Peridinin Chlorophyll Research Articles

Dynamics of Protein Elements of Hybrid Nanostructures - Molecular Dynamics Simulations of Light Harvesting Peridinin-Chlorophyll a-Protein Model

Hybrid nanostructures are often composed of inorganic parts and biological ones. Optimized through million years of evolution light harvesting proteins are hard to mimic synthetically. Promising strategy in search for e cient solar cells is an attachment of selected natural protein systems to inorganic quantum dots. Such experimental hybrid structures should have improved charge separation properties. Among the most promising proteins is peridinin chlorophyll protein from Amphidinium carterae (PCP). It has a wide absorption spectrum (420 550 nm), optimized for sunlight. The dynamics of this protein, used in modern nanotechnology has been not addressed yet. In this work we present results of PCP computer modeling using a well established molecular dynamics methodology. The CHARMM27 force eld parameters were prepared for this protein and all chromophore components. The system was embedded in a box of water, with proper counter ions, and a number of 10 ns molecular dynamics simulations were run using the NAMD code. It has been found that peridinine chromophores exhibit substantial orientational exibility but a pair Per612 and Per613 is more rigid than the remaining two carotenoids. Orientation and dynamics of absorption and emission electric dipole moments have been also analyzed. Apparently, the architecture of PCP is not optimized for e cient Per Chl a energy transfer by the Forster mechanism. Several practical issues related to molecular dynamics simulation of similar hybrid nanostructures are discussed.

Natural pigments from six species of Thai plants extracted by water for hair dyeing product application

Abstract Pigments from six species of Thai plants were water extracted at various temperatures and pH levels to generate eco-friendly dyes intended for application in alternative natural hair dyeing products. Sappan tree extracts gave greater yield than beleric myrobalan, false daisy, thao yanang, kae lae, and turmeric. False daisy, kae lae and thao yanang extracted with water at 100 °C-pH 9, sappan tree extracted with water at 25 °C-pH 9, beleric myrobalan extracted with water at 25 °C-pH 7, and turmeric extracted with water at 4 °C-pH 5 obtained the greatest yields within its species. The hue angle of extracts from all species ranged from yellowish, brownish and greenish color, and their absorption wavelengths ranged from 400 to 666 nm. The extracted pigments were peridinin, 19-but-fucoxanthin, fucoxanthin, diadinoxanthin, violaxanthin, antheraxanthin, zeaxanthin and DV chlorophyll b. When these extracts were applied for hair dyeing, the extracts of false daisy, kae lae, beleric myrobalan, and sappan tree mixed with ascorbic acid (a natural developer) and ferrous sulfate (a mordant agent), gave a dark reddish-brown to orangish-brown color. The dyed hair revealed excellent color strength, a smooth hair surface morphology, high-affinity interaction, and color fastness up to fifteen shampoos.

A NUP98-HOXD13 leukemic fusion gene leads to impaired class switch recombination and antibody production

Myelodysplastic syndrome is a clonal process characterized by ineffective hematopoiesis and progression to acute leukemia. Although many myelodysplastic syndrome and leukemic patients have compromised immunity, the role of underlying mutations in regulating immune function is poorly understood. Recent studies show that NUP98-HOXD13 (NHD13) fusion gene results in myelodysplastic syndrome and impairs lymphocyte differentiation in transgenic mice. In our studies, we sought to elucidate the mechanism by which NHD13 affects B-lymphocyte development and function. Based on our preliminary findings that transgenic mice had increased levels of IgM and reduced IgG1 and IgE, we hypothesized that the fusion gene might impair class switch recombination (CSR). Mice were immunologically challenged with dinitrophenol. NHD13 mice showed a marked reduction in B-lymphocyte differentiation in their bone marrow and spleen following dinitrophenol stimulation and had reduced production of dinitrophenol-specific antibodies. Spleen follicles from these mice were small and hypocellular, indicating failure of clonal expansion. When isolated NHD13 B lymphocytes were stimulated in vitro using Escherichia coli lipopolysaccharide or lipopolysaccharide + interleukin-4, they failed to undergo sufficient CSR and proliferation. Taken together, our findings show that expression of NUP98-HOXD13 impairs CSR and reduces the antibody-mediated immune response, in addition to its role in leukemia. Further delineation of the NUP98-HOXD13 transgene may reveal novel pathways involved in CSR.

Characterization of the peridinin–chlorophyll a-protein complex in the dinoflagellate Symbiodinium

The water-soluble peridinin–chlorophyll a-proteins (PCPs) are one of the major light harvesting complexes in photosynthetic dinoflagellates. PCP contains the carotenoid peridinin as its primary pigment. In this study, we identified and characterized the PCP protein and the PCP gene organization in Symbiodinium sp. CS-156. The protein molecular mass is 32.7kDa, revealing that the PCP is of the monomeric form. The intronless PCP genes are organized in tandem arrays. The PCP gene cassette is composed of 1095-bp coding regions and spacers in between. Despite the heterogeneity of PCP gene tandem repeats, we identified a single form of PCP, the sequence of which exactly matches the deduced sequence of PCP gene clone 7 (JQ395030) by LC–MS/MS analysis of tryptic digested PCP, revealing the mature PCP apoprotein is 312 amino acids in length. Pigment analysis showed a peridinin-to-Chl a ratio of 4. The peridinin-to-Chl a Qy energy transfer efficiency is 95% in this complex.

Do Regulatory T Cells Have a Role in Disease Progression and Autoimmune Cytopenias of Chronic Lymphocytic Leukemia?,

Abstract 3864 Introduction:Chronic lymphocytic leukemia (CLL) originates from mature B lymphocytes which avoid death through the intercession of external signals, including T cells. There is improved understanding of the relationship between different cell types in the normal immune response and the dynamic cross talk between tumors and immune system regulating tumor growth. Cancer and autoimmunity are considered as anti-thesis of each other. Regulatory T cells (Tregs) are shown to have a protective role in autoimmune diseases and conversely known to promote oncogenesis. The exact role of Tregs in disease biology of CLL is still unclear. Aims and Objectives : to define the role of Tregs in clinical presentation, disease progression and autoimmune cytopenias in CLL. Materials and Methods : 32 treatment naive CLL patients and 10 normal healthy volunteers were evaluated in this prospective study. Investigations included clinical examination, biochemical tests, complete blood counts, bone marrow examinaton and multicolor flowcytometry (FCM). More stringently defined specific markers (CD4+CD25highCD127low FoxP3+) were used to enumerate Tregs. Routine CLL panel for diagnosis and prognosis included 17 monoclonal antibodies (MoAbs) labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC) or peridinin chlorophyll protein (PerCP). FCM analysis was performed on FACS Calibur flowcytometer (BD Biosciences) using CellQuest® software. Diagnosis of autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia (ITP) and pure red cell aplasia (PRCA) was made according to standard criteria. Results : Study group included 20 male and 12 female CLL patients. The mean age was 64.2 years; with 8/32 (25%) patients being <55 years. 17/32 (53.12%) patients were categorized to Binet stage A and 20/32 (62.5%) were asymptomatic at diagnosis. Mean total leukocyte counts (TLC) and absolute lymphocyte counts (ALC) were 69,731/μl and 62,697/μl respectively. Mean hemoglobin and platelet counts were 12.1 g/dl and 178,087/μl respectively. 25 patients completed follow-up investigations at 6 months. Disease progression occurred in 12/25 (48%) patients, of which 5/25 (20%) patients progressed to Binet stage C and had symptoms other than lymphadenopathy while the number of asymptomatic patients decreased to 10/25 (40%). Disease progression was assessed according to NCI-WG guidelines. Autoimmune cytopenias occurred in 8/32(25%) patients. 3 of them at disease presentation and remaining 5 were diagnosed on follow-up. AIHA was diagnosed in one, ITP in 4 and PRCA in 3 patients. Mean Tregs % was lower in CLL patients (0.98+0.94) as compared to the controls (2.89+1.46) (p=0.0026). However, CLL patients had significantly higher absolute Treg cell count than the controls (581.63+734.5 vs. 83.89+55.45) (p=0.0006). While the mean % Treg increased by 46% (not significant, p >0.05) from stages A and B (early CLL) to stage C (advanced CLL), increase in mean ALC was 172% (p<0.005). The increase in mean absolute Treg count was further significantly higher 384% (p<0.005). There was no significant difference between the mean absolute Tregs count at baseline of CLL patients with disease progression (progressors) and without disease progression (nonprogressors) at follow-up. Absolute Treg count was significantly higher in patients having autoimmune cytopenias at presentation as well as on follow-up. Mean absolute Treg counts were not significantly different between ZAP70 positive and negative CLL patients (p=0.1), as well as between CD38 positive and negative CLL (p=0.15). There was a strong inverse correlation between lymphocyte doubling time and absolute Treg count (p=0.03) as well as lymphocyte doubling time and % Treg cells (p=0.006). No correlation could be found between absolute Treg count and CD4: CD8 ratio, r= 0.29 (p=0.1). Conclusion : Absolute Treg cell counts (rather than % Treg) are increased in CLL patients compared to controls and also increased with disease severity. Absolute Treg counts may be more important than % Tregs in CLL pathogenesis. Tregs also have a strong inverse relation with lymphocyte doubling time, also indirectly confirming the role of Tregs in disease progression. We also found Tregs to be increased in patients with autoimmune cytopenias. Autoimmune cytopenias may be viewed as a continuum of CLL disease itself rather than a complication of advanced disease. Disclosures:No relevant conflicts of interest to declare.

Conservation of Spin Polarization during Triplet–Triplet Energy Transfer in Reconstituted Peridinin–Chlorophyll–Protein Complexes

Peridinin-chlorophyll-protein (PCP) complexes, where the N-terminal domain of native PCP from Amphidinium carterae has been reconstituted with different chlorophyll (Chl) species, have been investigated by time-resolved EPR in order to elucidate the details of the triplet-triplet energy transfer (TTET) mechanism. This spectroscopic approach exploits the concept of spin conservation during TTET, which leads to recognizable spin-polarization effects in the observed time-resolved EPR spectra. The spin polarization produced at the acceptor site (peridinin) depends on the initial polarization of the donor (chlorophyll) and on the relative geometric arrangement of the donor-acceptor spin axes. A variation of the donor triplet state properties in terms of population probabilities or triplet spin axis directions, as produced by replacement of chlorophyll a (Chl a) with non-native chlorophyll species (ZnChl a and BacterioChl a) in the reconstituted complexes, is unambiguously reflected in the polarization pattern of the carotenoid triplet state. For the first time, in the present investigation spin-polarization conservation has been shown to occur among natural cofactors in protein complexes during the TTET process. Proving the validity of the assumption of spin conservation adopted in the EPR spectral analysis, the results reinforce the hypothesis that in PCP proteins peridinin 614, according to X-ray nomenclature (Hofmann, E.; et al. Science 1996, 272, 1788-1791), is the carotenoid of election in the photoprotection mechanism based on TTET.

Absorption Enhancement in Peridinin–Chlorophyll–Protein Light-Harvesting Complexes Coupled to Semicontinuous Silver Film

We report on experimental and theoretical studies of plasmon-induced effects in a hybrid nanostructure composed of light-harvesting complexes and metallic nanoparticles in the form of semicontinuous silver film. The results of continuous-wave and time-resolved spectroscopy indicate that absorption of the light-harvesting complexes is strongly enhanced upon coupling with the metallic film spaced by 25 nm of a dielectric silica layer. This conclusion is corroborated by modeling, which confirms the morphology of the silver island film.

Sphingosine-1-phosphate Lyase Deficiency Produces a Pro-inflammatory Response While Impairing Neutrophil Trafficking

Sphingosine-1-phosphate (S1P) lyase catalyzes the degradation of S1P, a potent signaling lysosphingolipid. Mice with an inactive S1P lyase gene are impaired in the capacity to degrade S1P, resulting in highly elevated S1P levels. These S1P lyase-deficient mice have low numbers of lymphocytes and high numbers of neutrophils in their blood. We found that the S1P lyase-deficient mice exhibited features of an inflammatory response including elevated levels of pro-inflammatory cytokines and an increased expression of genes in liver associated with an acute-phase response. However, the recruitment of their neutrophils into inflamed tissues was impaired and their neutrophils were defective in migration to chemotactic stimulus. The IL-23/IL-17/granulocyte-colony stimulating factor (G-CSF) cytokine-controlled loop regulating neutrophil homeostasis, which is dependent on neutrophil trafficking to tissues, was disturbed in S1P lyase-deficient mice. Deletion of the S1P4 receptor partially decreased the neutrophilia and inflammation in S1P lyase-deficient mice, implicating S1P receptor signaling in the phenotype. Thus, a genetic block in S1P degradation elicits a pro-inflammatory response but impairs neutrophil migration from blood into tissues.

Factors Associated with Heterocellular Aggregate Formation In Sickle Cell Disease

AbstractAbstract 2669White blood cells (WBCs) have emerged as key contributors to the pathophysiology of sickle cell disease (SCD), in part because they become activated and can form heterocellular aggregates with sickle red blood cells (SS RBCs) and platelets. These heterocellular aggregates are potentially detrimental to individuals with SCD because they may obstruct blood vessels and be themselves both pro-inflammatory and pro-coagulant. In non-SCD individuals, these aggregates are almost undetectable. In order to detect heterocellular aggregates, we collected blood into EDTA, and 10 μl of the each individual's whole blood was added to 190 μl of Dulbecco's phosphate buffered saline with Ca++, Mg++, and 2% (w/v) albumin in each tube. A 4-color flow cytometric assay was used to detect heterocellular aggregates as events positive for both erythrocyte and leukocyte markers, as well as to detect component cells within those aggregates. RBCs were detected using a fluorescein isothiocyanate (FITC)-conjugated antibody against the RBC-specific protein glycophorin A (CD235a). WBCs were detected using an allophycocyanin (APC)-conjugated antibody against the common leukocyte antigen (CD45). Platelets were detected using a phycoerythrin (PE)-conjugated antibody against the platelet-specific antigen GPIb (CD41a). To quantify leukocyte subsets, monocytes were detected using a PE-conjugated antibody against the lipopolysaccharide receptor antigen CD14, and lymphocytes were detected using a peridinin chlorophyll protein (PerCP)-conjugated antibody against the pan-lymphocyte marker CD3. We found that the mean percent of all events that contained both WBCs and RBCs was 0.40% for 33 SCD individuals in steady state and only 0.05% for 9 non-SCD individuals. In order to obtain a comprehensive picture of the factors associated with an increase in the number of aggregates in the blood of SCD individuals, we examined the association of reticulocyte count, WBC count, monocyte count, neutrophil count, lymphocyte count, platelet count, hydroxyurea treatment, and RBC exposure of phosphatidylserine with aggregate formation. Information regarding patients' cell counts was obtained from hospital records. The percent of WBCs in heterocellular aggregates was calculated by dividing the number of aggregates by the total WBC count. In our study of 33 patients, we found the strongest associations between percent events representing aggregates and total WBC count, monocyte count, and neutrophil count. In general, the higher the total WBC count, the greater was the percent of heterocellular aggregates. For the 8 patients in the quartile with the highest WBC count, the mean percent of heterocellular aggregates was 0.51% ± 0.06%, while the mean percent of heterocellular aggregates for the 8 patients with the lowest WBC count was 0.31% ± 0.06% (p = 0.028). Similar statistically significant associations were seen with both monocyte and neutrophil counts. Platelet counts were associated with the percent of leukocytes in heterocellular aggregates (p = 0.037); the higher the platelet count, the greater was the percent of WBCs in aggregates. The mean percent of WBCs in aggregates was 78.9% ± 3.5% and 62.2% ± 6.3%, for the uppermost and lowermost quartiles of platelet counts, respectively. On average, 61.8% of aggregates included platelets. There was no association between levels of annexin V binding or reticulocyte count and aggregate counts. We also examined the effect of hydroxyurea (HU) treatment on aggregate formation. Hydroxyurea treatment increases the production of hemoglobin F, reduces hemolysis and reticulocyte counts, and reduces white blood cell and platelet counts (Platt 2008). Hydroxyurea has also been associated with reduced RBC adhesivity. In our study, individuals not on hydroxyurea treatment had a higher percentage of aggregates than those on hydroxyurea (0.46 ± 0.05 without HU vs 0.37 ± 0.05 on HU, n=33), but this difference was not statistically significant. In summary, heterocellular aggregate formation in SCD involves many different cell types, but the factors promoting their participation in aggregate formation in SCD remain to be completely elucidated. Hydroxyurea may downregulate heterocellular aggregate formation, but this remains to be confirmed. Disclosures:Telen:GlycoMimetics, Inc.: Consultancy.

Affordable Technology for Enumeration of the Absolute CD4 T-Lymphocyte Count by Cell Bead Assay

The quantitative BD Trucount (San Jose, CA) tube method is the conventional but expensive method to quantitate CD4+T-lymphocyte (CD4) counts, and this may be beyond the means of countries with limited resources. In this study, we validated a quantitative method known as a cell-bead (CB) assay to quantitate CD4 counts in the peripheral blood of healthy subjects. The absolute CD4 count obtained from the CB method was highly correlated with those obtained from the Trucount tube ( r2 =0.98, y = 26.73 +1.01x, P <0.0001 and a mean bias of 34.8 cell/μL, limit of agreement [LOA] −34.8-104.4 cell/μL) and flow rate-based assay method ( r2 = 0.97; y =69.51 + 0.88x, P <0.0001 and a mean bias −53.5 cell/μL, LOA −149.4-42.3 cell/μL). This study demonstrates that the CB method is suitable and more affordable for CD4 quantitation. This method is inexpensive and interchangeable with the latex bead-based methods for generating absolute counts in resource-limited areas. * CB : cell-bead LOA : limit of agreement FCM : flow cytometry DP : dual-platform SP : single-platform WBC : white blood cell FR : flow rate-based calibration RBCs : red blood cells MP : microparticles EDTA : ethylene diamine tetraacetic acid FITC : fluorescein isothiocyanate PE : phycoerythrin PerCP : peridinin chlorophyll protein FSC-H : forward scatter SSC-H : side scatter CV : coefficient of variation SD : standard deviation

Natural Killer Cell Killing of Acute Myelogenous Leukemia and Acute Lymphoblastic Leukemia Blasts by Killer Cell Immunoglobulin-Like Receptor–Negative Natural Killer Cells after NKG2A and LIR-1 Blockade

Although the study of natural killer (NK) cell alloreactivity has been dominated by studies of killer cell immunoglobulin-like receptors (KIRs), we hypothesized that NKG2A and LIR-1, present on 53% +/- 13% and 36% +/- 18% of normal NK cells, respectively, play roles in the NK cell killing of primary leukemia targets. KIR(-) cells, which compose nearly half of the circulating NK cell population, exhibit tolerance to primary leukemia targets, suggesting signaling through other inhibitory receptors. Both acute myelogenous leukemia and acute lymphoblastic leukemia targets were rendered susceptible to lysis by fresh resting KIR(-) NK cells when inhibitory receptor-major histocompatibility class I interactions were blocked by pan-HLA antibodies, demonstrating that these cells are functionally competent. Blockade of a single inhibitory receptor resulted in slightly increased killing, whereas combined LIR-1 and NKG2A blockade consistently resulted in increased NK cell cytotoxicity. Dual blockade of NKG2A and LIR-1 led to significant killing of targets by resting KIR(-) NK cells, demonstrating that this population is not hyporesponsive. Together these results suggest that alloreactivity of a significant fraction of KIR(-) NK cells is mediated by NKG2A and LIR-1. Thus strategies to interrupt NKG2A and LIR-1 in combination with anti-KIR blockade hold promise for exploiting NK cell therapy in acute leukemias.

X-ray structures of the peridinin–chlorophyll-protein reconstituted with different chlorophylls

The peridinin–chlorophyll a-protein (PCP) from dinoflagellates is a soluble light harvesting antenna which gathers incoming photons mainly by the carotenoid peridinin. In PCPs reconstituted with different chlorophylls, the peridinin to chlorophyll energy transfer rates are well predicted by a Förster-like theory, but only if the pigment arrangements are identical in all PCPs. We have determined the X-ray structures of PCPs reconstituted with Chlorophyll- b (Chl- b), Chlorophyll- d (Chl- d) and Bacteriochlorophyll- a (BChl- a) to resolutions ⩽2 Å. In all three cases the pigment arrangements are essentially the same as in native PCP . Hydrogen bonding is not responsible for preferential incorporation of “non-native” chlorophylls over Chl -a.

Identification of excited-state energy transfer and relaxation pathways in the peridinin–chlorophyll complex: an ultrafast mid-infrared study

The peridinin chlorophyll-a protein (PCP) is a water-soluble, trimeric light harvesting complex found in marine dinoflagellates that binds peridinin and Chl-a in an unusual stoichiometric ratio of 4:1. In this paper, the pathways of excited-state energy transfer and relaxation in PCP were identified by means of femtosecond visible-pump, mid-infrared probe spectroscopy. In addition, excited-state relaxation of peridinin dissolved in organic solvent (CHCl(3) and MeOH) was investigated. For peridinin in solution, the transient IR signatures of the low-lying S(1) and intramolecular charge transfer (ICT) states were similar, in line with a previous ultrafast IR study. In PCP, excitation of the optically allowed S(2) state of peridinin results in ultrafast energy transfer to Chl-a, in competition with internal conversion to low-lying optically forbidden states of peridinin. After vibrational relaxation of the peridinin hot S(1) state in 150 fs, two separate low-lying peridinin singlet excited states are distinguished, assigned to an ICT state and to a slowly transferring, vibrationally relaxed S(1) state. These states exhibit different lactone bleaches, indicating that the ICT and S(1) states localize on distinct peridinins. Energy transfer from the peridinin ICT state to Chl-a constitutes the dominant energy transfer channel and occurs with a time constant of 2 ps. The peridinin S(1) state mainly decays to the ground state through internal conversion, in competition with slow energy transfer to Chl-a. The singlet excited state of Chl-a undergoes intersystem crossing (ISC) to the triplet state on the nanosecond timescale, followed by rapid triplet excitation energy transfer (TEET) from Chl-a to peridinin, whereby no Chl-a triplet is observed but rather a direct rise of the peridinin triplet. The latter contains some Chl-a features due to excitonic coupling of the pigments. The peridinin triplet state shows a lactone bleach mode at 1748 cm(-1), while that of the peridinin ICT state is located at 1745 cm(-1), indicating that the main channels of singlet and triplet energy transfer in PCP proceed through distinct peridinins. Our results are consistent with an energy transfer scheme where the ICT state mainly localizes on Per621/611 and Per623/613, the S(1) state on Per622/612 and the triplet state on Per624/614.

Four-wave-mixing spectroscopy of peridinin in solution and in the peridinin–chlorophyll- a protein

A model for the third order optical response of carotenoids is used to analyse transient grating and pump–probe data of peridinin in solution and bound in the peridinin–chlorophyll protein (PCP). For peridinin in solution, the transient grating signal detected at 505 nm exhibits a bi-exponential recovery whose fast phase is assigned to relaxation from the S 2 state that has a lifetime of 75 ± 25 fs. The slower, solvent-dependent rise component is assigned to equilibration of the (S 1/ICT) state, taking place on a time scale of 0.6 and ∼2.5 ps in acetontrile and benzene, respectively. These dynamics match those obtained from pump–probe measured in the spectral region of the ICT state, implying that the ICT state contributes to the signal at 505 nm. In PCP, the transient grating signal shows distinctly different kinetics, and the signal shows no recovery. This difference is explained by energy transfer from peridinin to chlorophyll- a.

CD33/CD10 Ratios in the Blast Region Evaluated by Flow Cytometry Predicts the Response of Chronic Myelogeneous Leukemia to Imatinib.

Abstract 2595▪▪This icon denotes an abstract that is clinically relevant.Poster Board II-571 Background:The prediction of the response of chronic myelogeneous leukemia (CML) to imatinib is clinically important. However, no reliable markers are known. Methods:From January 2005 to April 2007, 32 patients with newly diagnosed CML were treated at our hospital. The diagnosis was made based on the existence of the Philadelphia chromosome. A routine bone marrow aspiration was performed to evaluate bone marrow smears, karyotypes, phenotypes, and bcr/abl message levels before the start of imatinib. Phenotypic analysis was conducted using three-color flow cytometry as follows: bone marrow mononuclear cells were stained with fluorescein isothiocyanate-conjugated monoclonal antibody, phycoerythrin-conjugated monoclonal antibody, and peridinin chlorophyll protein-conjugated CD45. A gate was set to identify blasts characterized by intermediate CD45 expression and low side-scatter properties. Phenotypes of the cells in the blast region were analyzed using a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA, USA). As a control, bone marrow samples were obtained from 8 healthy volunteers and 35 patients with refractory anemia of myelodysplastic syndrome (MDS-RA). Results:All patients were diagnosed as having CML in the chronic phase and given imatinib alone. According to the response after one year of treatment with imatinib, patients showing and not showing a complete cytogenetic response were designated as group A (26 patients) and group B (6 patients), respectively. There were no significant differences between the two groups in terms of the age, peripheral blood cell counts, peripheral blood blast percentages, peripheral blood basophil percentages, bone marrow cell differentials, bone marrow blast percentages, additional chromosomal abnormalities, Sokal's score, Hasford's score, and spleen size before the start of imatinib. The mean imatinib dose per day was higher in group A (349 mg) than in group B (284.4 mg); however, the results were not significant (p=0.051). Group B showed higher percentages of myeloid cells in the blast region than group A, while the former showed lower percentages of B lymphoid cells in the blast region than the latter. CD33/CD10, CD33/CD19, CD13/CD10, and CD13/CD19 ratios in the blast region in group B were significantly higher than in group A: 56.6 vs. 6.0 (p<0.01), 34.5 vs. 6.0 (p<0.01), 33.1 vs. 7.7 (p<0.01), and 23.8 vs. 4.9 (p<0.05), respectively. There were no differences between the two groups regarding the percentage of CD34+ and CD117+ cells. Similar results were obtained, when patients were evaluated after six months of treatment with imatinib. After the six-month imatinib treatment, patients were divided into two groups: one group (group C) showed undetectable bcr/abl messages (17 patients), and the other (group D) showed detectable bcr/abl messages (6 patients). There were no significant differences between the two groups in terms of imatinib dose per day and other clinical data. CD33/CD10, CD33/CD19, CD13/CD10, and CD13/CD19 ratios in the blast region in the group D and in the group C were 11.6 vs. 6.4 (p=0.05), 10.5 vs. 6.1 (p<0.05), 13.9 vs. 8.1 (p<0.05), and 9.4 vs. 5.7 (p<0.05), respectively. CD33/CD10, CD33/CD19, CD13/CD10, and CD13/CD19 ratios in the blast region in the MDS-RA patients and healthy volunteers were 3.2 vs. 0.7, 3.2 vs. 0.5, 2.5 vs. 0.6, and 3.0 vs. 0.6, respectively. Conclusion:An increase in CD33/CD10 ratios in the blast region in CML before the start of imatinib is associated with resistance to the drug. A cut-off value of 30 for CD33/CD10 ratios in the blast region in CML at diagnosis is useful for predicting the response of CML to imatinib after one year of treatment with the drug. Disclosures:No relevant conflicts of interest to declare.

Pulsed EPR and ENDOR on the Peridinin Triplet State Involved in the Photoprotective Mechanism in Peridinin–Chlorophyll a–Proteins

The photoexcited triplet state of the carotenoid peridinin in the peridinin–chlorophyll a–protein (PCP) of the dinoflagellate Heterocapsa pygmaea has been investigated by pulsed electron paramagnetic resonance (EPR) and pulsed electron-nuclear double resonance (ENDOR) spectroscopies. The α- and β-protons hyperfine couplings of the peridinin-conjugated chain have been derived from Davies and Mims ENDOR experiments. The spectroscopic results have been compared to those obtained for the main form of the PCP complex and for the high-salt PCP form from Amphidinium carterae. The EPR features of the peridinin triplet state are very similar in the antenna complexes belonging to the two different dinoflagellate species, proving that the triplet formation pathway and the triplet localization on one specific peridinin per subcluster are common features of different PCP antennas. No significant variation of the hyperfine couplings of the peridinin triplet state has been detected between the main form of the PCP complex from A. carterae and H. pygmaea. The spectroscopic results confirm the close relationship between the Amphidinium PCP and the corresponding Heterocapsa complex at least in terms of mutual arrangement of the chlorophyll a–peridinin pair involved in photoprotection and in terms of conformation of the peridinin-conjugated chain.

Disruption of the meprin α and β genes in mice alters homeostasis of monocytes and natural killer cells

Meprin metalloproteases are implicated in inflammatory bowel disease, which involves dysfunction of immune cells. However, the roles of meprins in the immune and hematological system remain uncharacterized. In this report, we demonstrate that meprins were expressed in the hematological system, and meprin alpha/beta null (alpha(-/-)/beta(-/-)) mice had decreased prevalence of resident monocytes and natural killer (NK) cells in blood, with a concomitant accumulation of inflammatory monocytes and NK cells in bone marrow. In contrast, T and B lymphocytes were not affected by meprin deficiency. In response to acute inflammation induced by intraperitoneal injection of thioglycollate, meprin-deficient mice exhibited higher body temperature than wild-type mice, which was correlated with retention of inflammatory monocytes, but persistent low prevalence of NK cells in blood. These results indicate that meprin metalloproteases play important roles in the homeostasis of monocytes and NK cells, and possibly are involved in egress of these two type cells from bone marrow and homing to the periphery. Our findings are the first report to demonstrate that metalloproteases affect homeostasis of leukocytes, which have important implications for understanding physiology of and pathogenesis in the hematological system.

Factors Associated with Peripheral Blood Stem Cell Yield in Volunteer Donors Mobilized with Granulocyte Colony-Stimulating Factors: The Impact of Donor Characteristics and Procedural Settings

Peripheral blood stem cells (PBSCs) are increasingly used as the source of hematopoietic stem cells, but there are large variations in harvest outcome between individuals mobilized by granulocyte colony-stimulating factor (G-CSF). We examined the effects of donor characteristics and procedure factors on the day 1 CD34+ cell yield in 373 unrelated healthy donors. G-CSF was administered subcutaneously at a planned dose of 8.3 to 11 microg/kg daily for 5 days, followed by harvest started on day 5 of G-CSF treatment. Of the 373 donors, 159 (42.6%) had the radial artery as the inlet access for harvest. Poor day 1 cell yield was defined as <10x10(6) CD34+ cells/L of processed blood for the first apheresis; 62 donors (16.6%) did not attain this threshold. The male donors had significantly higher yields at harvest compared with the female donors. The female donors had higher CD34+ cell yields if the circulation access was through an artery than if is was through a vein. In a multiple regression analysis, donor age, sex, body mass index (BMI), preharvest white blood cell and circulating immature cell counts, access type, and flow rate correlated with day 1 yield. Female sex, older age, venous access, and a higher flow rate were significantly associated with greater risk for a day 1 poor yield of CD34+ cells (odds ratio=3.0074, 1.045, 4.3362, and 1.1131, respectively). A higher BMI may decrease the risk (odds ratio=0.8472). In donors at higher risk for poor CD34+ cell yield, strategies for increasing CD34+ cells must be considered.

Inter-pigment interactions in the peridinin chlorophyll protein studied by global and target analysis of time resolved absorption spectra

Inter-pigment interactions define the functioning of light-harvesting protein complexes. To describe the particularly complex molecular dynamics and interactions of peridinin and chlorophyll in the peridinin chlorophyll protein of Amphidinium carterae, we applied global and target analysis to a series of ultrafast transient absorption experiments. We have created and validated a model that consistently describes and characterizes the interactions and evolution of excited and ground-state populations after excitation in all different experiments. The series of energy transfer steps that follow excitation are described by our model of cascading populations and numerous rate constants that correspond to intra-molecular thermal relaxation, fast and slow peridinin-to-chlorophyll energy transfer steps, and chlorophyll excited-state annihilation. By analyzing the spectral response of ground-state peridinins to excited chlorophylls we have identified which specific peridinin molecule is most closely coupled to the chlorophylls. No evidence was found that the intra-molecular charge transfer (ICT) state of peridinin, identified in studies of peridinin in solution, is a separate entity in the protein. The peridinin that exhibited slow peridinin-to-chlorophyll energy transfer was characterized by a difference spectrum free from ICT features, consistent with the importance of coupled ICT and S 1 states for energy transfer.

CCR2 Receptor Is Essential to Activate Microbicidal Mechanisms to Control Toxoplasma gondii Infection in the Central Nervous System

Chemokines comprise a structurally related family of cytokines that regulate leukocyte trafficking. Because infection with Toxoplasma gondii can induce an important inflammatory reaction that, if left uncontrolled, can lead to death, we investigated the role of the chemokine receptor CCR2 in T. gondii infection. We orally infected CCR2(-/-) mice with five ME-49 T. gondii cysts and monitored morbidity, survival, and immune response thereafter. The CCR2(-/-) mice displayed higher susceptibility to infection as all mice died on day 28 after infection. Despite similar Th1 responses, a more evident anti-inflammatory response was induced in the peripheral organs of CCR2(-/-) mice compared with wild-type C57BL/6 mice. Additionally, CCR2(-/-) mice presented greater parasitism and a milder inflammatory reaction in their peripheral organs with lesser CD4(+) and MAC-1(+) and greater CD8(+) cell migration. The parasite load decreased in these organs in CCR2(-/-) mice but remained uncontrolled in the central nervous system. Additionally, we observed down-regulated inducible nitric oxide synthase expression in peripheral organs from CCR2(-/-) mice that was associated with a small nitric oxide production by spleen macrophages. In conclusion, in the absence of CCR2, another mechanism is activated to control tissue parasitism in peripheral organs. Nevertheless, CCR2 is essential for the activation of microbicidal mediators that control T. gondii replication in the central nervous system.