New perspectives of quercetin and vitamin C effects on fibronectin-binding integrins and chemokine receptors in prostate cancer cell lines.

OBJECTIVES: The aim of this study is to investigate the effect of two abundant dietary supplements, quercetin and vitamin C on some factors involved in metastasis and proliferation of prostate cancer, which are resistant to conventional chemotherapies in late stages. BACKGROUND: Bone and brain are two common sites of metastases in prostate cancer, nevertheless the factors involved in their metastatic pathways are not well understood. METHODS: The effect of quercetin (75µM) and vitamin C (100 µM) on CXCR4, CXCR7 chemokine receptors, α4, α5 and ß1 integrins, ki-67 proliferation marker and Vascular endothelial growth factor, VEGF was evaluated using Quantitative Reverse Transcription PCR (RT-qPCR). RESULTS: The effect of quercetin and vitamin C alone was different on PC3 and DU145 prostate cancer cell lines, but sequential combination reduced significantly the expression of CXCR and CXCR7 chemokine receptors, α4, α5 and ß1 integrin subunits, VEGF and Ki-67 proliferation markers in PC3 and DU145 cell lines. CONCLUSION: Our results indicated the beneficial effect of quercetin and vitamin C on prostate cancer cells with different metastatic sites and their differential response to the treatment which in turn may lead us to reach suitable therapeutic outcomes to combat cancer (Fig. 3, Ref. 36).

Antioxidant ameliorating effects against H2O2-induced cytotoxicity in primary endometrial cells.

Oxidative stress and a disrupted antioxidant system are involved in a variety of pregnancy complications. In the present study, the role of vitamin E (Vit E) and folate as radical scavengers on the GSH homeostasis in stress oxidative induced in rat endometrial cells was investigated. Primary endometrial stromal cell cultures treated with 50 and 200 µM of H2O2 and evaluated the cytoprotective effects of Vit E (5 µM) and folate (0.01 µM) in H2O2-treated cells for 24 h. Following the exposure of endometrial cells to H2O2 alone and in the presence of Vit E and/or folate, cell survival, glutathione peroxidase (GPx) and glutathione reductase activities and the level of reduced glutathione (GSH) were measured. Cell adhesions comprise of cell attachment and spreading on collagen were determined. Flow cytometric analysis using annexin V was used to measure apoptosis. H2O2 treatment showed a marked decrease in cell viability, GPx and GR activities and the level of GSH. Although Vit E or folate had some protective effect, combination therapy with Vit E and folate attenuated all the changes due to H2O2 toxicity. An increasing number of alive cells was showed in the cells exposed to H2O2 (50 µM) accompanied by co-treatment with Vit E and folic acid. The present findings indicate that co-administration of Vit E and folate before and during pregnancy may maintain a viable pregnancy and contribute to its clinical efficacy for the treatment of some idiopathic infertility.

Quercetin potentiates transdifferentiation of bone marrow mesenchymal stem cells into the beta cells in vitro.

PURPOSE: Type 1 diabetes is an autoimmune disease caused by the destruction of ß-cells in the pancreas. Bone marrow mesenchymal stem cells are multipotent and easy accessible adult stem cells that may provide options in the treatment of type 1 diabetes. Injured pancreatic extract can promote the differentiation of rat bone marrow mesenchymal stem cells into ß-cells. We aimed to observe the effect of quercetin in differentiation and insulin secretion in ß-cells. METHODS: Bone marrow mesenchymal stem cells were obtained from the tibiae of rats. Cell surface markers were analyzed by flow cytometry. The cells were treated with rat injured pancreatic extract and quercetin for 2 weeks. Insulin secretion was measured by ELISA. Insulin expression and some islet factors were evaluated by RT-PCR. PDX1, a marker for ß-cell function and differentiation, was evaluated by both immunocytochemistry and Western blot. ß-cell count was determined by stereology and cell count assay. RESULTS: ELISA showed significant differences in insulin secretion in the cells treated with RIPE + 20 µM quercetin (0.55 ± 0.01 µg/L) compared with the cells treated with RIPE alone (0.48 ± 0.01 µg/L) (P = 0.026). RT-PCR results confirmed insulin expression in both groups. PDX1 protein was detected in both groups by Western blot and immunocytochemistry. Stereology results showed a significant increase in ß-cell number in the RIPE + quercetin-treated cells (47 ± 2.0) when compared with RIPE treatment alone (44 ± 2.5) (P = 0.015). CONCLUSIONS: Quercetin has a strengthening effect on the differentiation of rat bone marrow mesenchymal stem cells into ß-cells and increases insulin secretion from the differentiated ß-cells in vitro.

Supplementation with a new therapeutic oxygen carrier reduces chronic fibrosis and organ dysfunction in kidney static preservation.

Static preservation is currently the most widely used organ preservation strategy; however, decreased donor organ quality is impacting outcome negatively. M101 is an O2 carrier with high-oxygen affinity and the capacity to function at low temperatures. We tested the benefits of M101 both in vitro, on cold preserved LLC-PK1, as well as in vivo, in a large white pig kidney autotransplantation model. In vitro, M101 supplementation reduced cold storage-induced cell death. In vivo, early follow-up demonstrated superiority of M101-supplemented solutions, lowering the peak of serum creatinine and increasing the speed of function recovery. On the longer term, supplementation with M101 reduced kidney inflammation levels and maintained structural integrity, particularly with University of Wisconsin (UW). At the end of the 3-month follow-up, M101 supplementation proved beneficial in terms of survival and function, as well as slowing the advance of interstitial fibrosis. We show that addition of M101 to classic organ preservation protocols with UW and Histidine-Tryptophane-Ketoglutarate, the two most widely used solutions worldwide in kidney preservation, provides significant benefits to grafts, both on early function recovery and outcome. Simple supplementation of the solution with M101 is easily translatable to the clinic and shows promises in terms of outcome.

Composition and Anti-Toxicity Effects of Cichorium intybus Distillate on Serum Antioxidant Status in Carbon Tetrachloride-Treated Rats.

The role of oxidative stress in female fertility is a compelling area for research. According to traditional medicine, Cichorium intybus, known as Kasni, is believed to improve fertility. For this purpose, the effects of C. intybus distillate (CI) on blood antioxidant status were assessed in rats with carbon tetrachloride (CCl4)-induced toxicity. The rats were assigned to four experimental groups of Control, CI, CCl4, and CI+CCl410 (n=10 in each group). The level of antioxidant enzymes, such as glutathione peroxidase (GPx), glutathione reductase (GR), and catalase (CAT), as well as lipid peroxidation and reduced glutathione (GSH) level, were measured in serum samples. In the second part of the study, the antioxidant activity and phytochemical composition of the hydrodistillate of C. intybus aerial parts were determined by DPPH radical scavenging and gas chromatography-mass spectrometry analysis, respectively. The administration of CCl4 decreased the enzyme activities of GPx, GR, and CAT which were significantly ameliorated after CI administration. The decreased level of serum GSH following CCl4 administration was not considerably elevated in the CI+CCl4 group. Furthermore, the level of malondialdehyde in the serum of CI+CCl4 rats was decreased, compared to the CCl4 group. The main compositions of the essential oil from the C. intybus distillate were the antioxidants of Pulegone (8.10%), Piperitenone (7.68%), dihydroactinidiolide (5.0%), and carvone (4.18%). The antioxidant activity of the distillate was obtained at 75µg/l using the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) test. In general, the results of the present study demonstrated that C. intybus distillate, as a safe herbal remedy, can attenuate CCl4-induced oxidative damages via boosting the endogenous antioxidant defense system.

Stereological and histopathological evaluation of doxorubicin-induced toxicity in female rats' ovary and uterus and palliative effects of quercetin and vitamin E.

Doxorubicin (DOX) is a widely used chemotherapeutic agent with demonstrated reproductive toxicity. This study sought to determine the DOX-induced toxicity in the ovary and uterus and the preventive effects of quercetin (QCT) and vitamin E (Vit.E). Female rats were divided into six groups as follows: control, QCT (20 mg/kg), Vit.E (200 mg/kg), DOX (accumulative 15 mg/kg), DOX/QCT, and DOX/Vit.E. After 3 weeks, the toxicity of DOX in ovarian and uterine tissues and the potential palliative effects of QCT and Vit.E were evaluated by histopathological-stereological methods. The findings indicate a dramatic decline in the number of ovarian follicles (p < 0.001), ovarian and its associated structures volume, the volume of the uterus, its layers, and related structures (p < 0.05). Coadministration of QCT and Vit.E with DOX-treated rats demonstrated an alleviative effect on most of the studied parameters. Nevertheless, few adverse effects were recognized concerning these antioxidants administration (p < 0.05). In conclusion, the findings of this study support the protective role of these dietary supplements in the prevention of DOX-induced toxicity in uterine and ovarian tissues.

Prevention of ischemia-reperfusion lung injury during static cold preservation by supplementation of standard preservation solution with HEMO2life® in pig lung transplantation model.

We describe the results of adding a new biological agent HEMO2life® to a standard preservation solution for hypothermic static lung preservation aiming to improve early functional parameters after lung transplantation. HEMO2life® is a natural oxygen carrier extracted from Arenicola marina with high oxygen affinity developed as an additive to standard organ preservation solutions. Standard preservation solution (Perfadex®) was compared with Perfadex® associated with HEMO2life® and with sham animals after 24 h of hypothermic preservation followed by lung transplantation. During five hours of lung reperfusion, functional parameters and biomarkers expression in serum and in bronchoalveolar lavage fluid (BALF) were measured. After five hours of reperfusion, HEMO2life® group led to significant improvement in functional parameters: reduction of graft vascular resistance (p < .05) and increase in graft oxygenation ratio (p < .05). Several ischemia-reperfusion related biomarkers showed positive trends in the HEMO2life® group: expression of HMG B1 in serum tended to be lower in comparison (2.1 ± 0.8 vs. 4.6 ± 1.5) with Perfadex® group, TNF-α and IL-8 in BALF were significantly higher in the two experimental groups compared to control (p < .05). During cold ischemia, expression of HIF1α and histology remained unchanged and similar to control. Supplementation of the Perfadex® solution by an innovative oxygen carrier HEMO2life® during hypothermic static preservation improves early graft function after prolonged cold ischemia in lung transplantation.

Application of HBOCs electrophoretic method to detect a new blood substitute derived from the giant extracellular haemoglobin of lugworm.

Manipulation of blood and blood components is prohibited in sports by the World Anti-Doping Agency (WADA). This includes the use of blood substitutes to increase oxygen transport, like haemoglobin-based oxygen carriers (HBOCs), which are compounds derived from haemoglobin. Despite their medical interest, the first generation of HBOCs had serious adverse effects and was abandoned. However, new studies are now exploiting the properties of marine worm haemoglobins, which circulate as giant extracellular complexes with high oxygen-binding capacities. HEMOXYCarrier® (HC), developed by Hemarina, is one of the most advanced and promising HBOCs, and HC may become a tempting doping tool for athletes in the future. Here, HC detection in plasma/serum was evaluated with the method used to detect the first HBOCs, based on electrophoresis and heme peroxidase properties. An HC-derived product was identified in human plasma up to 72 h after in vitro incubation at 37 °C. HC degradation also induced methemalbumin formation. After injecting HC at the effective dose of 200 mg/kg into mice, the HC-derived product was detected only for a few hours and no accumulation of methemalbumin was observed. Due to this limited detection window in vivo, measuring specific worm globin degradation products by mass spectrometry might be an alternative for future anti-doping analyses. Copyright © 2016 John Wiley & Sons, Ltd.

Adaptive Response Induced by Pre-Exposure to 915 MHz Radiofrequency: A Possible Role for Antioxidant Enzyme Activity.

BACKGROUND: Over the past few years, the rapid use of high frequency electromagnetic fields like mobile phones has raised global concerns about the negative health effects of its use. Adaptive response is the ability of a cell or tissue to better resist stress damage by prior exposure to a lesser amount of stress. This study aimed to assess whether radiofrequency radiation can induce adaptive response by changing the antioxidant balance. MATERIALS AND METHODS: In order to assess RF-induced adaptive response in tissues, we evaluated the level of GSH and the activity of GR in liver. 50 rats were divided into 5 groups. Three groups were pre-exposed to 915 MHz RF radiation, 4 hours per day for one week at different powers, as low, medium and high. 24 hours after the last exposure to radiation, they were exposed to 4 Gy sublethal dose of gamma radiation and then sacrificed after 5 hours. Their livers were removed, washed and were kept at -80o C until used. RESULTS: Our finding showed that pre-exposure to 915 MHz radiofrequency radiation with specific power could induce adaptive response in liver by inducing changes in the activity and level of antioxidant enzymes. CONCLUSION: It can be concluded that pre-exposure to microwave radiation could increase the level of GSH and the activity of GR enzyme, although these increases were seen just in low power group, and the GR activity was indicated in medium power group. This increase protects tissue from oxidative damage induced by sublethal dose of gamma radiation.

Observation of large, non-covalent globin subassemblies in the approximately 3600 kDa hexagonal bilayer hemoglobins by electrospray ionization time-of-flight mass spectrometry.

A non-covalent globin subassembly comprising 12 globin chains (204 to 214 kDa) was observed directly by electrospray ionization time-of-flight mass spectrometry in the native hexagonal bilayer hemoglobins from the oligochaetes Lumbricus terrestris and Tubifex tubifex, the polychaetes Tylorrhynchus heterochaetus, Arenicola marina, Amphitrite ornata and Alvinella pompejana, the leeches Macrobdella decora, Haemopis grandis and Nephelopsis oscura and the chlorocruorin from the polychaete Myxicola infundibulum, over the pH range 3.5-7.0. The Hb from the deep-sea polychaete Alvinella exhibited in addition, peaks at approximately 107 kDa and at approximately 285 kDa, which were assigned to subassemblies of six globin chains and of 12 globin chains with three non-globin linker chains, respectively. The experimental masses decreased slightly with increased de-clustering potential (60 to 160 V) and were generally 0.1 to 0.2 % higher than the calculated masses, due probably to complexation with cations and water molecules.

Three-dimensional reconstruction by cryoelectron microscopy of the giant hemoglobin of the polychaete worm Alvinella pompejana.

A frozen-hydrated specimen of the hexagonal bilayer hemoglobin (HBL Hb) from the deep-sea hydrothermal vent polychaete worm Alvinella pompejana, the most thermophilic metazoan known to date, was observed in the electron microscope and subjected to three-dimensional (3D) reconstruction by the method of random conical tilt series. At a resolution of 34.6 A by the differential phase residual method and 27.7 A by the Fourier shell correlation method, the 3D volume possesses a D6 point-group symmetry. While in previous 3D reconstructions of annelid and vestimentiferan Hbs the vertices of the upper layer were 16 degrees rotated compared with those of the lower layer, in Alvinella Hb the vertices of the two hexagonal layers are almost perfectly eclipsed when viewed along the 6-fold axis. As in the HBL Hbs of Riftia pachyptila and Macrobdella decora, a central linker complex is decorated by 12 hollow globular substructures (HGS). The linker complex comprises (1) a central hexagonal toroid, (2) two internal bracelets onto which the HGSs are built, and (3) six connections between the two hexagonal layers. Each HGS is composed of six masses, which are separated when the volume is displayed at high threshold, plus one additional mass involved in the bracelet connecting the six HGSs in both hexagonal layers. The HGSs have a local pseudo 3-fold symmetry and a disposition of the high-density masses different from those of Riftia V1 Hb.

Effect of 900 MHz Electromagnetic Radiation on the Induction of ROS in Human Peripheral Blood Mononuclear Cells.

BACKGROUND: Despite numerous studies over a decade, it still remains controversial about the biological effects of RF EMF emitted by mobile phone telephony. OBJECTIVE: Here we investigated the effect of 900 MHz GSM on the induction of oxidative stress and the level of intracellular reactive oxygen species (ROS) in human mononuclear cells, monocytes and lymphocytes as defence system cells. METHOD: 6 ml Peripheral Blood samples were obtained from 13 healthy volunteers (21-30 year-old). Each sample was devided into 2 groups: one was exposed RF radiation emitted from a mobile phone simulator for 2 hour and the other used as control group which was not exposed to any fields. After that, mononuclear cells were isolated from peripheral blood by density gradient centrifugation in Ficoll-Paque. The intracellular ROS content in monocytes and lymphocytes was measured by the CM-H2DCFDA fluorescence probe using flowcytometry technique. RESULTS: Our results showed significant increase in  ROS production after exposure in population rich in monocytes. This effect was not significant in population rich in lymphocytes in comparison with non exposed cells. CONCLUSION: The results obtained in this study clearly showed the oxidative stress induction capability of RF electromagnetic field in the portion of PBMCs mostly in monocytes, like the case of exposure to micro organisms, although the advantages or disadvantages of this effect should be evaluated.

Quaternary structure of the extracellular haemoglobin of the lugworm Arenicola marina: a multi-angle-laser-light-scattering and electrospray-ionisation-mass-spectrometry analysis.

To elucidate the quaternary structure of the extracellular haemoglobin (Hb) of the marine polychaete Arenicola marina (lugworm) it was subjected to multi-angle laser-light scattering (MALLS) and to electrospray-ionisation mass spectrometry (ESI-MS). It was also subjected to SDS/PAGE analysis for comparative purposes. MALLS analysis gave a molecular mass of 3648 +/- 24 kDa and a gyration radius of 11.3 +/- 1.7 nm. Maximum entropy analysis of the multiply charged electrospray spectra of the native, dehaemed, reduced and carbamidomethylated Hb forms, provided its complete polypeptide chain and subunit composition. We found, in the reduced condition, eight globin chains of molecular masses 15952.5 Da (a1), 15974.8 Da (a2), 15920.9 Da (b1), 16020.1 Da (b2), 16036.2 Da (b3), 16664.8 Da (c), 16983.2 Da (d1), 17033.1 Da (d2) and two linker chains L1, 25174.1 Da, and L2, 26829.7 Da. In the native Hb, chains b, c, d occur as five disulphide-bonded trimer subunits T with masses of 49560.4 Da (T1), 49613.9 Da (T2), 49658.6 Da (T3), 49706.8 Da (T4), 49724.5 Da (T5). Linker chains L1 and L2 occur as one disulphide-bonded homodimer 2L1 (D1) of 50323.1 Da and one disulphide-bonded heterodimer L1-L2 (D2) of 51 981.5 Da. Polypeptide chains a and d possess one free cysteine residue and chains d possess an unusual total of five cysteine residues. Semi-quantitative analysis of ESI-MS data allowed us to propose the following model for the one-twelfth protomer: [(3a1)(3a2)2T] (T corresponding to either T3, T4 or T5). From electron micrograph data T1 and T2 are probably located at the centre of the molecule as mentioned in previous studies. The Hb would thus be composed of 198 polypeptide chains with 156 globin chains and 42 linker chains, each twelfth being in contact with 3.5 linker subunits, providing a total mass of 3682 kDa including haems in agreement with the experimental molecular mass determined by MALLS. From ESI-MS relative intensities and the model proposed above, the globin/linker ratio gave 0.71:0.29 and 0.73:0.27, respectively. The estimation of haem content by pyridine haemochromogen and by cyanmethaemoglobin (HiCN) methods also support the globin chain number provided by ESI-MS.

Investigation by electrospray ionization mass spectrometry of the extracellular hemoglobin from the polychaete annelid Alvinella pompejana: an unusual hexagonal bilayer hemoglobin.

Alvinella pompejana inhabits deep-sea hydrothermal vent sites along the East-Pacific Rise, where it colonizes the walls of actively venting high-temperature chimneys. This worm is the most thermophilic metazoan known to date. In Alvinella, as in other alvinellids, oxygen transport is mainly achieved by an extracellular Hb dissolved in the vascular blood. This Hb has a molecular mass of 3833 +/- 14 kDa as revealed by multiangle laser light scattering (MALLS). Native and derivative Hb (reduced, carbamidomethylated, and deglycosylated) were analyzed by electrospray ionization mass spectrometry (ESI-MS). The data were processed by the maximum entropy deconvolution system (MaxEnt). We identified three groups of peaks for Alvinella Hb, at ca. 16, 23-26, and 50 kDa corresponding to (i) four monomeric globin chains, a1 (16 633.4), a2(16 532.4), a3 (16 419.6), and a4(16 348.9); (ii) four linker chains, L1-L4 (22 887. 1, 24 230.5, 26 233.6, and 25 974.4); and (iii) one disulfide-bonded trimer T (51 431.9) composed of globin chains b (16 477.5), c (16 916.1), and d (18 048.8). These Hbs were also subjected to SDS-PAGE analysis for comparative purposes. In addition, using the ESI-MS data we propose two alternative models for the quaternary structure of Alvinella's Hb.

Three-dimensional reconstruction of the hexagonal bilayer hemoglobin of the hydrothermal vent tube worm Riftia pachyptila by cryoelectron microscopy.

A frozen-hydrated specimen of the V1 hemoglobin of the hydrothermal vent tube worm Riftia pachyptila was observed in the electron microscope and subjected to three-dimensional reconstruction by the method of random conical tilt series. The 3D volume possesses a D6 point-group symmetry. When viewed along its 6-fold axis the vertices of its upper hexagonal layer are 16 degrees clockwise rotated compared to those of the lower layer. A central linker complex is decorated by 12 hollow globular substructures. The linker complex comprises (i) a central hexagonal toroid, (ii) two internal bracelets onto which the hollow globular substructures are built, and (iii) six structures connecting the two hexagonal layers. The hollow globular substructures, related to the dodecamers of globin chains resulting from the dissociation of the hexagonal bilayer hemoglobin, have a local pseudo 3-fold symmetry and are composed each of three elongated structures visible when the volume is displayed at high threshold. At a resolution of 36 A, the 3D volumes of the hexagonal bilayer hemoglobins of Riftia pachyptyla and of the leech Macrobdella decora look almost perfectly identical.

Primary structure of the common polypeptide chain b from the multi-hemoglobin system of the hydrothermal vent tube worm Riftia pachyptila: an insight on the sulfide binding-site.

The deep-sea tube worm Riftia pachyptila Jones possesses a multi-hemoglobin system with three different extracellular Hbs: two dissolved in the vascular blood, V1 (ca. 3,500 kDa) and V2 (ca. 400 kDa), and one in the coelomic fluid, C1 (ca. 400 kDa). V1 Hb consists of four heme-containing, globin chains (b-e) and four linker chains (L1-L4). V2 and C1 Hbs are exclusively built from globin chains, six for V2 (a-f) and five for C1 (a-e). The complete amino acid sequence of the isolated monomeric globin chain b, common to all Riftia Hbs, has been determined by automated Edman degradation sequencing of the peptides derived by digestion with trypsin, chymotrypsin, thermolysin, and CNBr. This polypeptide chain is composed of 144 amino acid residues, providing a M(r) of 16, 135.0 Da. Moreover, the primary sequence of chain b revealed 3 Cys residues at position 4, 75, and 134. Cys-4 and Cys-134 are located at positions where an intra-chain disulfide bridge is formed in all annelid, vestimentiferan, or pogonophoran chains, but Cys-75 is located at a unique position only found in three globin chains belonging to Lamellibrachia and Oligobrachia, a vestimentiferan and a pogonophoran. In both groups, Hbs can bind sulfide reversibly to fuel the chemosynthetic process of the symbiotic bacteria they harbor. Sulfide-binding experiments performed on purified Hb fractions (i.e., V1, V2, and C1 Hbs) suggest that free Cys residues on globin chains, and the numerous Cys found in linker chains, as determined previously by ESI-MS, may be the sulfide binding-sites. Blocking the free Cys by N-ethylmaleimide, we confirmed that free cysteines were involved in sulfide-binding but did not account for the whole sulfide-binding capacity of V1 Hb. Furthermore, a phylogenetic tree was constructed from 18 globin-like chains of annelid, vestimentiferan, and pogonophoran extracellular Hbs to clarify the systematic position of tubeworms. Riftia chain b clearly belongs to the "strain A" family with 30 to 80% identity with the other sequences analyzed. Its position in the tree confirmed a close relationship between vestimentiferan, pogonophoran, and annelid Hbs.

Partially glucose-capped oligosaccharides are found on the hemoglobins of the deep-sea tube worm Riftia pachyptila.

We report here the structural determination of N-linked oligosaccharides found on extracellular hemoglobins of the hydrothermal vent tube worm Riftia pachyptila. Structures were elucidated by a combination of electrospray ionization tandem mass spectrometry, matrix-assisted laser desorption/ionization mass spectrometry, normal-phase high performance liquid chromatography, and exoglycosidase digestion. The sugar chains were found to consist mainly of high-mannose-type glycans with some structures partially capped by one or two terminal glucose residues. The present study represents the first report of the occurrence of glucose capping of N-linked carbohydrates in a secreted glycoprotein of a metazoan. Previously, glucose capping has only been described for a membrane-bound surface glycoprotein from the unicellular parasite Leishmania mexicana amazonensis.

The multi-hemoglobin system of the hydrothermal vent tube worm Riftia pachyptila. I. Reexamination of the number and masses of its constituents.

The deep-sea tube worm Riftia pachyptila Jones possesses a well developed circulatory system and a large coelomic compartment, both containing extracellular hemoglobins. Fresh vascular blood is heterogeneous and contains two different hemoglobins (V1 and V2), whereas the coelomic fluid is homogeneous and comprises only one hemoglobin (C1). Their molecular weights have been determined by scanning transmission electron microscopy mass mapping (STEM) and by multi-angle laser light scattering (MALLS). Both methods yielded approximately the same molecular weights with masses significantly higher than the literature data for V1. V1, V2, and C1 had Mr of 3396 +/- 540 x 10(3), 393 +/- 71 x 10(3), and 410 +/- 51 x 10(3) by STEM, and 3503 +/- 13 x 10(3), 433 +/- 8 x 10(3), and 380 +/- 4 x 10(3) by MALLS, respectively. Transmission electron micrographs of V1 are typical of an hexagonal bilayer hemoglobin (HBL Hb). When submitted to dilution or osmotic shock, V1 dissociates into halves and one-twelfth subunits like annelid HBL Hbs. V1 is resistant to urea treatment, indicating that hydrophobic interactions play a small role in its quaternary structure. Conversely, V1 Hb is rather unstable in solution without denaturant, a property which seems to be characteristic of vestimentiferan HBL Hbs and could be explained by an important number of hydrogen bonds.

The multi-hemoglobin system of the hydrothermal vent tube worm Riftia pachyptila. II. Complete polypeptide chain composition investigated by maximum entropy analysis of mass spectra.

The deep-sea tube worm Riftia pachyptila Jones possesses a complex of three extracellular Hbs: two in the vascular compartment, V1 (approximately 3500 kDa) and V2 (approximately 400 kDa), and one in the coelomic cavity, C1 (approximately 400 kDa). These native Hbs, their dissociation products and derivatives were subjected to electrospray ionization mass spectrometry (ESI-MS). The data were analyzed by the maximum entropy deconvolution system. We identified three groups of peaks for V1 Hb, at approximately 16, 23 27, and 30 kDa, corresponding to (i) two monomeric globin chains, b (Mr 16,133.5) and c (Mr 16,805.9); (ii) four linker subunits, L1 L4 (Mr 23,505.2, 23,851.4, 26,342.4, and 27,425.8, respectively); and (iii) one disulfide-bonded dimer D1 (Mr 31,720.7) composed of globin chains d (Mr 15,578.5) and e (Mr 16, 148.3). V2 and C1 Hbs had no linkers and contained a glycosylated monomeric globin chain, a (Mr 15,933.4) and a second dimer D2 (Mr 32,511.7) composed of chains e and f (Mr 16,368.1). The dimer D1 was absent from C1 Hb, clearly differentiating V2 and C1 Hbs. These Hbs were also subjected to SDS-PAGE analysis for comparative purposes. The following models are proposed ((cD1)(bD1)3) for the one-twelfth protomer of V1 Hb, ((cD)(bD)6(aD)) (D corresponding to either D1 or D2) for V2 and C1 Hbs. HBL V1 Hb would be composed of 180 polypeptide chains with 144 globin chains and 36 linker chains, each twelfth being in contact with three linker subunits, providing a total molecular mass = 3285 kDa. V2 and C1 would be composed of 24 globin chains providing a total molecular mass = 403 kDa and 406 kDa, respectively. These results are in excellent agreement with experimental Mr determined by STEM mass mapping and MALLS.

Structural comparison of cephalopod hemocyanins: phylogenetic significance.

Hemocyanins, the respiratory molecules of cephalopod mollusks, are hollow cylinders with five internal arches. Three hemocyanins representative of three orders of cephalopods (Benthoctopus species, Octopoda; Vampyroteuthis infernalis, Vampyromorpha; Sepia officinalis, Sepioidea) were subjected to cryoelectron microscopy and three-dimensional (3D) reconstruction. The structure of Benthoctopus hemocyanin, solved at 26.4-A resolution, possesses arches comprising two identical functional units. The similarity between these functional units and the structure recently observed in X-ray crystallography for Octopus by Cuff et al. (J. Mol. Biol., 1998, 232, 522-529) allows the identification of their N- and C-terminal domains in the 3D reconstruction volume. Conversely, arches present in the 3D reconstruction volume of Sepia hemocyanin (21.8 A resolution) contain four functional units that are disposed differently. The strong resemblance between the reconstruction volumes of Vampyroteuthis (21.4-A resolution) and Benthoctopus hemocyanins suggests that Sepioidea diverged from a group containing Octopoda and Vampyromorpha.