The "prismane" protein resolved: X-ray structure at 1.7 Å and multiple spectroscopy of two novel 4Fe clusters

The three-dimensional structure of the native "putative prismane" protein from Desulfovibrio vulgaris (Hildenborough) has been solved by X-ray crystallography to a resolution of 1.72?Å. The molecule does not contain a [6Fe-6S] prismane cluster, but rather two 4Fe clusters some 12?Å apart and situated close to the interfaces formed by the three domains of the protein. Cluster 1 is a conventional [4Fe-4S] cubane bound, however, near the N-terminus by an unusual, sequential arrangement of four cysteine residues (Cys 3, 6, 15, 21). Cluster 2 is a novel 4Fe structure with two μ₂-sulfido bridges, two μ₂-oxo bridges, and a partially occupied, unidentified μ₂ bridge X. The protein ligands of cluster 2 are widely scattered through the second half of the sequence and include three cysteine residues (Cys 312, 434, 459), one persulfido-cysteine (Cys 406), two glutamates (Glu 268, 494), and one histidine (His 244). With this unusual mixture of bridging and external type of ligands, cluster 2 is named the "hybrid" cluster, and its asymmetric, open structure suggests that it could be the site of a catalytic activity. X-ray absorption spectroscopy at the Fe K-edge is readily interpretable in terms of the crystallographic model when allowance is made for volume contraction at 10?K; no Fe··Fe distances beyond 3.1?Å could be identified. EPR, Mössbauer and MCD spectroscopy have been used to define the oxidation states and the magnetism of the clusters in relation to the crystallographic structure. Reduced cluster 1 is a [4Fe-4S]¹⁺ cubane with S?=?3/2; it is the first biological example of a "spin-admixed" iron-sulfur cluster. The hybrid cluster 2 has four oxidation states from (formally) all Fe^III to three Fe^II plus one Fe^III. The four iron ions are exchange coupled resulting in the system spins S?=?0, 9/2, 0 (and 4), 1/2, respectively, for the four redox states. Resonance Raman spectroscopy suggests that the bridging ligand X which could not be identified unambiguously in the crystal structure is a solvent-exchangeable oxygen.     

Interaction of serum amyloid A with human cystatin C—assessment of amino acid residues crucial for hCC–SAA formation (part II)

Secondary amyloid A (AA) amyloidosis is an important complication of some chronic inflammatory diseases, primarily rheumatoid arthritis (RA). It is a serious, potentially life‐threatening disorder caused by the deposition of AA fibrils, which are derived from the circulatory, acute‐phase‐reactant, serum amyloid A protein (SAA). Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been proved. Using a combination of selective proteolytic excision and high‐resolution mass spectrometry, the binding sites in the SAA and hCC sequences were assessed as SAA(86–104) and hCC(96–102), respectively. Here, we report further details concerning the hCC–SAA interaction. With the use of affinity tests and florescent ELISA‐like assays, the amino acid residues crucial for the protein interaction were determined. It was shown that all amino acid residues in the SAA sequence, essential for the formation of the protein complex, are basic ones, which suggests an electrostatic interaction character. The idea is corroborated by the fact that the most important residues in the hCC sequence are Ser‐98 and Tyr‐102; these residues are able to form hydrogen bonds via their hydroxyl groups. The molecular details of hCC–SAA complex formation might be helpful for the design of new compounds modulating the biological role of both proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Amplification of CRAC current by STIM1 and CRACM1 (Orai1)

Depletion of intracellular calcium stores activates store-operated calcium entry across the plasma membrane in many cells. STIM1, the putative calcium sensor in the endoplasmic reticulum, and the calcium release-activated calcium (CRAC) modulator CRACM1 (also known as Orai1) in the plasma membrane have recently been shown to be essential for controlling the store-operated CRAC current (I(CRAC)). However, individual overexpression of either protein fails to significantly amplify I(CRAC). Here, we show that STIM1 and CRACM1 interact functionally. Overexpression of both proteins greatly potentiates I(CRAC), suggesting that STIM1 and CRACM1 mutually limit store-operated currents and that CRACM1 may be the long-sought CRAC channel.     

Immunocytochemistry of the neuromuscular systems of Loxosomella vivipara and L. parguerensis (Entoprocta: Loxosomatidae)

Little detailed information exists on the anatomy of the nervous system and the musculature of Entoprocta. Herein we describe the distribution of the neurotransmitters RFamide and serotonin as well as the myo-anatomy of adults and asexually produced budding stages of the solitary entoproct species Loxosomella vivipara and L. parguerensis using immunocytochemistry and epifluorescence as well as confocal microscopy. The development of the RFamidergic and serotonergic nervous system starts in early budding stages. In the adults, RFamide is present in the bilateral symmetric cerebral ganglion, a pair of oral nerves that innervate two pairs of nerve cell clusters in the heel of the foot, a pair of aboral nerves, the paired lateral nerves, the calyx nerves, the atrial ring nerve, the tentacle nerves, the stomach nerves, and the rectal nerves. Serotonin is only found in the cerebral ganglion, the oral nerves, and in the tentacle nerves. Some differences in the distribution of both neurotransmitters were found between L. vivipara and L. parguerensis and are most obvious in the differing number of large serotonergic perikarya associated with the oral nerves. Nerves arising from the cerebral ganglion and running in a ventral direction have not been described for Entoprocta before, and the homology of these to the ventral nerve cords of other Spiralia is considered possible. The body musculature of both Loxosomella species comprises longitudinal and diagonal muscles in the foot, the stalk, and the calyx. We found several circular muscles in the calyx. The stalk and parts of the foot and the calyx are surrounded by a fine outer layer of ring muscles. In addition to the congruent details regarding the myo-anatomy of both species, species-specific muscle structures could be revealed. The comparison of our data with recent findings of the myo-anatomy of two Loxosoma species indicates that longitudinal and diagonal body muscles, atrial ring muscles, tentacle muscles, esophageal and rectal ring muscles, as well as intestinal and anal sphincters are probably part of the ancestral entoproct muscle bauplan.     

Phyto-extraction of heavy metals and biochemical changes with Brassica nigra L. grown in rayon grade paper mill effluent irrigated soil

In this study, distribution of metal accumulation and their biological changes of Indian mustard plants (Brassica nigra L.) grown in soil irrigated with different concentration of rayon grade paper effluent (RGPE, 25%, 50%, 75%, 100%, v/v) were studied. A pronounced effect was recorded at 50% (v/v) RGPE on germination of seeds, amylase activity and other growth parameters in Indian mustard plants. An increase in the chlorophyll and protein contents was also recorded at <50% (v/v) RGPE followed by a decrease at higher concentrations of RGPE (>75%). A significant increase lipid peroxidation was recorded, which was evidenced by the increased malondialdehyde (MDA) content in shoot, leaves and seeds in tested plant at all the concentrations of RGPE. This Indian mustard plants (Brassica nigra L.) are well adapted for tolerance of significant amount of heavy metals due to increased level of antioxidants (cysteine and ascorbic acid) in root shoot and leaves of treated plants at all concentration of RGPE. Moreover, it is also important that RGPE should be treated to bring down the metal concentration well within the prescribed limit prior to use in agricultural soil for ferti-irrigation.     

Nutrient uptake and utilization can be used to select viable day 7 bovine blastocysts after cryopreservation

The ability of individual bovine blastocysts to survive freezing and thawing procedures was assessed by measuring glucose and pyruvate uptake and lactate production immediately before and after cryopreservation. Using glucose and pyruvate uptake and lactate production it was not possible to determine, prior to freezing, which blastocysts would be viable after thawing. However, in the 5 hr immediately after thawing, those blastocysts which expanded their blastocoel had significantly greater glucose and pyruvate uptake and lactate production (P < 0.01) than those embryos which failed to develop after a 14 hr overnight incubation. Interestingly, after thawing, two distinct populations of blastocysts existed with respect to glucose uptake and lactate production, indicating that it is possible to identify those blastocysts immediately after thawing which will reexpand. In contrast, there was a considerable degree of overlap in pyruvate uptakes between the viable and nonviable groups of embryos, indicating that this parameter could not be used to select viable embryos after thawing. There was an increase in the calculated oxidation of carbohydrates after thawing, consistent with a partial uncoupling of the inner mitochondrial membrane. In conclusion, glucose uptake and lactate production can be used to select prospectively viable blastocysts immediately after thawing, indicating that glycolysis is a major energy-generating pathway for the embryo at this time. © 1996 Wiley-Liss, Inc.     

A Natural Combination Extract of Viscum album L. Containing Both Triterpene Acids and Lectins Is Highly Effective against AML In Vivo

Aqueous Viscum album L. extracts are widely used in complementary cancer medicine. Hydrophobic triterpene acids also possess anti-cancer properties, but due to their low solubility they do not occur in significant amounts in aqueous extracts. Using cyclodextrins we solubilised mistletoe triterpenes (mainly oleanolic acid) and investigated the effect of a mistletoe whole plant extract on human acute myeloid leukaemia cells in vitro, ex vivo and in vivo. Single Viscum album L. extracts containing only solubilised triterpene acids (TT) or lectins (viscum) inhibited cell proliferation and induced apoptosis in a dose-dependent manner in vitro and ex vivo. The combination of viscum and TT extracts (viscumTT) enhanced the induction of apoptosis synergistically. The experiments demonstrated that all three extracts are able to induce apoptosis via caspase-8 and -9 dependent pathways with down-regulation of members of the inhibitor of apoptosis and Bcl-2 families of proteins. Finally, the acute myeloid leukaemia mouse model experiment confirmed the therapeutic effectiveness of viscumTT-treatment resulting in significant tumour weight reduction, comparable to the effect in cytarabine-treated mice. These results suggest that the combination viscumTT may have a potential therapeutic value for the treatment AML.     

Hemopoietic cell expression of the chemokine decoy receptor D6 is dynamic and regulated by GATA1

D6 scavenges inflammatory chemokines and is essential for the regulation of inflammatory and immune responses. Mechanisms explaining the cellular basis for D6 function have been based on D6 expression by lymphatic endothelial cells. In this study, we demonstrate that functional D6 is also expressed by murine and human hemopoietic cells and that this expression can be regulated by pro- and anti-inflammatory agents. D6 expression was highest in B cells and dendritic cells (DCs). In myeloid cells, LPS down-regulated expression, while TGF-beta up-regulated expression. Activation of T cells with anti-CD3 and soluble CD28 up-regulated mRNA expression 20-fold, while maturation of human macrophage and megakaryocyte precursors also up-regulated D6 expression. Competition assays demonstrated that chemokine uptake was D6 dependent in human leukocytes, whereas mouse D6-null cells failed to uptake and clear inflammatory chemokines. Furthermore, we present evidence indicating that D6 expression is GATA1 dependent, thus explaining D6 expression in myeloid progenitor cells, mast cells, megakaryocytes, and DCs. We propose a model for D6 function in which leukocytes, within inflamed sites, activate D6 expression and thus trigger resolution of inflammatory responses. Our data on D6 expression by circulating DCs and B cells also suggest alternative roles for D6, perhaps in the coordination of innate and adaptive immune responses. These data therefore alter our models of in vivo D6 function and suggest possible discrete, and novel, roles for D6 on lymphatic endothelial cells and leukocytes.     

Effect of prostaglandin E2 and tumor necrosis factor alpha on the VEGF-receptor system expression in cultured porcine luteal cells

The purpose of the present study was to investigate the effects of prostaglandin (PG) E(2) and tumor necrosis factor (TNF) alpha on expression of vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR), in cultured porcine luteal cells. Real-time PCR was used for quantification of VEGF and its receptors mRNA, whereas VEGF release by luteal cells was determined by radioimmunoassay (RIA). Only the highest dose of PGE(2) (1 microM) after 6 hr of incubation stimulated VEGF release by luteal cells collected in the mid-luteal phase (P < 0.05). Moreover, PGE(2) (100 nM, 1 microM) significantly stimulated VEGF secretion by luteal cells in the late phase and during pregnancy on Days 10-12 (P < 0.05). Elevated mRNA expression of both VEGF 120 and VEGF 164 isoforms was found in luteal cells cultured with PGE(2). The lack of an effect of PGE(2) on VEGF receptors mRNA expression was observed. TNFalpha was able to significantly stimulate VEGF release from cells obtained in the mid- and late luteal phase or during early pregnancy. All tested doses enhanced mRNA levels of VEGF 120 isoform, but not VEGF 164. Additionally, TNFalpha was able to decrease Flk-1/KDR mRNA expression, whereas Flt-1 mRNA levels were not affected. These results indicated that PGE(2) and TNFalpha influenced VEGF ligand-receptor system expression in porcine luteal cells and may therefore play an important role in regulation of luteal functions during the estrous cycle and pregnancy in pigs.     

Molecular identification, immunolocalization, and functional activity of a vacuolar-type H+-ATPase in bovine rumen epithelium

In this study, we have studied the expression, localization, and functionality of vacuolar-type H⁺-ATPase (vH⁺-ATPase) and Na⁺/K⁺-ATPase in the bovine rumen epithelium. Compared with the intracellular pH (pH_i) of control rumen epithelial cells (REC; 7.06 ± 0.07), application of inhibitors selective for vH⁺-ATPase (foliomycin) and Na⁺/K⁺-ATPase (ouabain) reduced pH_i by 0.10 ± 0.03 and 0.18 ± 0.03 pH-units, respectively, thereby verifying the existence of both functional proteins. Results from qRT-PCR and immunoblotting clearly confirm the expression of vH⁺-ATPase B subunit in REC. However, the amount of Na⁺/K⁺-ATPase mRNA and protein is tenfold and 11-fold of those of vH⁺-ATPase subunit B, respectively, reflecting a lower overall abundance of the latter in REC. Na⁺/K⁺-ATPase immunostaining has revealed the protein in the plasma membrane of all REC from the stratum basale to stratum granulosum, with the highest abundance in basal cells. In contrast, the vH⁺-ATPase B subunit has been detected in groups of cells only, mainly localized in the stratum spinosum and stratum granulosum of the epithelium. Furthermore, vH⁺-ATPase has been detected in the cell membrane and in intracellular pools. Thus, functional vacuolar-type H⁺ pumps are expressed in REC and probably play a role in the adaptation of epithelial transport processes.