Active carbon uptake in Laminaria digitata and L. saccharina (Phaeophyta) is driven by a proton pump in the plasma membrane

Mechanisms for inorganic carbon acquisition in Laminaria digitata (Hudson) Lamour and L. saccharina (L.) Lamour from the Swedish west coast were studied in pH-drift experiments, using several inhibitors for different types of carbon uptake across the cell membrane. Throughout the experiments total carbon decreased in concert with a pH increase while alkalinity stayed relatively stable. Addition of acetazolamide had a strong inhibitory effect on the carbon uptake rate in L. digitata and the anion exchange protein inhibitor DIDS had a small inhibitory effect above pH 9.5. These results indicate that carbon uptake in L. digitata depends on the presence of an external carbonic anhydrase while direct bicarbonate uptake may contribute at high pH. These two mechanisms have previously been shown to occur in L. saccharina. We show that two inhibitors of H⁺-ATPases, vanadate and erythrosin B, also decreased carbon uptake rates in both Laminaria species. The effect of erythrosin B was immediate and it probably acts on the outside of the cell membrane. Contrarily, vanadate needs to be transported into the cell, where it competes with the phosphate from ATP for the aspartic acid phosphorylation site on the plasma membrane P-type H⁺-ATPase. Therefore, 1–2 h of pH drift were usually required before an inhibitory effect became apparent. Additional experiments with P-enriched and P-starved L. saccharina corroborated this process. Based on these results we suggest that the investigated Laminaria species, besides external carbonic anhydrase and DIDS-sensitive anion exchange, also possess a mechanism for the active uptake of carbon, which is dependent on plasma membrane P-type H⁺-ATPase activity. This paper also reports on the buffering capacity of the inhibitors when used in natural seawater, an aspect that has been neglected in previous studies.     

A novel approach to specific allergy treatment: the recombinant allergen-S-layer fusion protein rSbsC-Bet v 1 matures dendritic cells that prime Th0/Th1 and IL-10-producing regulatory T cells

An ideal vaccine for allergen-specific immunotherapy of type I allergies should display reduced mediator-releasing capacity, induce maturation of APC, and modify the disease-eliciting Th2-dominated allergen-specific response to a more physiological response. We have previously shown that rSbsC-Bet v 1, the recombinant fusion protein of a bacterial surface (S-layer) protein of Geobacillus stearothermophilus ATCC 12980 and the major birch pollen allergen Bet v 1, exhibited reduced allergenicity and induced IFN-gamma and IL-10 synthesis in Bet v 1-specific Th2 clones. In this study, we characterized the effects of rSbsC-Bet v 1 on immature monocyte-derived dendritic cells (mdDC) and the consequences for the polarization of naive CD4(+) T lymphocytes isolated from the blood of birch pollen-allergic patients. mdDC responded to rSbsC-Bet v 1 with a significant up-regulation of costimulatory molecules, functional maturation, and the synthesis of IL-10 and IL-12. mdDC matured with rSbsC-Bet v 1 induced the differentiation of naive T cells into IFN-gamma-producing cells. This effect was IL-12 dependent. In parallel, a substantial number of naive T cells developed into IL-10-producing CD25(+)Foxp3(+)CLTA-4(+) cells capable of active suppression. Thus, rSbsC-Bet v 1 showed immune stimulatory capacity on DC, which then promoted the simultaneous differentiation of Th0/Th1 cells and regulatory T cells. These data further support that the concept of conjugating allergens to bacterial agents is a promising approach to improve vaccines for specific immunotherapy of atopic allergies.     

Activation of TLR2 and TLR4 by glycosylphosphatidylinositols derived from Toxoplasma gondii

GPIs isolated from Toxoplasma gondii, as well as a chemically synthesized GPI lacking the lipid moiety, activated a reporter gene in Chinese hamster ovary cells expressing TLR4, while the core glycan and lipid moieties cleaved from the GPIs activated both TLR4- and TLR2-expressing cells. MyD88, but not TLR2, TLR4, or CD14, is absolutely needed to trigger TNF-alpha production by macrophages exposed to T. gondii GPIs. Importantly, TNF-alpha response to GPIs was completely abrogated in macrophages from TLR2/4-double-deficient mice. MyD88(-/-) mice were more susceptible to death than wild-type (WT), TLR2(-/-), TLR4(-/-), TLR2/4(-/-), and CD14(-/-) mice infected with the ME-49 strain of T. gondii. The cyst number was higher in the brain of TLR2/4(-/-), but not TLR2(-/-), TLR4(-/-), and CD14(-/-), mice, as compared with WT mice. Upon infection with the ME-49 strain of T. gondii, we observed no decrease of IL-12 and IFN-gamma production in TLR2-, TLR4-, or CD14-deficient mice. Indeed, splenocytes from T. gondii-infected TLR2(-/-) and TLR2/4(-/-) mice produced more IFN-gamma than cells from WT mice in response to in vitro stimulation with parasite extracts enriched in GPI-linked surface proteins. Together, our results suggest that both TLR2 and TLR4 receptors may participate in the host defense against T. gondii infection through their activation by the GPIs and could work together with other MyD88-dependent receptors, like other TLRs or even IL-18R or IL-1R, to obtain an effective host response against T. gondii infection.     

Oncolytic virotherapy synergism with signaling inhibitors: Rapamycin increases myxoma virus tropism for human tumor cells

Myxoma virus is a rabbit-specific poxvirus pathogen that also exhibits a unique tropism for human tumor cells and is dramatically oncolytic for human cancer xenografts. Most tumor cell lines tested are permissive for myxoma infection in a fashion intimately tied to the activation state of Akt kinase. A host range factor of myxoma virus, M-T5, directly interacts with Akt and mediates myxoma virus tumor cell tropism. mTOR is a regulator of cell growth and metabolism downstream of Akt and is specifically inhibited by rapamycin. We report that treatment of nonpermissive human tumor cell lines, which normally restrict myxoma virus replication, with rapamycin dramatically increased virus tropism and spread in vitro. This increased myxoma replication is concomitant with global effects on mTOR signaling, specifically, an increase in Akt kinase. In contrast to the effects on human cancer cells, rapamycin does not increase myxoma virus replication in rabbit cell lines or permissive human tumor cell lines with constitutively active Akt. This indicates that rapamycin increases the oncolytic capacity of myxoma virus for human cancer cells by reconfiguring the internal cell signaling environment to one that is optimal for productive virus replication and suggests the possibility of a potentially therapeutic synergism between kinase signaling inhibitors and oncolytic poxviruses for cancer treatment.     

Assessment of DNA damage at the dimer level: measurement of the formamide lesion

UVC-radiation-induced DNA damage was measured in mouse fibroblast cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in conjunction with isotopically labeled internal standards. The thymine glycol and formamide lesions were assayed in the form of modified dinucleoside monophosphates. The 8-oxo-7,8-dihydroguanine lesion was measured as the modified nucleoside. DNA damage in cells treated with tirapazamine was also measured. Tirapazamine is a chemotherapeutic agent that acts via a free radical mechanism. The two agents, UVC radiation and tirapazamine, produce markedly different profiles of DNA damage, reflecting their respective mechanisms of action. Both agents produce significant amounts of thymine glycol and formamide damage, but only the former produced a measurable amount of the 8-oxo-7,8-dihydroguanine lesion. The merits of measuring DNA damage at the dimer level are discussed.     

Parvicapsula bicornis n. sp. and P. limandae n. sp. (Myxozoa, Parvicapsulidae) in Pleuronectidae (Teleostei, Heterosomata) from Denmark

Two species of Parvicapsula were found in the kidney tubules and the urinary bladder of 2 pleuronectid fish from the northern Oresund, Denmark. The coelozoic, spherical, disporic trophozoites of both species are 10 to 12 pm in diameter. The myxospores of both species are elongate, asymmetrical and slightly curved, and have spherical polar capsules. Parvicapsula bicornis n. sp. (6-8 x 5-6 microm, polar capsule 2.5 microm in diameter) occurs in Pleuronectes platessa. The polar capsules of P. bicornis are arranged symmetrically on either side of the longitudinal axis and its spores differ from other species of Parvicapsula in having two 2-3 microm long posterior processes of different length. Parvicapsula limandae n. sp. (8-11 x 4-5 pm, polar capsule 1.6 microm in diameter) is found in Limanda limanda. The polar capsules are arranged along the longitudinal axis. It differs from Parvicapsula unicornis Kabata, 1962, recorded from L. limanda, in the arrangement of the polar capsules and in the absence of a posterior horn-like projection. The phylogenetic relationship between P. bicornis n. sp., P. limandae n. sp. and other Parvicapsula spp. was examined with their partial small subunit rDNA (SSU rDNA) sequences. P. limandae n. sp. and P. asymmetrica appear to be closely related, while P. bicornis n. sp. and P. minibicornis are the most divergent members of the genus.     

Iron absorption by Belgrade rat pups during lactation

Divalent metal transporter-1 (DMT1) mediates dietary nonheme iron absorption. Belgrade (b) rats have defective iron metabolism due to a mutation in the DMT1 gene. To examine the role of DMT1 in neonatal iron assimilation, b/b and b/+ pups were cross-fostered to F344 Fischer dams injected with (59)FeCl(3) twice weekly during lactation. Tissue distribution of the radioisotope in the pups was determined at weaning (day 21). The b/b pups had blood (59)Fe levels significantly lower than b/+ controls but significantly higher (59)Fe tissue levels in heart, bone marrow, skeletal muscle, kidney, liver, spleen, stomach, and intestines. To study the pharmacokinetics of nonheme iron absorption at the time of weaning, (59)FeCl(3) was administered to 21-day-old b/b and b/+ rats by intragastric gavage. Blood (59)Fe levels measured 5 min to 4 h postgavage were significantly lower in b/b rats, consistent with impaired DMT1 function in intestinal iron absorption. Tissue (59)Fe levels were also lower in b/b rats postgavage. Combined, these data suggest that DMT1 function is not essential for iron assimilation from milk during early development in the rat.     

Identification of a novel 82 kDa proMMP-9 species associated with the surface of leukaemic cells: (auto-)catalytic activation and resistance to inhibition by TIMP-1

MMP-9 (matrix metalloproteinase 9) plays a critical role in tumour progression. Although the biochemical properties of the secreted form of proMMP-9 are well characterized, little is known about the function and activity of cell surface-associated proMMP-9. We purified a novel 82 kDa species of proMMP-9 from the plasma membrane of THP-1 leukaemic cells, which has substantial differences from the secreted 94 kDa proMMP-9. The 82 kDa form was not detected in the medium even upon stimulation with a phorbol ester. It is truncated by nine amino acid residues at its N-terminus, lacks O-linked oligosaccharides present in the 94 kDa proMMP-9, but retains N-linked carbohydrates. Incubation of 94 kDa proMMP-9 with MMP-3 generated the well-known 82 kDa active form, but the 82 kDa proMMP-9 was converted into an active species of 35 kDa, which was also produced by autocatalytic processing in the absence of activating enzymes. The activated 35 kDa MMP-9 efficiently degraded gelatins, native collagen type IV and fibronectin. The enzyme was less sensitive to TIMP-1 (tissue inhibitor of metalloproteinase 1) inhibition with IC50 values of 82 nM compared with 1 nM for the 82 kDa active MMP-9. The synthetic MMP inhibitor GM6001 blocked the activity of both enzymes, with similar IC50 values below 1 nM. The 82 kDa proMMP-9 is also produced in HL-60 and NB4 leukaemic cell lines as well as ex vivo leukaemic blast cells. It is, however, absent from neutrophils and mononuclear cells isolated from peripheral blood of healthy individuals. Thus, the 82 kDa proMMP-9 expressed on the surface of malignant cells may escape inhibition by natural TIMP-1, thereby facilitating cellular invasion in vivo.