoris allowed easy production and purification of the recombinant active enzyme, without overglycosylation. Its biotechnological interest was further evaluated by measuring kinetics of ferrocinium-methanol (FM_ox) reduction, which is commonly used for electron transfer to the electrode surface. Despite their homologies in sequence and structure, pH-dependant FM_ox reduction was different between the two enzymes. At physiological pH and temperature, we observed that electron transfer to the redox mediator is also more efficient for yGOXpenag than for nGOXaspng(k_cat/K_M(FM_ox) = 27 μM⁻¹ s⁻¹ and 17 μM⁻¹ s⁻¹ respectively). In our model system, the catalytic current observed in the presence of blood glucose concentration (5 mM) was two times higher with yGOXpenag than with nGOXaspng. All our results indicated that yGOXpenag is a better candidate for industrial development of efficient bioelectrochemical devices used in physiological conditions.     

Intracellular expression of Vitreoscilla hemoglobin improves S-adenosylmethionine production in a recombinant Pichia pastoris

To develop an efficient way to produce S-adenosylmethionine (SAM), methionine adenosyltransferase gene (mat) from Streptomyces spectabilis and Vitreoscilla hemoglobin gene (vgb) were coexpressed intracellularly in Pichia pastoris, both under control of methanol-inducible promoter. Expression of mat in P. pastoris resulted in about 27 times higher specific activity of methionine adenosyltransferase (SMAT) and about 19 times higher SAM production relative to their respective control, suggesting that overexpression of mat could be used as an efficient method for constructing SAM-accumulating strain. Under induction concentration of 0.8 and 2.4% methanol, coexpression of vgb improved, though to different extent, cell growth, SAM production, and respiratory rate. However, the effects of VHb on SAM content (specific yield of SAM production) and SMAT seemed to be methanol concentration-dependent. When cells were induced with 0.8% methanol, no significant effects of VHb expression on SAM content and specific SMAT could be detected. When the cells were induced with 2.4% methanol, vgb expression increased SAM content significantly and depressed SMAT remarkably. We suggested that under our experimental scheme, the presence of VHb might improve ATP synthesis rate and thus improve cell growth and SAM production in the recombinant P. pastoris.     

Improved production of monoclonal antibodies through oxygen-limited cultivation of glycoengineered yeast

Glycoengineering technology can elucidate and exploit glycan related structure-function relationships for therapeutic proteins. Glycoengineered yeast has been established as a safe, robust, scalable, and economically viable expression platform. It has been found that specific productivity of antibodies in glycoengineered Pichia pastoris is a non-linear function of specific growth rate that is dictated by a limited methanol feed rate. The optimal carbon-limited cultivation requires an exponential methanol feed rate with an increasing biomass concentration and more significantly an increase in heat and mass transfer requirements that often become the limiting factor in scale-up. Both heat and mass transfer are stoichiometrically linked to the oxygen uptake rate. Consequently an oxygen-limited cultivation approach was evaluated to limit the oxygen uptake rate and ensure robust and reliable scale-up. The oxygen-limited process not only limited the maximum oxygen uptake rate (and consequently the required heat removal rate) in mut+ P. pastoris strains but also enabled extension of the induction phase leading to an increased antibody concentration (1.9 g L⁻¹ vs. 1.2 g L⁻¹), improved N-glycan composition and galactosylation, and reduced antibody fragmentation. Furthermore, the oxygen-limited process was successfully scaled to manufacturing pilot scale and thus presents a promising process option for the glycoengineered yeast protein expression platform.     

Optimized production of scygonadin in Pichia pastoris and analysis of its antimicrobial and antiviral activities

The crab antimicrobial peptide scygonadin is confirmed to have antimicrobial activity against bacteria and it is probably associated with the reproductive immunity in Scylla paramamosain. To obtain large quantity of scygonadin for further biological assays, a 306 bp cDNA sequence encoding the mature peptide of scygonadin was cloned into a secretion vector of pPIC9K, and a high-level of the recombinant scygonadin was achieved in Pichia pastoris. The optimal expression condition was determined as incubation with 0.5% methanol for 48 h at 28 °C under pH 6.0, and a total of 70 mg scygonadin was expressed in 1 L culture medium. The recombinant product was purified and 97% pure scygonadin was obtained using immobilized metal affinity chromatography with a yield of 46 mg/L. The recombinant scygonadin was confirmed using SDS-PAGE analysis and MS-fingerprinting. P. pastoris-derived scygonadin exhibited relatively higher antimicrobial activities against bacteria than Escherichia coli-derived scygonadin. The antimicrobial activity of the recombinant scygonadin against pathogenic Aeromonas hydrophila showed salt resistant and the killing kinetics of A. hydrophila was time dependent. Besides, the antiviral assay demonstrated that scygonadin could interfere with white spot syndrome virus (WSSV) replication in vitro-cultured crayfish haematopoietic (Hpt) cells. Taken together, this is the first report on the heterologous expression of scygonadin in P. pastoris, and P. pastoris is an effective expression system for producing large quantities of biological active scygonadin for both research and agricultural application.     

Effect of NaCl on photosynthesis,salt accumulation and ion compartmentation in two mangrove species, Kandelia candel and Bruguiera gymnorhiza

In a 4-week study, we investigated the effects of increasing soil NaCl (100–400 mM) on photosynthesis, salt uptake and transport, and intracellular compartmentation of Na⁺ and Cl⁻ in 1-year-old seedlings of Kandelia candel (L.) Druce and Bruguiera gymnorhiza (L.) Savigny. Increasing NaCl stress significantly elevated Na⁺ and Cl⁻ in root and shoot tissues (stem + leaf) of both species, but B. gymnorhiza showed a rapid Na⁺ accumulation upon the initiation of salt stress and leaves contained 90% more Na⁺ and 40% more Cl⁻ than K. candel at the end of experiment. Net photosynthetic rate (Pn) declined with increasing salinity, and the most marked reduction occurred after exposure of mangrove seedlings to a severe salinity, 400 mM NaCl. However, the inhibitory effects of severe stress varied with species: Pn decreased by 80% in K. candel whereas in B. gymnorhiza the decline was 60%. The quantum yield (AQY) and carboxylation efficiency (CE) response to severe salinity showed a trend similar to Pn, in which a lesser reduction of AQY and CE was observed in B. gymnorhiza (33–35%), as compared to K. candel (43–52%). X-ray microanalysis of leaf mesophyll cells showed evidence of distinct vacuolar compartmentation of Na⁺ in K. candel but Cl⁻ in B. gymnorhiza after seedlings were subjected to 100 mM NaCl for 7 d. Moreover, Na⁺ within cell wall, cytoplasm, vacuole and chloroplast remained 23–72% lower in stressed B. gymnorhiza as compared to K. candel. In conclusion, B. gymnorhiza exhibited effective salt exclusion from chloroplasts although increasing salt stress caused a rapid and higher build up of Na⁺ and Cl⁻ in the leaves. We suggest that the salt-induced Pn reduction in the two mangrove species is correlated with the ability to exclude Na⁺ and Cl⁻ from the chloroplast, rather than with the bulk leaf salt concentration.     

Development of anaerobically inducible nar promoter expression vectors for the expression of recombinant proteins in Escherichia coli

Dissolved oxygen (DO)-controlled nar promoter expression vectors were constructed, and their expression efficiency was compared with that of the T7 promoter pET22 expression vector by expressing human growth hormone (hGH), enhanced green fluorescence protein (EGFP), and β-tyrosinase in Escherichia coli cells. The nar promoter expression vector pRBS, which was engineered with a 5′-untranslated region and ribosomal binding site for the T7 promoter, expressed hGH at a rate of up to 32% of the total cellular proteins (TCP) in E. coli W3110narL⁻. The expression level of hGH was further enhanced, up to ∼42% of the TCP, by adding the N-terminal peptide tag of β-galactosidase to hGH, which was comparable to the expression of ∼43% of the TCP in pET-lac:hGH/BL21(DE3). A further engineered expression vector, pRBS(fnr), which coexpressed fumarate/nitrate reductase (fnr), expressed more EGFP than pET22 in BL21(DE3). In addition, recombinant β-tyrosinase was successfully expressed at a rate of up to ∼45% of the TCP in pRBS(fnr) in W3110narL⁻. From these results, the DO-controlled nar promoter system developed in this study can be considered a reliable and cost-effective expression system for protein production, especially in large-scale fermentation, as an alternative to the pET/BL(DE3) system.     

Ethanol production from syngas by Clostridium strain P11 using corn steep liquor as a nutrient replacement to yeast extract

The feasibility of replacing yeast extract (YE) by corn steep liquor (CSL), a low cost nutrient source, for syngas fermentation to produce ethanol using Clostridium strain P11 was investigated. About 32% more ethanol (1.7 g L⁻¹) was produced with 20 g L⁻¹ CSL media in 250-mL bottle fermentations compared to media with 1 g L⁻¹ YE after 360 h. Maximum ethanol concentrations after 360 h of fermentation in a 7.5-L fermentor with 10 and 20 g L⁻¹ CSL media were 8.6 and 9.6 g L⁻¹, respectively, which represent 57% and 60% of the theoretical ethanol yields from CO. Only about 6.1 g L⁻¹ of ethanol was obtained in the medium with 1 g L⁻¹ YE after 360 h, which represents 53% of the theoretical ethanol yield from CO. The use of CSL also enhanced butanol production by sevenfold compared to YE in bottle fermentations. These results demonstrate that CSL can replace YE as the primary medium component and significantly enhance ethanol production by Clostridium strain P11.     

Functional expression of FIP-fve,a fungal immunomodulatory protein from the edible mushroom Flammulina velutipes in Pichia pastoris GS115

FIP-fve is a bioactive protein isolated from the mushroom Flammulina velutipes, which belongs to the fungal immunomodulatory protein (FIP) family and demonstrates several kinds of biological activities including anti-allergy, anti-tumor and immunomodulation. In the current study, the FIP-fve gene was cloned and expressed in the yeast Pichia pastoris GS115, and its correctness was confirmed by SDS-PAGE and Western blot. Optimal expression of rFIP-fve was observed when the P. pastoris cells were cultured in 1% methanol for 96 h, which resulted in a yield of 258.2 mg l⁻¹. The rFIP-fve protein was subsequently purified via ammonium sulfate precipitation and Sephadex G-100 gel chromatography. In vitro bioactivity examination showed that rFIP-fve could agglutinate human red blood cells and stimulate the cell viability of murine splenocytes. The immunomodulatory capacity and anti-tumor activity of rFIP-fve were demonstrated by enhanced interleukin-2 secretion and interferon-γ release from the murine lymphocytes, similar to the biological FIP-fve. In conclusion, the FIP-fve gene was functionally and effectively expressed in P. pastoris, and rFIP-fve displayed biological activities similar to those of native FIP-fve. These results indicated the potential use of rFIP-fve from P. pastoris as an effective and feasible source for therapeutic studies and medical applications.     

α2-3 Sialic acid glycoconjugate loss and its effect on infection with Toxoplasma parasites

Recognition of sialylated glycoconjugates is important for host cell invasion by Apicomplexan parasites. Toxoplasma gondii parasites penetrate host cells via interactions between their microneme proteins and sialylated glycoconjugates on the surface of host cells. However, the role played by sialic acids during infection with T. gondii is not well understood. Here, we focused on the role of α2-3 sialic acid linkages as they appear to be widely expressed in vertebrates. Removal of α2-3 sialic acid linkages on macrophages by neuraminidase treatment did not influence the rate of infection or growth of T. gondii, nor did it affect phagocytosis in vitro. Sialyltransferase ST3Gal-I deficient mice (ST3Gal-I^−/− mice) lost α2-3 sialic acid linkages in macrophages and spleen cells. The numbers of T. gondii-infected CD11b⁺ cells in peritoneal cavities of the infected ST3Gal-I^−/− mice were relatively lower than those of the infected wild type animals. In addition, CD8⁺ T cell populations and numbers in the spleens and peritoneal cavities of the ST3Gal-I^−/− mice were significantly lower than those in the wild type animals before and after the T. gondii infection. ST3Gal-I^−/− mice had severe liver damage and reduced survival rates following peritoneal infection with T. gondii. Furthermore, adoptive transfer of immune CD8⁺ cells from wild type mice to ST3Gal-I^−/− mice increased their survival during infection with T. gondii. Our data show that parasite invasion via α2-3 sialic acid linkages might not contribute on host survival and indicate the impact that loss of α2-3 sialic acid linkages has on CD8⁺ T cell populations, which are necessary for effective immune responses against infection with T. gondii.     

Different sensitivity of Phragmites australis and Glyceria maxima to high availability of ammonium-N

The ability to cope with NH₄⁺-N was studied in the littoral helophytes Phragmites australis and Glyceria maxima, species commonly occupying fertile habitats rich in NH₄⁺ and often used in artificial wetlands. In the present study, Glyceria growth rate was reduced by 16% at 179 μM NH₄⁺-N, and the biomass production was reduced by 47% at 3700 μM NH₄⁺-N compared to NO₃⁻-N. Similar responses were not found in Phragmites. The amounts (mg g⁻¹ dry wt) of starch and total non-structural carbohydrates (TNC) in rhizomes were significantly lower in NH₄⁺ (8.9; 12.2 starch; 20.1; 41.9 TNC) compared to NO₃⁻ treated plants (28.0; 15.6 starch; 58.5; 56.3 TNC) in Phragmites and Glyceria, respectively. In addition, Glyceria showed lower amounts (mg g⁻¹ dry wt) of soluble sugars, TNC, K⁺, and Mg²⁺ in roots under NH₄⁺ (5.6; 14.3; 20.6; 1.9) compared to NO₃⁻ nutrition (11.6; 19.9; 37.9; 2.9, for soluble sugars, TNC, K⁺, and Mg²⁺, respectively), while root internal levels of NH₄⁺ and Ca²⁺ (0.29; 4.6 mg g⁻¹ dry wt, mean of both treatments) were only slightly affected. In Phragmites, no changes in soluble sugars, TNC, Ca²⁺, K⁺, and Mg²⁺ contents of roots (7.3; 14.9; 5.1; 17.3; 2.6 mg g⁻¹ dry wt, means of both treatments) were found in response to treatments. The results, therefore, indicate a more pronounced tolerance towards high NH₄⁺ supply in Phragmites compared to Glyceria, although the former may be susceptible to starch exhaustion in NH₄⁺-N nutrition. In contrast, Glyceria's ability to colonize fertile habitats rich in NH₄⁺ is probably related to the avoidance strategy due to shallow rooting or to the previously described ability to cope with high NH₄⁺ levels when P availability is high and NO₃⁻ is also provided.