Chromophore-Independent Roles of Opsin Apoproteins in Drosophila Mechanoreceptors.

Rhodopsins, the major light-detecting molecules of animal visual systems [1], consist of opsin apoproteins that covalently bind a retinal chromophore with a conserved lysine residue [1, 2]. In addition to capturing photons, this chromophore contributes to rhodopsin maturation [3, 4], trafficking [3, 4], and stabilization [5], and defects in chromophore synthesis and recycling can cause dysfunction of the retina and dystrophy [6-9]. Indications that opsin apoproteins alone might have biological roles have come from archaebacteria and platyhelminths, which present opsin-like proteins that lack the chromophore binding site and are deemed to function independently of light [10, 11]. Light-independent sensory roles have been documented for Drosophila opsins [12-15], yet also these unconventional opsin functions are thought to require chromophore binding [12, 13, 15]. Unconjugated opsin apoproteins act as phospholipid scramblases in mammalian photoreceptor disks [16], yet chromophore-independent roles of opsin apoproteins outside of eyes have, to the best of our knowledge, hitherto not been described. Drosophila chordotonal mechanoreceptors require opsins [13, 15], and we find that their function remains uncompromised by nutrient carotenoid depletion. Disrupting carotenoid uptake and cleavage also left the mechanoreceptors unaffected, and manipulating the chromophore attachment site of the fly's major visual opsin Rh1 impaired photoreceptor, but not mechanoreceptor, function. Notwithstanding this chromophore independence, some proteins that process and recycle the chromophore in the retina are also required in mechanoreceptors, including visual cycle components that recycle the chromophore upon its photoisomerization. Our results thus establish biological function for unconjugated opsin apoproteins outside of eyes and, in addition, document chromophore-independent roles for chromophore pathway components.

Alveolus-like organoid from isolated tip epithelium of embryonic mouse lung.

Embryonic lungs were obtained from embryonic day 13.5 ICR mice. The lung-tip epithelium isolated using dispase treatment was embedded in low-growth factor Matrigel, cultured in DMEM/F12 medium containing 0.1% bovine serum albumin, supplemented with insulin, transferrin, and selenium (ITS), with or without fibroblast growth factor 7 (FGF7), and were observed for 14 days. With the addition of FGF7, the tip epithelium grew to form a cyst by culture day 7. Then, tubular tufts-like alveolus appeared around the cyst surface. Reverse transcription-polymerase chain reaction revealed that, with the addition of FGF7, the cultured lung explants expressed alveolar-type 1 cell markers, such as HopX and Aquaporin5, and type 2 cell markers, such as Lamp3 and Surfactant apoproteins (Sftp) C and D. Paraffin-embedded sections were stained with hematoxylin and eosin, and alveolar structures at culture day 14 were composed of squamous and cuboidal epithelial cells. Immunohistochemical studies showed that the squamous epithelial cells were positive for HopX, and the cuboidal epithelial cells were positive for pro-SftpC. Furthermore, transmission electron microscopic observation confirmed that the squamous epithelial cells were alveolar-type 1 cells and the cuboidal cells were type 2 cells, because they had many lamellar inclusion bodies. Embryonic lung-tip epithelium forms an alveolus-like organoid through the self organization with the aid of Matrigel, ITS, and FGF7. This method to make alveolus-like organoid in vitro is easy, reproducible, and economical. This method could have potential to solve many issues in alveolar epithelial cells in normal and pathological conditions.

Computational comparison of a calcium-dependent jellyfish protein (apoaequorin) and calmodulin-cholesterol in short-term memory maintenance.

Memory reconsolidation and maintenance depend on calcium channels and on calcium/calmodulin-dependent kinases regulating protein turnover in the hippocampus. Ingestion of a jellyfish protein, apoaequorin, reportedly protects and/or improves verbal learning in adults and is currently widely advertised for use by the elderly. Apoaequorin is a member of the EF-hand calcium binding family of proteins that includes calmodulin. Calmodulin-1 (148 residues) differs from Apoaequorin (195 residues) in that it contains four rather than three Ca2+-binding sites and three rather than four cholesterol-binding (CRAC, CARC) domains. All three cholesterol-binding CARC domains in calmodulin have a high interaction affinity for cholesterol compared to only two high affinity CARC domains in apoaequorin. Both calmodulin and apoaequorin can form dimers with a potential of eight bound Ca2+ ions and six high affinity-bound cholesterol molecules in calmodulin with six bound Ca2+ ions and a mixed population of eight cholesterols bound to both CARC and CRAC domains in apoaqueorin. MEMSAT-SVM analysis indicates that both calmodulin and apoaqueorin have a pore-lining region. The Peptide-Cutter algorithm predicts that calmodulin-1 contains 11 trypsin-specific cleavage sites (compared to 21 in apoaqueorin), four of which are potentially blocked by cholesterol and three are within the Ca-binding domains and/or the pore-lining region. Three are clustered between the third and fourth Ca2+-binding sites. Only calmodulin pore-lining regions contain Ca2+ binding sites and as dimers may insert into the plasma membrane of neural cells and act as Ca2+ channels. In a dietary supplement, bound cholesterol may protect both apoaequorin and calmodulin from proteolysis in the gut as well as facilitate uptake across the blood-brain barrier. Our results suggest that a physiological calmodulin-cholesterol complex, not cholesterol-free jellyfish protein, may better serve as a dietary supplement to facilitate memory maintenance.

Nitration of plant apoplastic proteins from cell suspension cultures.

Nitric oxide causes numerous protein modifications including nitration of tyrosine residues. This modification, though one of the greatest biological importance, is poorly recognized in plants and is usually associated with stress conditions. In this study we analyzed nitrotyrosines from suspension cultures of Arabidopsis thaliana and Nicotiana tabacum, treated with NO modulators and exposed to osmotic stress, as well as of BY2 cells long-term adapted to osmotic stress conditions. Using confocal microscopy, we showed that the cell wall area is one of the compartments most enriched in nitrotyrosines within a plant cell. Subsequently, we analyzed nitration of ionically-bound cell-wall proteins and identified selected proteins with MALDI-TOF spectrometry. Proteomic analysis indicated that there was no significant increase in the amount of nitrated proteins under the influence of NO modulators, among them 3-morpholinosydnonimine (SIN-1), considered a donor of nitrating agent, peroxynitrite. Moreover, osmotic stress conditions did not increase the level of nitration in cell wall proteins isolated from suspension cells, and in cultures long-term adapted to stress conditions; that level was even reduced in comparison with control samples. Among identified nitrotyrosine-containing proteins dominated the ones associated with carbon circulation as well as the numerous proteins responding to stress conditions, mainly peroxidases. BIOLOGICAL SIGNIFICANCE: High concentrations of nitric oxide found in the cell wall and the ability to produce large amounts of ROS make the apoplast a site highly enriched in nitrotyrosines, as presented in this paper. Analysis of ionically bound fraction of the cell wall proteins indicating generally unchanged amounts of nitrotyrosines under influence of NO modulators and osmotic stress, is noticeably different from literature data concerning, however, the total plant proteins analysis. This observation is supplemented by further nitroproteome analysis, for cells long-term adapted to stressful conditions, and results showing that such conditions did not always cause an increase in nitrotyrosine content. These findings may be interpreted as characteristic features of apoplastic protein nitration.

Effects of mild and moderate hypothemia therapy on expression of cerebral neuron apoptosis related proteins and glial fiber acidic protein after rat cardio-pulmonary resuscitation.

To explore the effects of different degrees of hypothermia on brain tissue apoptosis after cardio-pulmonary resuscitation (CPR). Cardiac arrest for 5 min induced by asphyxia method was used to create CPR model. 30 SD rats were randomly divided into control group (normothermia), 33 °C hypothermia group and 30 °C hypothermia group with ten rats in each. Rats in control group received routine treatment at 25 °C room temperature after CPR; Rats in mild hypothermia and moderate hypothermia groups were given hypothermia treatment 0.5 h after CPR. Brain tissue in all groups was taken 24 h after CPR, and immunohistochemistry was used to detect the caspase-3 in cerebral cortex and glial fiber acidic protein (GFAP) expression in astrocyte. Western blotting was used to detect Bcl-2 and Bax protein expression, and histopathological change was observed in brain tissue. Compare to the control group, caspase-3 expression in cerebral neurons in hypothermia group was significantly decreased (p<0.01), which was significantly lower in 30 °C group than that in 33 °C group (p > 0.05); GFAP level in hypothermia groups was significantly increased (p < 0.01), which was higher in 30 °C hypothermia group than that in 33 °C hypothermia group (p < 0.05); Bcl-2 expression level in hypothermia group was significantly increased (p < 0.01), which was higher in 30 °C hypothermia group than that in 33 °C hypothermia group (p < 0.05); The level of Bax had no significant difference among the three groups. Hypothermia-regulated GFAP expression by decreasing caspase-3 expression and increasing Bcl-2 expression to promote brain cell signaling transduction, and further inhibited cell apoptosis and reduced brain injury. Moderate hypothermia therapy is more effective than mild hypothermia in preventing brain injure.

During hormone depletion or tamoxifen treatment of breast cancer cells the estrogen receptor apoprotein supports cell cycling through the retinoic acid receptor α1 apoprotein.

INTRODUCTION: Current hormonal adjuvant therapies for breast cancer including tamoxifen treatment and estrogen depletion are overall tumoristatic and are severely limited by the frequent recurrence of the tumors. Regardless of the resistance mechanism, development and progression of the resistant tumors requires the persistence of a basal level of cycling cells during the treatment for which the underlying causes are unclear. METHODS: In estrogen-sensitive breast cancer cells the effects of hormone depletion and treatment with estrogen, tamoxifen, all-trans retinoic acid (ATRA), fulvestrant, estrogen receptor α (ER) siRNA or retinoic acid receptor α (RARα) siRNA were studied by examining cell growth and cycling, apoptosis, various mRNA and protein expression levels, mRNA profiles and known chromatin associations of RAR. RARα subtype expression was also examined in breast cancer cell lines and tumors by competitive PCR. RESULTS: Basal proliferation persisted in estrogen-sensitive breast cancer cells grown in hormone depleted conditioned media without or with 4-hydroxytamoxifen (OH-Tam). Downregulating ER using either siRNA or fulvestrant inhibited basal proliferation by promoting cell cycle arrest, without enrichment for ErbB2/3+ overexpressing cells. The basal expression of RARα1, the only RARα isoform that was expressed in breast cancer cell lines and in most breast tumors, was supported by apo-ER but was unaffected by OH-Tam; RAR-ß and -γ were not regulated by apo-ER. Depleting basal RARα1 reproduced the antiproliferative effect of depleting ER whereas its restoration in the ER depleted cells partially rescued the basal cycling. The overlapping tamoxifen-insensitive gene regulation by apo-ER and apo-RARα1 comprised activation of mainly genes promoting cell cycle and mitosis and suppression of genes involved in growth inhibition; these target genes were generally insensitive to ATRA but were enriched in RAR binding sites in associated chromatin regions. CONCLUSIONS: In hormone-sensitive breast cancer, ER can support a basal fraction of S-phase cells (i) without obvious association with ErbB2/3 expression, (ii) by mechanisms unaffected by hormone depletion or OH-Tam and (iii) through maintenance of the basal expression of apo-RARα1 to regulate a set of ATRA-insensitive genes. Since isoform 1 of RARα is genetically redundant, its targeted inactivation or downregulation should be further investigated as a potential means of enhancing hormonal adjuvant therapy.

The interaction between retinol-binding protein and transthyretin analyzed by fluorescence anisotropy.

The retinol carrier retinol-binding protein (RBP) forms in blood a complex with the thyroid hormone carrier transthyretin (TTR). The interactions of retinoid-RBP complexes, as well as of unliganded RBP, with TTR can be investigated by means of fluorescence anisotropy. RBP represents the prototypic lipocalin, in the internal cavity of which the retinol molecule is accommodated. Due to the tight binding of retinol within a substantially apolar binding site, an intense fluorescence emission characterizes the RBP-bound vitamin. The addition of TTR to the retinol-RBP complex (holoRBP) causes a marked increase in the fluorescence anisotropy of the RBP-bound retinol within the system, due to the formation of the holoRBP-TTR complex, which allows the interaction between the two proteins to be monitored. The fluorescence anisotropy technique is also suitable to study the interaction of TTR with apoRBP and RBP in complex with non-fluorescent retinoids. In the latter cases, the fluorescence signal is provided by a fluorescent probe covalently linked to TTR rather than by RBP-bound retinol. We report here on the preparation of recombinant human RBP and TTR, the covalent labeling of TTR with the fluorescent dansyl probe, and fluorescence anisotropy titrations for RBP and TTR.

In silico cloning, expression of Rieske-like apoprotein gene and protein subcellular localization in the Pacific oyster, Crassostrea gigas.

Rieske protein gene in the Pacific oyster Crassostrea gigas was obtained by in silico cloning for the first time, and its expression profiles and subcellular localization were determined, respectively. The full-length cDNA of Cgisp is 985 bp in length and contains a 5'- and 3'-untranslated regions of 35 and 161 bp, respectively, with an open reading frame of 786 bp encoding a protein of 262 amino acids. The predicted molecular weight of 30 kDa of Cgisp protein was verified by prokaryotic expression. Conserved Rieske [2Fe-2S] cluster binding sites and highly matched-pair tertiary structure with 3CWB_E (Gallus gallus) were revealed by homologous analysis and molecular modeling. Eleven putative SNP sites and two conserved hexapeptide sequences, box I (THLGC) and II (PCHGS), were detected by multiple alignments. Real-time PCR analysis showed that Cgisp is expressed in a wide range of tissues, with adductor muscle exhibiting the top expression level, suggesting its biological function of energy transduction. The GFP tagging Cgisp indicated a mitochondrial localization, further confirming its physiological function.

Structural insights into the binding of uranyl with human serum protein apotransferrin structure and spectra of protein-uranyl interactions.

Ab initio quantum mechanical computational studies for the structure and IR spectra of the uranyl complex with human serum apotransferrin (TF) protein are carried out to model uranyl intake into the human cell through endocytosis and formation of a coordination complex with the protein binding sites. The computed IR spectra and structure of the uranyl-protein complex facilitate interpretation of the observed spectra and confirm the primary binding sites of the transferrin protein with the uranyl ion. Our computed equilibrium geometry and the IR spectra of the uranyl-TF complex reveal that uranyl ion is bound to two tyrosines, one aspartate group, and one carbonate ion. Our IR spectra indicate that histidine is not involved in binding to uranyl with transferrin protein. Our computations reveal a short, strong hydrogen bond, which could play an important role in the stabilization and formation of the uranyl-TF complex. Computed Laplacian charge plots indicate high chemical reactivity on this complex as both an electrophile and a nucleophile, facilitating binding to different receptors and thus entry into a number of target organs and the blood-brain barrier. The Mulliken charge density plots and the three-dimensional charge density plots suggest a donor-acceptor mechanism in the complex formation.

Resonant x-ray scattering and absorption for the global and local structures of Cu-modified metallothioneins in solution.

With Cd and Zn metal ions removed from the native rabbit-liver metallothionein upon unfolding, Cu-modified metallothioneins (Cu-MTs) were obtained during refolding in solutions containing Cu(I) or Cu(II) ions. X-ray absorption near-edge spectroscopic results confirm the respectively assigned oxidation states of the copper ions in Cu(I)-MT and Cu(II)-MT. Global and local structures of the Cu-MTs were subsequently characterized by anomalous small-angle x-ray scattering (ASAXS) and extended x-ray absorption fine structure. Energy-dependent ASAXS results indicate that the morphology of Cu(II)-MT resembles that of the native MT, whereas Cu(I)-MT forms oligomers with a higher copper content. Both dummy-residue simulation and model-shape fitting of the ASAXS data reveal consistently rodlike morphology for Cu(II)-MT. Clearly identified Cu-S, Cu-O, and Cu-Cu contributions in the extended x-ray absorption fine structure analysis indicate that both Cu(I) and Cu(II) ions are bonded with O and S atoms of nearby amino acids in a four-coordination environment, forming metal clusters smaller than metal thiolate clusters in the native MT. It is demonstrated that a combination of resonant x-ray scattering and x-ray absorption can be particularly useful in revealing complementary global and local structures of metalloproteins due to the atom specific characteristics of the two techniques.

Fluorescence assay of non-transferrin-bound iron in thalassemic sera using bacterial siderophore.

Transfusional iron overload associated with thalassemia leads to the appearance of non-transferrin-bound iron (NTBI) in blood that is toxic and causes morbidity and mortality via tissue damage. Hence, a highly sensitive and accurate assay of NTBI, with broad clinical application in both diagnosis and validation of treatment regimens for iron overload, is important. An assay based on iron chelation by a high-affinity siderophore, azotobactin, has been developed. The steps consist of blocking of native apotransferrin iron binding sites, mobilization of NTBI, ultrafiltration of all serum proteins, and finally the addition of the probe, which has a chromophore that fluoresces at 490 nm. Binding of Fe3+ to azotobactin quenches the fluorescence in a concentration-dependent manner. Measured NTBI levels in 63 sera ranged from 0.07 to 3.24 microM (0.375+/-0.028 microM [means+/-SEM]). It correlated well with serum iron and percentage transferrin saturation but not with serum ferritin. Pearson's correlation coefficients were found to be 0.6074 (P<0.0001) and 0.6102 (P<0.0001) for percentage transferrin saturation and total serum iron, respectively. The low values are due to the patients being under regular chelation therapy even prior to sampling, indicating that the method is sensitive to very low levels of NTBI, allowing a much lower detection limit than the available methods.

Covalent heme attachment to the protein in human heme oxygenase-1 with selenocysteine replacing the His25 proximal iron ligand.

To characterize heme oxygenase with a selenocysteine (SeCys) as the proximal iron ligand, we have expressed truncated human heme oxygenase-1 (hHO-1) His25Cys, in which Cys-25 is the only cysteine, in the Escherichia coli cysteine auxotroph strain BL21(DE3)cys. Selenocysteine incorporation into the protein was demonstrated by both intact protein mass measurement and mass spectrometric identification of the selenocysteine-containing tryptic peptide. One selenocysteine was incorporated into approximately 95% of the expressed protein. Formation of an adduct with Ellman's reagent (DTNB) indicated that the selenocysteine in the expressed protein was in the reduced state. The heme-His25SeCys hHO-1 complex could be prepared by either (a) supplementing the overexpression medium with heme, or (b) reconstituting the purified apoprotein with heme. Under reducing conditions in the presence of imidazole, a covalent bond is formed by addition of the selenocysteine residue to one of the heme vinyl groups. No covalent bond is formed when the heme is replaced by mesoheme, in which the vinyls are replaced by ethyl groups. These results, together with our earlier demonstration that external selenolate ligands can transfer an electron to the iron [Y. Jiang, P.R. Ortiz de Montellano, Inorg. Chem. 47 (2008) 3480-3482 ], indicate that a selenyl radical is formed in the hHO-1 His25SeCys mutant that adds to a heme vinyl group.

Identification of the functional expression of adenosine A3 receptor in pancreas using transgenic mice expressing jellyfish apoaequorin.

To investigate the functional expression of adenosine A3 receptor (A3AR) in mammalian living tissues, we generated an apoaequorin-transgenic mouse that expresses jellyfish apoaequorin throughout its body. The expression of apoaequorin under the control of a strong CAG promoter was detected in various tissues, including the abdominal skin, adipose, ear, brain, esophagus, heart, inferior vena cava vessel, kidney, lens, liver, lung, pancreas, skeletal muscle, spleen, tail, testis, and thymus. The transgene was mapped to the C1-2 region of chromosome 16 by Fluorescence in situ hybridization analysis. Among these transgenic mouse tissues, we succeeded in detecting elevated responses of intracellular Ca2+ as a light emission of aequorin induced by the A3AR agonist in the pancreas, brain, and testis, the last two of which are known to be main tissues abundantly expressing A3AR. The A3AR agonist led to the phosphorylation of both extracellular signal-regulated kinase 1/2 and protein kinase B in mouse pancreas, and all the intracellular responses via A3AR were antagonized by the A3AR-specific antagonist. In addition, the mRNA expression of A3AR and the A3AR-induced intracellular responses were also found in the rat pancreatic acinar cell line AR42J. These results suggest that pancreas is one of the main tissues functionally expressing A3AR in mammalians in vivo, and that the present approach using transgenic mice that express apoaequorin throughout their bodies will facilitate the functional analysis of proteins of interest.

Structural consequences of binding of UO2(2+) to apotransferrin: can this protein account for entry of uranium into human cells?

It has been established that transferrin binds a variety of metals. These include toxic uranyl ions which form rather stable uranyl-transferrin derivatives. We determined the extent to which the iron binding sites might accommodate the peculiar topographic profile of the uranyl ion and the consequences of its binding on protein conformation. Indeed, metal intake via endocytosis of the transferrin/transferrin receptor depends on the adequate coordination of the metal in its site, which controls protein conformation and receptor binding. Using UV-vis and Fourier transform infrared difference spectroscopy coupled to a microdialysis system, we showed that at both metal binding sites two tyrosines are uranyl ligands, while histidine does not participate with its coordination sphere. Analysis by circular dichroism and differential scanning calorimetry (DSC) showed major differences between structural changes associated with interactions of iron or uranyl with apotransferrin. Uranyl coordination reduces the level of protein stabilization compared to iron, but this may be simply related to partial lobe closure. The lack of interaction between uranyl-TF and its receptor was shown by flow cytometry using Alexa 488-labeled holotransferrin. We propose a structural model summarizing our conclusion that the uranyl-TF complex adopts an open conformation that is not appropriate for optimal binding to the transferrin receptor.

Lipoproteins in inflammation and sepsis. II. Clinical aspects.

BACKGROUND: Systemic inflammation and sepsis are accompanied by severe metabolic alterations, including insulin resistance together with increased levels of triglycerides (TGs) and decreases in high- and low-density lipoproteins. Clinical studies have clearly established a link between lipid metabolism and systemic inflammation. Lipoproteins were shown to neutralize LPS and to exert direct anti-inflammatory actions. High- and low-density lipoproteins are thus thought to be important regulators of the host immune response during endotoxemia, which may also have the potential of improving the care of patients with Gram-negative sepsis. DISCUSSION: Nutritional lipids supplied during critical illness have been shown to modulate the host response to inflammation. In particular, inclusion of omega-3 fatty acids seems to have beneficial effects on cellular immunity and helps to maintain the balance between pro- and anti-inflammatory cytokines thereby preventing hyperinflammatory complications. In addition to improvements in the profile of lipid mediators generated, omega-3 fatty acids act as activating ligands of peroxisome proliferator-activated receptors and directly inhibit nuclear factor kappaB mediated proinflammatory signaling. We present an overview on the alterations in the metabolism of serum lipoproteins during sepsis and present data from clinical studies and discuss the significance of nutritional lipids and their role in immunomodulation with special emphasis on omega-3 fatty acids.

Dodecins: a family of lumichrome binding proteins.

Dodecin is a small dodecameric flavoprotein from Halobacterium salinarum that contains two flavins stacked between two tryptophan residues to form an aromatic tetrade. The functional properties of heterologously expressed dodecin were investigated by fluorescence spectroscopy, which allowed the determination of dissociation constants for a number of protein-ligand complexes. The values obtained were in the nanomolar to micromolar range and correlate positively with the ligand size. These data were supplemented by X-ray crystal structures of the apododecin and holocomplexes with lumichrome, lumiflavin, riboflavin and FMN at resolutions between 1.55 to 1.95 A to unravel a gating mechanism as the structural basis for the preferential binding of the small ligands lumichrome and lumiflavin. The detailed analysis of the dodecin manifold for preferential binding of lumichrome and lumiflavin provides insight on a subatom level into a protein's strategy to gain selectivity for low molecular mass compounds by steric restrictions rather than specific interactions. Investigations on the ligand composition of a wild-type dodecin crystal (1.32 A resolution) support conclusions of functional and structural investigations on heterologously expressed dodecin, and strongly suggest that lumichrome, a molecule associated with the flavin metabolism, is a ligand of dodecin in vivo. Studies on mutant protein and a Halorhodospira halophila homologue spread the idea of a lumichrome binding system as a possible "waste"-trapping device, widely distributed in prokaryotes.

Self-association and ligand-induced conformational changes of iron regulatory proteins 1 and 2.

Iron regulatory proteins (IRPs) regulate iron metabolism in mammalian cells. We used biophysical techniques to examine the solution properties of apo-IRP1 and apo-IRP2 and the interaction with their RNA ligand, the iron regulatory element (IRE). Sedimentation velocity and equilibrium experiments have shown that apo-IRP1 exists as an equilibrium mixture of monomers and dimers in solution, with an equilibrium dissociation constant in the low micromolar range and slow kinetic exchange between the two forms. However, only monomeric IRP1 is observed in complex with IRE. In contrast, IRP2 exists as monomer in both the apo-IRP2 form and in the IRP2/IRE complex. For both IRPs, sedimentation velocity and dynamic light-scattering experiments show a decrease of the Stokes radius upon binding of IRE. This conformational change was also observed by circular dichroism. Studies with an RNA molecule complementary to IRE indicate that, although specific base interactions can increase the stability of the protein/RNA complex, they are not essential for inducing this conformational change. The dynamic change of the IRP between different oligomeric and conformational states induced by interaction with IRE may play a role in the iron regulatory functions of IRPs.

Podophyllum hexandrum (Himalayan mayapple) extract provides radioprotection by modulating the expression of proteins associated with apoptosis.

Podophyllum hexandrum Royale (Himalayan mayapple), a high-altitude Himalayan plant, has been shown to provide over 80% whole-body radioprotection in mice. To investigate the radioprotective potential of P. hexandrum at the molecular level, expression patterns of various proteins associated with apoptosis were studied in the spleen of male Swiss albino strain A mice by immunoblotting. Treatment with P. hexandrum [200 mg/kg of body weight; an ethanolic 50% (w/v) extract delivered intraperitoneally] 2 h before irradiation resulted in MAPKAP (mitogen-activated protein kinase-activated protein) kinase-2 activation along with HSF-1 (heat-shock transcription factor-1), leading to up-regulation of HSP-70 (heat-shock protein-70) as compared with sham-irradiated (10 Gy) mice. Strong inhibition of AIF (apoptosis-inducing factor) expression was observed in the mice treated with P. hexandrum 2 h before irradiation as compared with the sham-irradiated group. Inhibition in the translocation of free NF-kappaB (nuclear factor kappaB) from cytoplasm to nucleus was observed upon P. hexandrum pretreatment 2 h before irradiation when compared with radiation-treated mice. P. hexandrum pre-treatment (2 h before irradiation) resulted in inhibition of NF-kappaB translocation, and the expression of tumour suppressor protein p53 was observed to be down-regulated as compared with sham-irradiated control. An increase in the expression of proteins responsible for cell proliferation [Bcl-2 (B-cell chronic lymphocytic lymphoma 2), Ras-GAP (Ras-GTPase-activating protein) and PCNA (proliferating cell nuclear antigen)] was observed in the P. hexandrum-pretreated irradiated mice as compared with sham-irradiated controls. Caspase 3 activation resulted PARP [poly(ADP-ribose) DNA polymerase] cleavage, and DNA degradation was strongly inhibited in the mice treated with P. hexandrm (+/-irradiation) as compared with the mice treated with radiation (+/-heat shock). The present study thus clearly demonstrated that P. hexandrum extract provides protection from gamma-radiation by the modulation of expression of proteins associated with cell death.

Solution structures of a cyanobacterial metallochaperone: insight into an atypical copper-binding motif.

The Atx1 copper metallochaperone from Synechocystis PCC 6803, ScAtx1, interacts with two P(1)-type copper ATPases to supply copper proteins within intracellular compartments, avoiding ATPases for other metals en route. Here we report NMR-derived solution structures for ScAtx1. The monomeric apo form has a betaalphabetabetaalpha fold with backbone motions largely restricted to loop 1 containing Cys-12 and Cys-15. The tumbling rate of Cu(I)ScAtx1 (0.1-0.8 mm) implies dimers. Experimental restraints are satisfied by symmetrical dimers with Cys-12 or His-61, but not Cys-15, invading the copper site of the opposing subunit. A full sequence of copper ligands from the cell surface to thylakoid compartments is proposed, considering in vitro homodimer liganding to mimic in vivo liganding in ScAtx1-ATPase heterodimers. A monomeric high resolution structure for Cu(I)ScAtx1, with Cys-12, Cys-15, and His-61 as ligands, is calculated without violations despite the rotational correlation time. (2)J(NH) couplings in the imidazole ring of His-61 establish coordination of N(epsilon2) to copper. His-61 is analogous to Lys-65 in eukaryotic metallochaperones, stabilizing Cu(I)S(2) complexes but by binding Cu(I) rather than compensating charge. Cys-Cys-His ligand sets are an emergent theme in some copper metallochaperones, although not in related Atx1, CopZ, or Hah1. Surface charge (Glu-13) close to the metal-binding site of ScAtx1 is likely to support interaction with complementary surfaces of copper-transporting ATPases (PacS-Arg-11 and CtaA-Lys-14) but to discourage interaction with zinc ATPase ZiaA and so inhibit aberrant formation of copper-ZiaA complexes.

Analysis of the transient increase in cytosolic Ca2+ during the action potential of higher plants with high temporal resolution: requirement of Ca2+ transients for induction of jasmonic acid biosynthesis and PINII gene expression.

Plants respond to various abiotic stimuli by activation and propagation of fast electrical signals, action potentials. To resolve the temporal increase in cytosolic Ca(2)(+) during the action potentials of higher plants, we regenerated transgenic potato plants that expressed the Ca(2)(+) photoprotein apoaequorin. These genetically engineered potato plants were used for simultaneous measurements of transient changes in the membrane potential and the Ca(2)(+) luminescence triggered by heat-induced action potentials. High temporal resolution for recording of the fast transient electrical and light signals was accomplished by a sampling rate of 1 kHz. Upon elicitation by heat the membrane potential depolarization preceded the rise of cytosolic Ca(2)(+) by 50-100 ms. Several Ca(2)(+) channel blockers were tested to inhibit the rise in cytosolic Ca(2)(+). Treatment of plants with Ruthenium Red blocked the elevation in cytosolic Ca(2)(+) that was associated with heat-stimulated action potentials. Furthermore, action potentials have been demonstrated to stimulate jasmonic acid biosynthesis and PINII gene expression. Therefore, we measured jasmonic acid and PINII gene expression levels subsequent to action potential initiation by a short heating pulse. As expected, jasmonic acid biosynthesis and PINII gene expression were induced by action potentials. Pretreatment of potato plants with Ruthenium Red inhibited induction of jasmonic acid biosynthesis and PINII gene expression that was generally triggered by heat-activated action potentials.