RBCS1 expression in coffee: Coffea orthologs, Coffea arabica homeologs, and expression variability between genotypes and under drought stress

PubMed Central

2011-01-01

Background In higher plants, the inhibition of photosynthetic capacity under drought is attributable to stomatal and non-stomatal (i.e., photochemical and biochemical) effects. In particular, a disruption of photosynthetic metabolism and Rubisco regulation can be observed. Several studies reported reduced expression of the RBCS genes, which encode the Rubisco small subunit, under water stress. Results Expression of the RBCS1 gene was analysed in the allopolyploid context of C. arabica, which originates from a natural cross between the C. canephora and C. eugenioides species. Our study revealed the existence of two homeologous RBCS1 genes in C. arabica: one carried by the C. canephora sub-genome (called CaCc) and the other carried by the C. eugenioides sub-genome (called CaCe). Using specific primer pairs for each homeolog, expression studies revealed that CaCe was expressed in C. eugenioides and C. arabica but was undetectable in C. canephora. On the other hand, CaCc was expressed in C. canephora but almost completely silenced in non-introgressed ("pure") genotypes of C. arabica. However, enhanced CaCc expression was observed in most C. arabica cultivars with introgressed C. canephora genome. In addition, total RBCS1 expression was higher for C. arabica cultivars that had recently introgressed C. canephora genome than for "pure" cultivars. For both species, water stress led to an important decrease in the abundance of RBCS1 transcripts. This was observed for plants grown in either greenhouse or field conditions under severe or moderate drought. However, this reduction of RBCS1 gene expression was not accompanied by a decrease in the corresponding protein in the leaves of C. canephora subjected to water withdrawal. In that case, the amount of RBCS1 was even higher under drought than under unstressed (irrigated) conditions, which suggests great stability of RBCS1 under adverse water conditions. On the other hand, for C. arabica, high nocturnal expression of RBCS1 could also explain the accumulation of the RBCS1 protein under water stress. Altogether, the results presented here suggest that the content of RBCS was not responsible for the loss of photosynthetic capacity that is commonly observed in water-stressed coffee plants. Conclusion We showed that the CaCe homeolog was expressed in C. eugenioides and non-introgressed ("pure") genotypes of C. arabica but that it was undetectable in C. canephora. On the other hand, the CaCc homeolog was expressed in C. canephora but highly repressed in C. arabica. Expression of the CaCc homeolog was enhanced in C. arabica cultivars that experienced recent introgression with C. canephora. For both C. canephora and C. arabica species, total RBCS1 gene expression was highly reduced with WS. Unexpectedly, the accumulation of RBCS1 protein was observed in the leaves of C. canephora under WS, possibly coming from nocturnal RBCS1 expression. These results suggest that the increase in the amount of RBCS1 protein could contribute to the antioxidative function of photorespiration in water-stressed coffee plants. PMID:21575242

Plastid transformation for Rubisco engineering and protocols for assessing expression.

PubMed

Whitney, Spencer M; Sharwood, Robert E

2014-01-01

The assimilation of CO2 within chloroplasts is catalyzed by the bi-functional enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, Rubisco. Within higher plants the Rubisco large subunit gene, rbcL, is encoded in the plastid genome, while the Rubisco small subunit gene, RbcS is coded in the nucleus by a multi-gene family. Rubisco is considered a poor catalyst due to its slow turnover rate and its additional fixation of O2 that can result in wasteful loss of carbon through the energy requiring photorespiratory cycle. Improving the carboxylation efficiency and CO2/O2 selectivity of Rubisco within higher plants has been a long-term goal which has been greatly advanced in recent times using plastid transformation techniques. Here we present experimental methodologies for efficiently engineering Rubisco in the plastids of a tobacco master-line and analyzing leaf Rubisco content.

Red Blood Cell Passage of Small Capillaries Is Associated with Transient Ca2+-mediated Adaptations.

PubMed

Danielczok, Jens G; Terriac, Emmanuel; Hertz, Laura; Petkova-Kirova, Polina; Lautenschläger, Franziska; Laschke, Matthias W; Kaestner, Lars

2017-01-01

When red blood cells (RBCs) pass constrictions or small capillaries they need to pass apertures falling well below their own cross section size. We used different means of mechanical stimulations (hypoosmotic swelling, local mechanical stimulation, passing through microfluidic constrictions) to observe cellular responses of human RBCs in terms of intracellular Ca 2+ -signaling by confocal microscopy of Fluo-4 loaded RBCs. We were able to confirm our in vitro results in a mouse dorsal skinfold chamber model showing a transiently increased intracellular Ca 2+ when RBCs were passing through small capillaries in vivo . Furthermore, we performed the above-mentioned in vitro experiments as well as measurements of RBCs filterability under various pharmacological manipulations (GsMTx-4, TRAM-34) to explore the molecular mechanism of the Ca 2+ -signaling. Based on these experiments we conclude that mechanical stimulation of RBCs activates mechano-sensitive channels most likely Piezo1. This channel activity allows Ca 2+ to enter the cell, leading to a transient activation of the Gardos-channel associated with K + , Cl - , and water loss, i.e., with a transient volume adaptation facilitating the passage of the RBCs through the constriction.

Red Blood Cell Passage of Small Capillaries Is Associated with Transient Ca2+-mediated Adaptations

PubMed Central

Danielczok, Jens G.; Terriac, Emmanuel; Hertz, Laura; Petkova-Kirova, Polina; Lautenschläger, Franziska; Laschke, Matthias W.; Kaestner, Lars

2017-01-01

When red blood cells (RBCs) pass constrictions or small capillaries they need to pass apertures falling well below their own cross section size. We used different means of mechanical stimulations (hypoosmotic swelling, local mechanical stimulation, passing through microfluidic constrictions) to observe cellular responses of human RBCs in terms of intracellular Ca2+-signaling by confocal microscopy of Fluo-4 loaded RBCs. We were able to confirm our in vitro results in a mouse dorsal skinfold chamber model showing a transiently increased intracellular Ca2+ when RBCs were passing through small capillaries in vivo. Furthermore, we performed the above-mentioned in vitro experiments as well as measurements of RBCs filterability under various pharmacological manipulations (GsMTx-4, TRAM-34) to explore the molecular mechanism of the Ca2+-signaling. Based on these experiments we conclude that mechanical stimulation of RBCs activates mechano-sensitive channels most likely Piezo1. This channel activity allows Ca2+ to enter the cell, leading to a transient activation of the Gardos-channel associated with K+, Cl−, and water loss, i.e., with a transient volume adaptation facilitating the passage of the RBCs through the constriction. PMID:29259557

Plastidial Glycolytic Glyceraldehyde-3-Phosphate Dehydrogenase Is an Important Determinant in the Carbon and Nitrogen Metabolism of Heterotrophic Cells in Arabidopsis1

PubMed Central

Anoman, Armand D.; Muñoz-Bertomeu, Jesús; Rosa-Téllez, Sara; Flores-Tornero, María; Serrano, Ramón; Bueso, Eduardo; Fernie, Alisdair R.; Segura, Juan; Ros, Roc

2015-01-01

This study functionally characterizes the Arabidopsis (Arabidopsis thaliana) plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in photosynthetic and heterotrophic cells. We expressed the enzyme in gapcp double mutants (gapcp1gapcp2) under the control of photosynthetic (Rubisco small subunit RBCS2B [RBCS]) or heterotrophic (phosphate transporter PHT1.2 [PHT]) cell-specific promoters. Expression of GAPCp1 under the control of RBCS in gapcp1gapcp2 had no significant effect on the metabolite profile or growth in the aerial part (AP). GAPCp1 expression under the control of the PHT promoter clearly affected Arabidopsis development by increasing the number of lateral roots and having a major effect on AP growth and metabolite profile. Our results indicate that GAPCp1 is not functionally important in photosynthetic cells but plays a fundamental role in roots and in heterotrophic cells of the AP. Specifically, GAPCp activity may be required in root meristems and the root cap for normal primary root growth. Transcriptomic and metabolomic analyses indicate that the lack of GAPCp activity affects nitrogen and carbon metabolism as well as mineral nutrition and that glycerate and glutamine are the main metabolites responding to GAPCp activity. Thus, GAPCp could be an important metabolic connector of glycolysis with other pathways, such as the phosphorylated pathway of serine biosynthesis, the ammonium assimilation pathway, or the metabolism of γ-aminobutyrate, which in turn affect plant development. PMID:26134167

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eckardt, N.A.; Pell, E.J.

Leaf senescence is characterized by loss of the major photosynthetic enzyme, Ribulose bisphosphate carboxylase (Rubisco). Exposure to ozone (O[sub 3]) is often associated with a premature decline in the quantity of this enzyme. Declines in Rubisco quantity could arise through inhibition of synthesis or enhancement of degradation. Several experiments were conducted to investigate the effect of O[sub 3] on these events in immature and mature leaves of potato. The effect of O[sub 3] on Rubisco synthesis was investigated indirectly by measuring the relative quantities of mRNA for the rubisco large (rbcL) and small (rbcS) subunits following a 5 hour exposuremore » to 0.309 [mu]L L[sup [minus]1] O[sup 3] or charcoal-filtered air. O[sup 3] treatment was associated with a significant loss in rbcS mRNA in immature and mature potato leaves sampled immediately following the exposure. After the O[sup 3] exposure, a set of plants was placed in the dark at 30 C for two days. Levels of rbcS mRNA declined rapidly during the first twelve hours of dark incubation, thus declines in Rubisco quantity following two days of dark incubation were ascribed to degradation. Enhanced degradation due to O[sub 3] during the dark incubation was observed in the mature leaves, but not in the immature leaves. We conclude that O[sub 3] can cause both inhibited synthesis and enhanced degradation of Rubisco, and the response in dependent on leaf age.« less

Microscopic Measurements of Axial Accumulation of Red Blood Cells in Capillary Flows Effects of Deformability

NASA Astrophysics Data System (ADS)

Sasaki, Takahiro; Seki, Junji; Itano, Tomoaki; Sugihara-Seki, Masako

2017-11-01

In the microcirculation, red blood cells (RBCs) are known to accumulate in the region near the central axis of microvessels, which is called the ``axial accumulation''. Although this behavior of RBCs is considered to originate from high deformability of RBCs, there have been few experimental studies on the mechanism. In order to elucidate the effect of RBC deformability on the axial accumulation, we measured the cross-sectional distributions of RBCs flowing through capillary tubes with a high spatial resolution by a newly devised observation system for intact and softened RBCs as well as hardened RBCs to various degrees. It was found that the intact and softened RBCs are concentrated in the small area centered on the tube axis, whereas the hardened RBCs are dispersed widely over the tube cross section dependent on the degree of hardness. These results demonstrate clearly the essential role of the deformability of RBCs in the ``axial accumulation'' of RBCs. JSPS KAKENHI Grant Number 17H03176, Kansai University ORDIST group funds.

Over-expression of LeNCED1 in tomato (Solanum lycopersicum L.) with the rbcS3C promoter allows recovery of lines that accumulate very high levels of abscisic acid and exhibit severe phenotypes.

PubMed

Tung, Swee Ang; Smeeton, Rachel; White, Charlotte A; Black, Colin R; Taylor, Ian B; Hilton, Howard W; Thompson, Andrew J

2008-07-01

Previous work where 9-cis-epoxycarotenoid dioxygenase (NCED) was over-expressed using the constitutive Gelvin Superpromoter resulted in mild increases in abscisic acid (ABA) accumulation, accompanied by stomatal closure and increased water-use efficiency (WUE), but with apparently little impact on long-term biomass production. However, one of the negative effects of the over-expression of NCED using constitutive promoters in tomato was increased seed dormancy. Here we report the use of the rbcS3C promoter, from a gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), to drive LeNCED1 transgene expression in tomato in a light-responsive and circadian manner. In comparison to the constitutive promoter, the rbcS3C promoter allowed the generation of transgenic plants with much higher levels of ABA accumulation in leaves and sap, but the effect on seed dormancy was diminished. These plants displayed the expected reductions in stomatal conductance and CO(2) assimilation, but they also exhibited a severe set of symptoms that included perturbed cotyledon release from the testa, increased photobleaching in young seedlings, substantially reduced chlorophyll and carotenoid content, interveinal leaf flooding, and greatly reduced growth. These symptoms illustrate adverse consequences of long-term, very high ABA accumulation. Only more moderate increases in ABA biosynthesis are likely to be useful in the context of agriculture. Implications are discussed for the design of transgenic 'high ABA' plants that exhibit increased WUE but have minimal negative phenotypic effects.

An engineered Streptomyces hygroscopicus aph 7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii.

PubMed

Berthold, Peter; Schmitt, Rüdiger; Mages, Wolfgang

2002-12-01

We have developed a positively selectable marker for the green alga Chlamydomonas reinhardtii using the Streptomyces hygroscopicus aminoglycoside phosphotransferase gene (aph7"). Its expression is controlled by C. reinhardtii regulatory elements, namely, the beta2-tubulin gene promoter in combination with the first intron and the 3' untranslated region of the small subunit of ribulose bisphosphate carboxylase, rbcS2. C. reinhardtii cell-wall deficient and wild-type strains were transformed at rates up to 5 x 10(-5) with two constructs, pHyg3 and pHyg4 (intron-less). Transformants selected on plates with 10 microg/ml hygromycin B exhibited diverse levels of resistance of up to 200 microg/ml that were stably maintained for at least seven months; they contained two to five copies of the construct integrated in their genomes. Transcription of the chimeric aph7" gene, correct splicing of the rbcS2 intron, and polyadenylation of the transcripts have been verified by sequencing of RT-PCR products. Average co-transformation rates using pHyg3 and a second selectable plasmid were about 11%. This advocates the hygromycin-resistance plasmid, pHyg3, as a new versatile tool for the transformation of a broad range of C. reinhardtii strains without the sustained need for using auxotrophic mutants as recipients.

Effects of 60 Hz sinusoidal magnetic field on in vitro establishment, multiplication, and acclimatization phases of Coffea arabica seedlings.

PubMed

Isaac Alemán, Elizabeth; Oliveira Moreira, Rafael; Almeida Lima, Andre; Chaves Silva, Samuel; González-Olmedo, Justo Lorenzo; Chalfun-Junior, Antonio

2014-09-01

The influence of extremely low frequency electromagnetic fields on net photosynthesis, transpiration, photosynthetic pigment concentration, and gene expression of ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit (RBCS1), during in vitro establishment, in vitro multiplication and acclimatization phases of coffee seedlings were investigated. Untreated coffee plants were considered as control, whereas treated plants were exposed to a 60 Hz sinusoidal magnetic field of 2 mT of magnetic induction during 3 min. This magnetic field was generated by an electromagnet, connected to a wave generator. The results revealed that magnetically treated plants showed a significant increase in net photosynthesis (85.4% and 117.9%, in multiplication and acclimatization phases, respectively), and in photosynthetic pigment concentration (66.6% for establishment phase, 79.9% for multiplication phase, and 43.8% for acclimatization phase). They also showed a differential RBCS1 gene expression (approximately twofold) and a decrease of transpiration rates in regard to their control plants. In conclusion, the findings suggest that the application of 60 Hz magnetic field to in vitro coffee plants may improve the seedlings quality by modifying some photosynthetic physiological and molecular processes, increasing their vigor, and ensuring better plant development in later stages. © 2014 Wiley Periodicals, Inc.

Inhibition of the light-independent synthesis of chlorophyll in pine cotyledons at low temperature.

PubMed

Muramatsu, S; Kojima, K; Igasaki, T; Azumi, Y; Shinohara, K

2001-08-01

Cotyledons of Japanese black pine (Pinus thunbergii) were yellow when they developed in darkness at 8 degrees C since the light-independent synthesis of chlorophyll was almost completely inhibited in these cotyledons. The level of chlorophyll in dark-grown cotyledons was less than one-twentieth of that in light-grown cotyledons at the same temperature. In the yellow cotyledons, levels of transcripts of cab, rbcS, rbcL and psbA genes were quite high. The large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase were also detected at relatively high levels in yellow cotyledons. However, the accumulation of the two apoproteins of the light-harvesting chlorophyll a/b-binding protein of PSII was limited because of the limited supply of chlorophyll.

Cultured blood versus donated blood: long-run perspectives of the economy of blood.

PubMed

Mercier Ythier, Jean

2015-01-01

Recent advances of fundamental research on the in vitro generation of red blood cells (RBCs) from hematopoietic stem cells in the laboratory open new possibilities of the utilization of cultured RBCs in transfusion medicine. We study the economic challenge of the setup and development of the mass industrial production of RBCs in mature transfusion organizations. We argue that: (i) RBC manufacturing could be set up and developed in the short-medium run for the treatment of the small proportion of transfused patients who have a rare blood type or are alloimmunized against blood antigens; (ii) manufactured RBCs could substitute for donated RBCs in the long run if the physical productivity of RBC engineering technology approaches that of bone marrow.

Demonstration of specific binding of heparin to Plasmodium falciparum-infected vs. non-infected red blood cells by single-molecule force spectroscopy

NASA Astrophysics Data System (ADS)

Valle-Delgado, Juan José; Urbán, Patricia; Fernàndez-Busquets, Xavier

2013-04-01

Glycosaminoglycans (GAGs) play an important role in the sequestration of Plasmodium falciparum-infected red blood cells (pRBCs) in the microvascular endothelium of different tissues, as well as in the formation of small clusters (rosettes) between infected and non-infected red blood cells (RBCs). Both sequestration and rosetting have been recognized as characteristic events in severe malaria. Here we have used heparin and pRBCs infected by the 3D7 strain of P. falciparum as a model to study GAG-pRBC interactions. Fluorescence microscopy and fluorescence-assisted cell sorting assays have shown that exogenously added heparin has binding specificity for pRBCs (preferentially for those infected with late forms of the parasite) vs. RBCs. Heparin-pRBC adhesion has been probed by single-molecule force spectroscopy, obtaining an average binding force ranging between 28 and 46 pN depending on the loading rate. No significant binding of heparin to non-infected RBCs has been observed in control experiments. This work represents the first approach to quantitatively evaluate GAG-pRBC molecular interactions at the individual molecule level.Glycosaminoglycans (GAGs) play an important role in the sequestration of Plasmodium falciparum-infected red blood cells (pRBCs) in the microvascular endothelium of different tissues, as well as in the formation of small clusters (rosettes) between infected and non-infected red blood cells (RBCs). Both sequestration and rosetting have been recognized as characteristic events in severe malaria. Here we have used heparin and pRBCs infected by the 3D7 strain of P. falciparum as a model to study GAG-pRBC interactions. Fluorescence microscopy and fluorescence-assisted cell sorting assays have shown that exogenously added heparin has binding specificity for pRBCs (preferentially for those infected with late forms of the parasite) vs. RBCs. Heparin-pRBC adhesion has been probed by single-molecule force spectroscopy, obtaining an average binding force ranging between 28 and 46 pN depending on the loading rate. No significant binding of heparin to non-infected RBCs has been observed in control experiments. This work represents the first approach to quantitatively evaluate GAG-pRBC molecular interactions at the individual molecule level. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr32821f

Mutant selection in the self-incompatible plant radish (Raphanus sativus L. var. sativus) using two-step TILLING

PubMed Central

Kohzuma, Kaori; Chiba, Motoko; Nagano, Soichiro; Anai, Toyoaki; Ueda, Miki U.; Oguchi, Riichi; Shirai, Kazumasa; Hanada, Kousuke; Hikosaka, Kouki; Fujii, Nobuharu

2017-01-01

Radish (Raphanus sativus L. var. sativus), a widely cultivated root vegetable crop, possesses a large sink organ (the root), implying that photosynthetic activity in radish can be enhanced by altering both the source and sink capacity of the plant. However, since radish is a self-incompatible plant, improved mutation-breeding strategies are needed for this crop. TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful method used for reverse genetics. In this study, we developed a new TILLING strategy involving a two-step mutant selection process for mutagenized radish plants: the first selection is performed to identify a BC1M1 line, that is, progenies of M1 plants crossed with wild-type, and the second step is performed to identify BC1M1 individuals with mutations. We focused on Rubisco as a target, since Rubisco is the most abundant plant protein and a key photosynthetic enzyme. We found that the radish genome contains six RBCS genes and one pseudogene encoding small Rubisco subunits. We screened 955 EMS-induced BC1M1 lines using our newly developed TILLING strategy and obtained six mutant lines for the six RsRBCS genes, encoding proteins with four different types of amino acid substitutions. Finally, we selected a homozygous mutant and subjected it to physiological measurements. PMID:28744180

The effects of an overnight holding of whole blood at room temperature on haemoglobin modification and in vitro markers of red blood cell aging.

PubMed

Eckstein, M; Zimmermann, R; Roth, T; Hauck-Dlimi, B; Strasser, E F; Xiang, W

2015-05-01

Some effects of the red blood cell (RBC) storage lesion are well documented whereas others are not. Whether a period of room temperature hold (RTH) during RBC production enhances the RBC storage lesion has remained controversial. In this study, we compared whole blood (WB)-derived RBCs produced after 24-h RTH with rapidly cooled (RC) RBCs and tested them for classical metabolic markers and signs of oxidative damage. SAGM-RBCs were prepared from mixed and split pairs (n = 12) of WB units. RBCs prepared after a 24-h period of RTH on day+1 after collection (RTH-RBCs) were compared with RC-RBCs. All RBCs were stored at 4°C for 42 days with assay of in vitro variables on days+1, +15, +22, +29 and +42. The study examined standard quality parameters, glutathione, catalase and superoxide dismutase (SOD) activities, and indicative markers of oxidative cell damage including post-translational haemoglobin modification, malondialdehyde (MDA), and phosphatidylserine expression. RTH-RBCs exhibited decreased levels of potassium (1·98 ± 0·26 vs. 5·23 ± 0·65 mmol/l) and of 2,3-diphosphoglycerate (2,3-DPG) on day+1 compared with RC-RBCs. Haemolysis rate on day+42 was higher in RTH-RBCs than in RC-RBCs (0·52 ± 0·13 vs. 0·37 ± 0·12%). The phosphatidylserine expression amounted to 0·25 ± 0·20% in RTH-RBCs and 0·07 ± 0·12% in RC-RBCs. Haemoglobin modification was not different between both RBC groups. RTH-RBCs showed slightly higher MDA concentration on days +29 and +42. RC-RBCs and RTH-RBCs show only small differences of classical in vitro parameters and no relevant differences in antioxidative metabolism and oxidative haemoglobin modification. These findings do not explain the loss observed in in vivo survival studies with RBCs. © 2015 International Society of Blood Transfusion.

A biomimetic microfluidic chip to study the circulation and mechanical retention of red blood cells in the spleen.

PubMed

Picot, Julien; Ndour, Papa Alioune; Lefevre, Sophie D; El Nemer, Wassim; Tawfik, Harvey; Galimand, Julie; Da Costa, Lydie; Ribeil, Jean-Antoine; de Montalembert, Mariane; Brousse, Valentine; Le Pioufle, Bruno; Buffet, Pierre; Le Van Kim, Caroline; Français, Olivier

2015-04-01

Red blood cells (RBCs) are deformable and flow through vessels narrower than their own size. Their deformability is most stringently challenged when they cross micrometer-wide slits in the spleen. In several inherited or acquired RBC disorders, blockade of small vessels by stiff RBCs can trigger organ damage, but a functional spleen is expected to clear these abnormal RBCs from the circulation before they induce such complications. We analyzed flow behavior of RBCs in a microfluidic chip that replicates the mechanical constraints imposed on RBCs as they cross the human spleen. Polymer microchannels obtained by soft lithography with a hydraulic diameter of 25 μm drove flow into mechanical filtering units where RBCs flew either slowly through 5- to 2-μm-wide slits or rapidly along 10-μm-wide channels, these parallel paths mimicking the splenic microcirculation. Stiff heated RBCs accumulated in narrow slits seven times more frequently than normal RBCs infused simultaneously. Stage-dependent retention of Plasmodium falciparum-infected RBCs was also observed in these slits. We also analyzed RBCs from patients with hereditary spherocytosis and observed retention for those having the most altered mechanical properties as determined by ektacytometry. Thus, in keeping with previous observations in vivo and ex vivo, the chip successfully discriminated poorly deformable RBCs based on their distinct mechanical properties and on the intensity of the cell alteration. Applications to the exploration of the pathogenesis of malaria, hereditary spherocytosis, sickle cell disease and other RBC disorders are envisioned. © 2015 Wiley Periodicals, Inc.

Optimization of Acidothermus Celluloyticus Endoglucanase (E1) Production in Transgenic Tobacco Plants by Transcriptional, Post-transcription and Post-Translational Modification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Ziyu; Hooker, Brian S.; Quesenberry, Ryan D.

2005-10-01

Biochemical characteristics of Acidothermus cellulolyticus endoglucanase (E1) and its physiological effects in transgenic tobacco (Nicotiana tabacum) has been studied previously. In an attempt to obtain a high level of production of intact E1 in transgenic plants, the E1 gene was expressed under the control of strong Mac promoter (a hybrid promoter of manopine synthase promoter and cauliflower mosaic virus 35S promoter enhancer region) or tomato Rubisco small subunit (RbcS-3C) promoter with different 5’ untranslated leader (UTL) sequence and targeted to different subcellular comartmentations with various transit peptides. The expression of E1 protein in transgenic tobacco plants was determined via E1more » activity, protein immunobloting, and RNA gel-blotting analyses. Effects of different transit peptides on E1 protein production and its stability were examined in transgenic tobacco plants carrying one of six transgene expression vectors with the same (Mac) promoter and transcription terminator (Tmas). Transgenic tobacco plants with apoplast transit peptide (Mm-apo) had the highest average E1 activity and protein accumulation , while E1 protein was more stable in transgenic plants with no transit peptide (Mm) than others. The E1 expression under tomato RbcS-3C promoter was higher than that under Mac promoter based on the average E1 activity, E1 protein accumulation, and RNA gel-blotting. The E1 expression was increased more than two fold when the 5’-UTL of alfalfa mosaic virus RNA4 gene replaced the UTL of RbcS-3C promoter, while the UTL of alfalfa mosaic virus RNA4 gene was less effective than the UTL of Mac promoter. The optimal combination of promoter, 5’-UTL, and subcellular compartmentation (transit peptide) for E1 protein production in transgenic tobacco plants are discussed.« less

Posttranscriptional Control of Photosynthetic mRNA Decay under Stress Conditions Requires 3′ and 5′ Untranslated Regions and Correlates with Differential Polysome Association in Rice1[W][OA

PubMed Central

Park, Su-Hyun; Chung, Pil Joong; Juntawong, Piyada; Bailey-Serres, Julia; Kim, Youn Shic; Jung, Harin; Bang, Seung Woon; Kim, Yeon-Ki; Do Choi, Yang; Kim, Ju-Kon

2012-01-01

Abiotic stress, including drought, salinity, and temperature extremes, regulates gene expression at the transcriptional and posttranscriptional levels. Expression profiling of total messenger RNAs (mRNAs) from rice (Oryza sativa) leaves grown under stress conditions revealed that the transcript levels of photosynthetic genes are reduced more rapidly than others, a phenomenon referred to as stress-induced mRNA decay (SMD). By comparing RNA polymerase II engagement with the steady-state mRNA level, we show here that SMD is a posttranscriptional event. The SMD of photosynthetic genes was further verified by measuring the half-lives of the small subunit of Rubisco (RbcS1) and Chlorophyll a/b-Binding Protein1 (Cab1) mRNAs during stress conditions in the presence of the transcription inhibitor cordycepin. To discern any correlation between SMD and the process of translation, changes in total and polysome-associated mRNA levels after stress were measured. Total and polysome-associated mRNA levels of two photosynthetic (RbcS1 and Cab1) and two stress-inducible (Dehydration Stress-Inducible Protein1 and Salt-Induced Protein) genes were found to be markedly similar. This demonstrated the importance of polysome association for transcript stability under stress conditions. Microarray experiments performed on total and polysomal mRNAs indicate that approximately half of all mRNAs that undergo SMD remain polysome associated during stress treatments. To delineate the functional determinant(s) of mRNAs responsible for SMD, the RbcS1 and Cab1 transcripts were dissected into several components. The expressions of different combinations of the mRNA components were analyzed under stress conditions, revealing that both 3′ and 5′ untranslated regions are necessary for SMD. Our results, therefore, suggest that the posttranscriptional control of photosynthetic mRNA decay under stress conditions requires both 3′ and 5′ untranslated regions and correlates with differential polysome association. PMID:22566494

The mechanical properties of stored red blood cells measured by a convenient microfluidic approach combining with mathematic model.

PubMed

Wang, Ying; You, Guoxing; Chen, Peipei; Li, Jianjun; Chen, Gan; Wang, Bo; Li, Penglong; Han, Dong; Zhou, Hong; Zhao, Lian

2016-03-01

The mechanical properties of red blood cells (RBCs) are critical to the rheological and hemodynamic behavior of blood. Although measurements of the mechanical properties of RBCs have been studied for many years, the existing methods, such as ektacytometry, micropipette aspiration, and microfluidic approaches, still have limitations. Mechanical changes to RBCs during storage play an important role in transfusions, and so need to be evaluated pre-transfusion, which demands a convenient and rapid detection method. We present a microfluidic approach that focuses on the mechanical properties of single cell under physiological shear flow and does not require any high-end equipment, like a high-speed camera. Using this method, the images of stretched RBCs under physical shear can be obtained. The subsequent analysis, combined with mathematic models, gives the deformability distribution, the morphology distribution, the normalized curvature, and the Young's modulus (E) of the stored RBCs. The deformability index and the morphology distribution show that the deformability of RBCs decreases significantly with storage time. The normalized curvature, which is defined as the curvature of the cell tail during stretching in flow, suggests that the surface charge of the stored RBCs decreases significantly. According to the mathematic model, which derives from the relation between shear stress and the adherent cells' extension ratio, the Young's moduli of the stored RBCs are also calculated and show significant increase with storage. Therefore, the present method is capable of representing the mechanical properties and can distinguish the mechanical changes of the RBCs during storage. The advantages of this method are the small sample needed, high-throughput, and easy-use, which make it promising for the quality monitoring of RBCs.

Estimation of adult and neonatal RBC lifespans in anemic neonates using RBCs labeled at several discrete biotin densities.

PubMed

Kuruvilla, Denison J; Widness, John A; Nalbant, Demet; Schmidt, Robert L; Mock, Donald M; An, Guohua; Veng-Pedersen, Peter

2017-06-01

Prior conclusions that autologous neonatal red blood cells (RBC) have substantially shorter lifespans than allogeneic adult RBCs were not based on direct comparison of autologous neonatal vs. allogeneic adult RBCs performed concurrently in the same infant. Biotin labeling of autologous neonatal RBCs and allogeneic adult donor RBCs permits concurrent direct comparison of autologous vs. allogeneic RBC lifespan. RBCs from 15 allogeneic adult donors and from 15 very-low-birth-weight (VLBW) neonates were labeled at separate biotin densities and transfused simultaneously into the 15 neonates. Two mathematical models that account for the RBC differences were employed to estimate lifespans for the two RBC populations. Mean ± SD lifespan for adult allogeneic RBC was 70.1 ± 19.1 d, which is substantially shorter than the 120 d lifespan of both autologous and adult allogeneic RBC in healthy adults. Mean ± SD lifespan for neonatal RBC was 54.2 ± 11.3 d, which is only about 30% shorter than that of the adult allogeneic RBCs. This study provides evidence that extrinsic environmental factors primarily determine RBC survival (e.g., small bore of the capillaries of neonates, rate of oxygenation/deoxygenation cycles) rather than factors intrinsic to RBC.

Computational study of a magnetic design to improve the diagnosis of malaria: 2D model

NASA Astrophysics Data System (ADS)

Vyas, Siddharth; Genis, Vladimir; Friedman, Gary

2017-02-01

This paper investigates the feasibility of a cost effective high gradient magnetic separation based device for the detection and identification of malaria parasites in a blood sample. The design utilizes magnetic properties of hemozoin present in malaria-infected red blood cells (mRBCs) in order to separate and concentrate them inside a microfluidic channel slide for easier examination under the microscope. The design consists of a rectangular microfluidic channel with multiple magnetic wires positioned on top of and underneath it along the length of the channel at a small angle with respect to the channel axis. Strong magnetic field gradients, produced by the wires, exert sufficient magnetic forces on the mRBCs in order to separate and concentrate them in a specific region small enough to fit within the microscope field of view at magnifications typically required to identify the malaria parasite type. The feasibility of the device is studied using a model where the trajectories of the mRBCs inside the channel are determined using first-order ordinary differential equations (ODEs) solved numerically using a multistep ODE solver available within MATLAB. The mRBCs trajectories reveal that it is possible to separate and concentrate the mRBCs in less than 5 min, even in cases of very low parasitemia (1-10 parasites/μL of blood) using blood sample volumes of around 3 μL employed today.

Biomechanics and biorheology of red blood cells in sickle cell anemia

PubMed Central

Li, Xuejin; Dao, Ming; Lykotrafitis, George; Karniadakis, George Em

2017-01-01

Sickle cell anemia (SCA) is an inherited blood disorder that causes painful crises due to vaso-occlusion of small blood vessels. The primary cause of the clinical phenotype of SCA is the intracellular polymerization of sickle hemoglobin resulting in sickling of red blood cells (RBCs) in deoxygenated conditions. In this review, we discuss the biomechanical and biorheological characteristics of sickle RBCs and sickle blood as well as their implications toward a better understanding of the pathophysiology and pathogenesis of SCA. Additionally, we highlight the adhesive heterogeneity of RBCs in SCA and their specific contribution to vaso-occlusive crisis. PMID:27876368

Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane

PubMed Central

Gokhin, David S.; Nowak, Roberta B.; Khoory, Joseph A.; de la Piedra, Alfonso; Ghiran, Ionita C.; Fowler, Velia M.

2015-01-01

Short, uniform-length actin filaments function as structural nodes in the spectrin-actin membrane skeleton to optimize the biomechanical properties of red blood cells (RBCs). Despite the widespread assumption that RBC actin filaments are not dynamic (i.e., do not exchange subunits with G-actin in the cytosol), this assumption has never been rigorously tested. Here we show that a subpopulation of human RBC actin filaments is indeed dynamic, based on rhodamine-actin incorporation into filaments in resealed ghosts and fluorescence recovery after photobleaching (FRAP) analysis of actin filament mobility in intact RBCs (∼25–30% of total filaments). Cytochalasin-D inhibition of barbed-end exchange reduces rhodamine-actin incorporation and partially attenuates FRAP recovery, indicating functional interaction between actin subunit turnover at the single-filament level and mobility at the membrane-skeleton level. Moreover, perturbation of RBC actin filament assembly/disassembly with latrunculin-A or jasplakinolide induces an approximately twofold increase or ∼60% decrease, respectively, in soluble actin, resulting in altered membrane deformability, as determined by alterations in RBC transit time in a microfluidic channel assay, as well as by abnormalities in spontaneous membrane oscillations (flickering). These experiments identify a heretofore-unrecognized but functionally important subpopulation of RBC actin filaments, whose properties and architecture directly control the biomechanical properties of the RBC membrane. PMID:25717184

A study of red blood cell deformability in diabetic retinopathy using optical tweezers

NASA Astrophysics Data System (ADS)

Smart, Thomas J.; Richards, Christopher J.; Bhatnagar, Rhythm; Pavesio, Carlos; Agrawal, Rupesh; Jones, Philip H.

2015-08-01

Diabetic retinopathy (DR) is a microvascular complication of diabetes mellitus (DM) in which high blood sugar levels cause swelling, leaking and occlusions in the blood vessels of the retina, often resulting in a loss of sight. The microvascular system requires red blood cells (RBCs) to undergo significant cellular deformation in order to pass through vessels whose diameters are significantly smaller than their own. There is evidence to suggest that DM impairs the deformability of RBCs, and this loss of deformability has been associated with diabetic kidney disease (or nephropathy) - another microvascular complication of DM. However, it remains unclear whether reduced deformability of RBCs correlates with the presence of DR. Here we present an investigation into the deformability of RBCs in patients with diabetic retinopathy using optical tweezers. To extract a value for the deformability of RBCs we use a dual-trap optical tweezers set-up to stretch individual RBCs. RBCs are trapped directly (i.e. without micro-bead handles), so rotate to assume a `side-on' orientation. Video microscopy is used to record the deformation events, and shape analysis software is used to determine parameters such as initial and maximum RBC length, allowing us to calculate the deformability for each RBC. A small decrease in deformability of diabetes cells subject to this stretching protocol is observed when compared to control cells. We also report on initial results on three dimensional imaging of individual RBCs using defocussing microscopy.

Multiscale Modeling of Hematologic Disorders

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fedosov, Dmitry A.; Pivkin, Igor; Pan, Wenxiao

Parasitic infectious diseases and other hereditary hematologic disorders are often associated with major changes in the shape and viscoelastic properties of red blood cells (RBCs). Such changes can disrupt blood flow and even brain perfusion, as in the case of cerebral malaria. Modeling of these hematologic disorders requires a seamless multiscale approach, where blood cells and blood flow in the entire arterial tree are represented accurately using physiologically consistent parameters. In this chapter, we present a computational methodology based on dissipative particle dynamics (DPD) which models RBCs as well as whole blood in health and disease. DPD is a Lagrangianmore » method that can be derived from systematic coarse-graining of molecular dynamics but can scale efficiently up to small arteries and can also be used to model RBCs down to spectrin level. To this end, we present two complementary mathematical models for RBCs and describe a systematic procedure on extracting the relevant input parameters from optical tweezers and microfluidic experiments for single RBCs. We then use these validated RBC models to predict the behavior of whole healthy blood and compare with experimental results. The same procedure is applied to modeling malaria, and results for infected single RBCs and whole blood are presented.« less

Influence of Pre-Storage Irradiation on the Oxidative Stress Markers, Membrane Integrity, Size and Shape of the Cold Stored Red Blood Cells

PubMed Central

Antosik, Adam; Czubak, Kamila; Gajek, Arkadiusz; Marczak, Agnieszka; Glowacki, Rafal; Borowczyk, Kamila; Zbikowska, Halina Malgorzata

2015-01-01

Background To investigate the extent of oxidative damage and changes in morphology of manually isolated red blood cells (RBCs) from whole blood, cold stored (up to 20 days) in polystyrene tubes and subjected to pre-storage irradiation (50 Gy) and to compare the properties of SAGM-preserved RBCs stored under experimental conditions (polystyrene tubes) with RBCs from standard blood bag storage. Methods The percentage of hemolysis as well as the extracellular activity of LDH, thiobarbituric acid-reactive substances, reduced glutathione (GSH), and total antioxidant capacity (TAC) were measured. Changes in the topology of RBC membrane, shape, and size were evaluated by flow cytometry and judged against microscopy images. Results Irradiation caused significant LDH release as well as increased hemolysis and lipid peroxidation, GSH depletion, and reduction of TAC. Prolonged storage of irradiated RBCs resulted in phosphatidylserine exposure on the cell surface. By day 20, approximately 60% of RBCs displayed non-discoid shape. We did not notice significant differences in percentage of altered cells and cell volume between RBCs exposed to irradiation and those not exposed. Conclusion Irradiation of RBC transfusion units with a dose of 50 Gy should be avoided. For research purposes such as studying the role of antioxidants, storage of small volumes of RBCs derived from the same donor would be more useful, cheaper, and blood-saving. PMID:26195927

Influence of Pre-Storage Irradiation on the Oxidative Stress Markers, Membrane Integrity, Size and Shape of the Cold Stored Red Blood Cells.

PubMed

Antosik, Adam; Czubak, Kamila; Gajek, Arkadiusz; Marczak, Agnieszka; Glowacki, Rafal; Borowczyk, Kamila; Zbikowska, Halina Malgorzata

2015-05-01

To investigate the extent of oxidative damage and changes in morphology of manually isolated red blood cells (RBCs) from whole blood, cold stored (up to 20 days) in polystyrene tubes and subjected to pre-storage irradiation (50 Gy) and to compare the properties of SAGM-preserved RBCs stored under experimental conditions (polystyrene tubes) with RBCs from standard blood bag storage. The percentage of hemolysis as well as the extracellular activity of LDH, thiobarbituric acid-reactive substances, reduced glutathione (GSH), and total antioxidant capacity (TAC) were measured. Changes in the topology of RBC membrane, shape, and size were evaluated by flow cytometry and judged against microscopy images. Irradiation caused significant LDH release as well as increased hemolysis and lipid peroxidation, GSH depletion, and reduction of TAC. Prolonged storage of irradiated RBCs resulted in phosphatidylserine exposure on the cell surface. By day 20, approximately 60% of RBCs displayed non-discoid shape. We did not notice significant differences in percentage of altered cells and cell volume between RBCs exposed to irradiation and those not exposed. Irradiation of RBC transfusion units with a dose of 50 Gy should be avoided. For research purposes such as studying the role of antioxidants, storage of small volumes of RBCs derived from the same donor would be more useful, cheaper, and blood-saving.

Biomechanics and biorheology of red blood cells in sickle cell anemia.

PubMed

Li, Xuejin; Dao, Ming; Lykotrafitis, George; Karniadakis, George Em

2017-01-04

Sickle cell anemia (SCA) is an inherited blood disorder that causes painful crises due to vaso-occlusion of small blood vessels. The primary cause of the clinical phenotype of SCA is the intracellular polymerization of sickle hemoglobin resulting in sickling of red blood cells (RBCs) in deoxygenated conditions. In this review, we discuss the biomechanical and biorheological characteristics of sickle RBCs and sickle blood as well as their implications toward a better understanding of the pathophysiology and pathogenesis of SCA. Additionally, we highlight the adhesive heterogeneity of RBCs in SCA and their specific contribution to vaso-occlusive crisis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mechanism of Enzyme Repair by the AAA+ Chaperone Rubisco Activase.

PubMed

Bhat, Javaid Y; Miličić, Goran; Thieulin-Pardo, Gabriel; Bracher, Andreas; Maxwell, Andrew; Ciniawsky, Susanne; Mueller-Cajar, Oliver; Engen, John R; Hartl, F Ulrich; Wendler, Petra; Hayer-Hartl, Manajit

2017-09-07

How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) in metabolic repair of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits containing eight catalytic sites. Rubisco is prone to inhibition by tight-binding sugar phosphates, whose removal is catalyzed by Rca. We engineered a stable Rca hexamer ring and analyzed its functional interaction with Rubisco. Hydrogen/deuterium exchange and chemical crosslinking showed that Rca structurally destabilizes elements of the Rubisco active site with remarkable selectivity. Cryo-electron microscopy revealed that Rca docks onto Rubisco over one active site at a time, positioning the C-terminal strand of RbcL, which stabilizes the catalytic center, for access to the Rca hexamer pore. The pulling force of Rca is fine-tuned to avoid global destabilization and allow for precise enzyme repair. Copyright © 2017 Elsevier Inc. All rights reserved.

Measurement of erythrocyte deformability by two laser diffraction methods.

PubMed

Wang, X; Zhao, H; Zhuang, F Y; Stoltz, J F

1999-01-01

The aim of this work is to study the deformability of red blood cells (RBC) by two laser diffraction methods: the Laser-assisted Optical Rotational Cell Analyser (LORCA, Mechatronics, Amsterdam, Netherlands) and a Shear Stress Diffractometer (RHEODYN SSD, Myrenne, Roetgen, Germany). Experiments were carried out on 46 healthy human subjects. The elongation index EI of normal and hardened RBCs (obtained by heating blood at 49 degrees C or by incubating RBCs in solutions of diamide) was measured. The results showed that the standard deviations of the experimental data for normal RBCs were relatively small, especially at high shear stresses (more than 3.0 Pa), but higher than those reported before. Some correlations between the results given by the two instruments were also found. It should be noted that for hardened RBCs, the standard deviations of the measurements were important compared with the mean values in the two instruments.

Flow of a circulating tumor cell and red blood cells in microvessels

NASA Astrophysics Data System (ADS)

Takeishi, Naoki; Imai, Yohsuke; Yamaguchi, Takami; Ishikawa, Takuji

2015-12-01

Quantifying the behavior of circulating tumor cells (CTCs) in the blood stream is of fundamental importance for understanding metastasis. Here, we investigate the flow mode and velocity of CTCs interacting with red blood cells (RBCs) in various sized microvessels. The flow of leukocytes in microvessels has been described previously; a leukocyte forms a train with RBCs in small microvessels and exhibits margination in large microvessels. Important differences in the physical properties of leukocytes and CTCs result from size. The dimensions of leukocytes are similar to those of RBCs, but CTCs are significantly larger. We investigate numerically the size effects on the flow mode and the cell velocity, and we identify similarities and differences between leukocytes and CTCs. We find that a transition from train formation to margination occurs when (R -a ) /tR≈1 , where R is the vessel radius, a is the cell radius, and tR is the thickness of RBCs, but that the motion of RBCs differs from the case of leukocytes. Our results also show that the velocities of CTCs and leukocytes are larger than the average blood velocity, but only CTCs move faster than RBCs for microvessels of R /a ≈1.5 -2.0 . These findings are expected to be useful not only for understanding metastasis, but also for developing microfluidic devices.

In premature infants there is no decrease in 24-hour posttransfusion allogeneic red blood cell recovery after 42 days of storage.

PubMed

Nalbant, Demet; Cancelas, José A; Mock, Donald M; Kyosseva, Svetlana V; Schmidt, Robert L; Cress, Gretchen A; Zimmerman, M Bridget; Strauss, Ronald G; Widness, John A

2018-02-01

Critically ill preterm very-low-birthweight (VLBW) neonates (birthweight ≤ 1.5 kg) frequently develop anemia that is treated with red blood cell (RBC) transfusions. Although RBCs transfused to adults demonstrate progressive decreases in posttransfusion 24-hour RBC recovery (PTR 24 ) during storage-to a mean of approximately 85% of the Food and Drug Administration-allowed 42-day storage-limited data in infants indicate no decrease in PTR 24 with storage. We hypothesized that PTR 24 of allogeneic RBCs transfused to anemic VLBW newborns: 1) will be greater than PTR 24 of autologous RBCs transfused into healthy adults and 2) will not decrease with increasing storage duration. RBCs were stored at 4°C for not more than 42 days in AS-3 or AS-5. PTR 24 was determined in 46 VLBW neonates using biotin-labeled RBCs and in 76 healthy adults using 51 Cr-labeled RBCs. Linear mixed-model analysis was used to estimate slopes and intercepts of PTR 24 versus duration of RBC storage. For VLBW newborns, the estimated slope of PTR 24 versus storage did not decrease with the duration of storage (p = 0.18) while for adults it did (p < 0.0001). These estimated slopes differed significantly in adults compared to newborns (p = 0.04). At the allowed 42-day storage limit, projected mean neonatal PTR 24 was 95.9%; for adults, it was 83.8% (p = 0.0002). These data provide evidence that storage duration of allogeneic RBCs intended for neonates can be increased without affecting PTR 24 . This conclusion supports the practice of transfusing RBCs stored up to 42 days for small-volume neonatal transfusions to limit donor exposure. © 2017 AABB.

Hydrogen sulphide improves adaptation of Zea mays seedlings to iron deficiency

PubMed Central

Chen, Juan; Wu, Fei-Hua; Shang, Yu-Ting; Wang, Wen-Hua; Hu, Wen-Jun; Simon, Martin; Liu, Xiang; Shangguan, Zhou-Ping; Zheng, Hai-Lei

2015-01-01

Hydrogen sulphide (H2S) is emerging as a potential molecule involved in physiological regulation in plants. However, whether H2S regulates iron-shortage responses in plants is largely unknown. Here, the role of H2S in modulating iron availability in maize (Zea mays L. cv Canner) seedlings grown in iron-deficient culture solution is reported. The main results are as follows: Firstly, NaHS, a donor of H2S, completely prevented leaf interveinal chlorosis in maize seedlings grown in iron-deficient culture solution. Secondly, electron micrographs of mesophyll cells from iron-deficient maize seedlings revealed plastids with few photosynthetic lamellae and rudimentary grana. On the contrary, mesophyll chloroplasts appeared completely developed in H2S-treated maize seedlings. Thirdly, H2S treatment increased iron accumulation in maize seedlings by changing the expression levels of iron homeostasis- and sulphur metabolism-related genes. Fourthly, phytosiderophore (PS) accumulation and secretion were enhanced by H2S treatment in seedlings grown in iron-deficient solution. Indeed, the gene expression of ferric-phytosiderophore transporter (ZmYS1) was specifically induced by iron deficiency in maize leaves and roots, whereas their abundance was decreased by NaHS treatment. Lastly, H2S significantly enhanced photosynthesis through promoting the protein expression of ribulose-1,5-bisphosphate carboxylase large subunit (RuBISCO LSU) and phosphoenolpyruvate carboxylase (PEPC) and the expression of genes encoding RuBISCO large subunit (RBCL), small subunit (RBCS), D1 protein (psbA), and PEPC in maize seedlings grown in iron-deficient solution. These results indicate that H2S is closely related to iron uptake, transport, and accumulation, and consequently increases chlorophyll biosynthesis, chloroplast development, and photosynthesis in plants. PMID:26208645

Hydrogen sulphide improves adaptation of Zea mays seedlings to iron deficiency.

PubMed

Chen, Juan; Wu, Fei-Hua; Shang, Yu-Ting; Wang, Wen-Hua; Hu, Wen-Jun; Simon, Martin; Liu, Xiang; Shangguan, Zhou-Ping; Zheng, Hai-Lei

2015-11-01

Hydrogen sulphide (H2S) is emerging as a potential molecule involved in physiological regulation in plants. However, whether H2S regulates iron-shortage responses in plants is largely unknown. Here, the role of H2S in modulating iron availability in maize (Zea mays L. cv Canner) seedlings grown in iron-deficient culture solution is reported. The main results are as follows: Firstly, NaHS, a donor of H2S, completely prevented leaf interveinal chlorosis in maize seedlings grown in iron-deficient culture solution. Secondly, electron micrographs of mesophyll cells from iron-deficient maize seedlings revealed plastids with few photosynthetic lamellae and rudimentary grana. On the contrary, mesophyll chloroplasts appeared completely developed in H2S-treated maize seedlings. Thirdly, H2S treatment increased iron accumulation in maize seedlings by changing the expression levels of iron homeostasis- and sulphur metabolism-related genes. Fourthly, phytosiderophore (PS) accumulation and secretion were enhanced by H2S treatment in seedlings grown in iron-deficient solution. Indeed, the gene expression of ferric-phytosiderophore transporter (ZmYS1) was specifically induced by iron deficiency in maize leaves and roots, whereas their abundance was decreased by NaHS treatment. Lastly, H2S significantly enhanced photosynthesis through promoting the protein expression of ribulose-1,5-bisphosphate carboxylase large subunit (RuBISCO LSU) and phosphoenolpyruvate carboxylase (PEPC) and the expression of genes encoding RuBISCO large subunit (RBCL), small subunit (RBCS), D1 protein (psbA), and PEPC in maize seedlings grown in iron-deficient solution. These results indicate that H2S is closely related to iron uptake, transport, and accumulation, and consequently increases chlorophyll biosynthesis, chloroplast development, and photosynthesis in plants. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Influence of polymer architecture on antigens camouflage, CD47 protection and complement mediated lysis of surface grafted red blood cells.

PubMed

Chapanian, Rafi; Constantinescu, Iren; Rossi, Nicholas A A; Medvedev, Nadia; Brooks, Donald E; Scott, Mark D; Kizhakkedathu, Jayachandran N

2012-11-01

Hyperbranched polyglycerol (HPG) and polyethylene glycol (PEG) polymers with similar hydrodynamic sizes in solution were grafted to red blood cells (RBCs) to investigate the impact of polymer architecture on the cell structure and function. The hydrodynamic sizes of polymers were calculated from the diffusion coefficients measured by pulsed field gradient NMR. The hydration of the HPG and PEG was determined by differential scanning calorimetry analyses. RBCs grafted with linear PEG had different properties compared to the compact HPG grafted RBCs. HPG grafted RBCs showed much higher electrophoretic mobility values than PEG grafted RBCs at similar grafting concentrations and hydrodynamic sizes indicating differences in the structure of the polymer exclusion layer on the cell surface. PEG grafting impacted the deformation properties of the membrane to a greater degree than HPG. The complement mediated lysis of the grafted RBCs was dependent on the type of polymer, grafting concentration and molecular size of grafted chains. At higher molecular weights and graft concentrations both HPG and PEG triggered complement activation. The magnitude of activation was higher with HPG possibly due to the presence of many hydroxyl groups per molecule. HPG grafted RBCs showed significantly higher levels of CD47 self-protein accessibility than PEG grafted RBCs at all grafting concentrations and molecular sizes. PEG grafted polymers provided, in general, a better shielding and protection to ABO and minor antigens from antibody recognition than HPG polymers, however, the compact HPGs provided greater protection of certain antigens on the RBC surface. Our data showed that HPG 20 kDa and HPG 60 kDa grafted RBCs exhibited properties that are more comparable to the native RBC than PEG 5 kDa and PEG 10 kDa grafted RBCs of comparable hydrodynamic sizes. The study shows that small compact polymers such as HPG 20 kDa have a greater potential in the generation of functional RBC for therapeutic delivery applications. The intermediate sized polymers (PEG or HPG) which showed greater antigen camouflage at lower grafting concentrations have significant potential in transfusion as universal red blood donor cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

Small molecule ice recrystallization inhibitors mitigate red blood cell lysis during freezing, transient warming and thawing

NASA Astrophysics Data System (ADS)

Briard, Jennie G.; Poisson, Jessica S.; Turner, Tracey R.; Capicciotti, Chantelle J.; Acker, Jason P.; Ben, Robert N.

2016-03-01

During cryopreservation, ice recrystallization is a major cause of cellular damage. Conventional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol function by a number of different mechanisms but do not mitigate or control ice recrystallization at concentrations utilized in cryopreservation procedures. In North America, cryopreservation of human red blood cells (RBCs) utilizes high concentrations of glycerol. RBC units frozen under these conditions must be subjected to a time-consuming deglycerolization process after thawing in order to remove the glycerol to <1% prior to transfusion thus limiting the use of frozen RBC units in emergency situations. We have identified several low molecular mass ice recrystallization inhibitors (IRIs) that are effective cryoprotectants for human RBCs, resulting in 70-80% intact RBCs using only 15% glycerol and slow freezing rates. These compounds are capable of reducing the average ice crystal size of extracellular ice relative to a 15% glycerol control validating the positive correlation between a reduction in ice crystal size and increased post-thaw recovery of RBCs. The most potent IRI from this study is also capable of protecting frozen RBCs against the large temperature fluctuations associated with transient warming.

Small molecule ice recrystallization inhibitors mitigate red blood cell lysis during freezing, transient warming and thawing

PubMed Central

Briard, Jennie G.; Poisson, Jessica S.; Turner, Tracey R.; Capicciotti, Chantelle J.; Acker, Jason P.; Ben, Robert N.

2016-01-01

During cryopreservation, ice recrystallization is a major cause of cellular damage. Conventional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol function by a number of different mechanisms but do not mitigate or control ice recrystallization at concentrations utilized in cryopreservation procedures. In North America, cryopreservation of human red blood cells (RBCs) utilizes high concentrations of glycerol. RBC units frozen under these conditions must be subjected to a time-consuming deglycerolization process after thawing in order to remove the glycerol to <1% prior to transfusion thus limiting the use of frozen RBC units in emergency situations. We have identified several low molecular mass ice recrystallization inhibitors (IRIs) that are effective cryoprotectants for human RBCs, resulting in 70–80% intact RBCs using only 15% glycerol and slow freezing rates. These compounds are capable of reducing the average ice crystal size of extracellular ice relative to a 15% glycerol control validating the positive correlation between a reduction in ice crystal size and increased post-thaw recovery of RBCs. The most potent IRI from this study is also capable of protecting frozen RBCs against the large temperature fluctuations associated with transient warming. PMID:27021850

Small molecule ice recrystallization inhibitors mitigate red blood cell lysis during freezing, transient warming and thawing.

PubMed

Briard, Jennie G; Poisson, Jessica S; Turner, Tracey R; Capicciotti, Chantelle J; Acker, Jason P; Ben, Robert N

2016-03-29

During cryopreservation, ice recrystallization is a major cause of cellular damage. Conventional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol function by a number of different mechanisms but do not mitigate or control ice recrystallization at concentrations utilized in cryopreservation procedures. In North America, cryopreservation of human red blood cells (RBCs) utilizes high concentrations of glycerol. RBC units frozen under these conditions must be subjected to a time-consuming deglycerolization process after thawing in order to remove the glycerol to <1% prior to transfusion thus limiting the use of frozen RBC units in emergency situations. We have identified several low molecular mass ice recrystallization inhibitors (IRIs) that are effective cryoprotectants for human RBCs, resulting in 70-80% intact RBCs using only 15% glycerol and slow freezing rates. These compounds are capable of reducing the average ice crystal size of extracellular ice relative to a 15% glycerol control validating the positive correlation between a reduction in ice crystal size and increased post-thaw recovery of RBCs. The most potent IRI from this study is also capable of protecting frozen RBCs against the large temperature fluctuations associated with transient warming.

Glycerol uptake is by passive diffusion in the heart but by facilitated transport in RBCs at high glycerol levels in cold acclimated rainbow smelt (Osmerus mordax).

PubMed

Clow, Kathy A; Driedzic, William R

2012-04-15

Rainbow smelt (Osmerus mordax) is a small fish that accumulates glycerol at low winter seawater temperatures. In laboratory-held fish, glycerol concentration typically reaches 225 mM in plasma and in all cells. Glycerol uptake by the heart and red blood cells (RBCs) was assessed by tracking [(14)C(U)]glycerol into the acid-soluble pool. In fish acclimated to 9-10°C a decrease in perfusion/incubation temperature from 8 to 1°C resulted in a decrease in glycerol uptake with a Q(10) of 3.2 in heart and 2.4 in RBCs. Acclimation to ∼1.5°C did not result in an adaptive enhancement of glycerol uptake as rates were unchanged in heart and RBCs. Glycerol uptake at 1°C was by passive diffusion in heart as evidenced by a linear relationship between glycerol uptake and extracellular glycerol concentration and a lack of inhibition by phloretin. In contrast, in RBCs, glycerol uptake with respect to glycerol concentration showed two linear relationships with a transition point around 50 mM extracellular glycerol. The slope of the second phase was much steeper and eliminated with the inclusion of phloretin. In RBCs from Atlantic salmon (Salmo salar), a related species that does not accumulate glycerol, glycerol uptake showed only a single linear curve and was not inhibited by phloretin. The data imply a strong facilitated component to glycerol uptake in rainbow smelt RBCs at high glycerol concentrations. We propose this is related to cyclic changes in RBC glycerol content involving a loss of glycerol at the gill and a reaccumulation during passage through the liver.

Vesiculation of healthy and defective red blood cells

NASA Astrophysics Data System (ADS)

Li, He; Lykotrafitis, George

2015-07-01

Vesiculation of mature red blood cells (RBCs) contributes to removal of defective patches of the erythrocyte membrane. In blood disorders, which are related to defects in proteins of the RBC membrane, vesiculation of the plasma membrane is intensified. Several hypotheses have been proposed to explain RBC vesiculation but the exact underlying mechanisms and what determines the sizes of the vesicles are still not completely understood. In this work, we apply a two-component coarse-grained molecular dynamics RBC membrane model to study how RBC vesiculation is controlled by the membrane spontaneous curvature and by lateral compression of the membrane. Our simulation results show that the formation of small homogeneous vesicles with a diameter less than 40 nm can be attributed to a large spontaneous curvature of membrane domains. On the other hand, compression on the membrane can cause the formation of vesicles with heterogeneous composition and with sizes comparable with the size of the cytoskeleton corral. When spontaneous curvature and lateral compression are simultaneously considered, the compression on the membrane tends to facilitate formation of vesicles originating from curved membrane domains. We also simulate vesiculation of RBCs with membrane defects connected to hereditary elliptocytosis (HE) and to hereditary spherocytosis (HS). When the vertical connectivity between the lipid bilayer and the membrane skeleton is elevated, as in normal RBCs, multiple vesicles are shed from the compressed membrane with diameters similar to the cytoskeleton corral size. In HS RBCs, where the connectivity between the lipid bilayer and the cytoskeleton is reduced, larger-size vesicles are released under the same compression ratio as in normal RBCs. Lastly, we find that vesicles released from HE RBCs can contain cytoskeletal filaments due to fragmentation of the membrane skeleton while vesicles released from the HS RBCs are depleted of cytoskeletal filaments.

Changes in rubisco, cysteine-rich proteins and antioxidant system of spinach (Spinacia oleracea L.) due to sulphur deficiency, cadmium stress and their combination.

PubMed

Bagheri, Rita; Ahmad, Javed; Bashir, Humayra; Iqbal, Muhammad; Qureshi, M Irfan

2017-03-01

Sulphur (S) deficiency, cadmium (Cd) toxicity and their combinations are of wide occurrence throughout agricultural lands. We assessed the impact of short-term (2 days) and long-term (4 days) applications of cadmium (40 μg/g soil) on spinach plants grown on sulphur-sufficient (300 μM SO 4 2- ) and sulphur-deficient (30 μM SO 4 2- ) soils. Compared with the control (+S and -Cd), oxidative stress was increased by S deficiency (-S and -Cd), cadmium (+S and +Cd) and their combination stress (-S and +Cd) in the order of (S deficiency) < (Cd stress) < (S deficiency and +Cd stress). SDS-PAGE profile of leaf proteins showed a high vulnerability of rubisco large subunit (RbcL) to S deficiency. Rubisco small subunit (RbcS) was particularly sensitive to Cd as well as dual stress (+Cd and -S) but increased with Cd in the presence of S. Cysteine content in low molecular weight proteins/peptide was also affected, showing a significant increase under cadmium treatment. Components of ascorbate-glutathione antioxidant system altered their levels, showing the maximum decline in ascorbate (ASA), dehydroascorbate (DHA), total ascorbate (ASA + DHA, hereafter TA), glutathione (GSH) and total glutathione (GSH + GSSG, hereafter TG) under S deficiency. However, total ascorbate and total glutathione increased, besides a marginal increase in their reduced and oxidized forms, when Cd was applied in the presence of sufficient S. Sulphur supply also helped in increasing the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and catalase (CAT) under Cd stress. However, their activity suffered by S deficiency and by Cd stress during S deficiency. Each stress declined the contents of soluble protein and photosynthetic pigments; the highest decline in contents of protein and pigments occurred under S deficiency and dual stress respectively. The fresh and dry weights, although affected adversely by every stress, declined most under dual stress. It may be concluded that an optimal level of S is required during Cd stress for better response of SOD, APX, GR and CAT activity, as well as synthesis of cysteine. RbcS is as highly sensitive to S deficiency as RbcL is to Cd stress.

Dissipative particle dynamics simulations of deformation and aggregation of healthy and diseased red blood cells in a tube flow

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ye, Ting; Phan-Thien, Nhan, E-mail: Nhan@nus.edu.sg; Khoo, Boo Cheong

In this paper, we report simulation results assessing the deformation and aggregation of mixed healthy and malaria-infected red blood cells (RBCs) in a tube flow. A three dimensional particle model based on Dissipative Particle Dynamics (DPD) is developed to predict the tube flow containing interacting cells. The cells are also modelled by DPD, with a Morse potential to characterize the cell-cell interaction. As validation tests, a single RBC in a tube flow and two RBCs in a static flow are simulated to examine the cell deformation and intercellular interaction, respectively. The study of two cells, one healthy and the othermore » malaria-infected RBCs in a tube flow demonstrates that the malaria-infected RBC (in the leading position along flow direction) has different effects on the healthy RBC (in the trailing position) at the different stage of parasite development or at the different capillary number. With parasitic development, the malaria-infected RBC gradually loses its deformability, and in turn the corresponding trailing healthy RBC also deforms less due to the intercellular interaction. With increasing capillary number, both the healthy and malaria-infected RBCs are likely to undergo an axisymmetric motion. The minimum intercellular distance becomes small enough so that rouleaux is easily formed, i.e., the healthy and malaria-infected RBCs are difficultly disaggregated.« less

Modeling of Red Blood Cells and Related Spleen Function

NASA Astrophysics Data System (ADS)

Peng, Zhangli; Pivkin, Igor; Dao, Ming

2011-11-01

A key function of the spleen is to clear red blood cells (RBCs) with abnormal mechanical properties from the circulation. These abnormal mechanical properties may be due to RBC aging or RBC diseases, e.g., malaria and sickle cell anemia. Specifically, 10% of RBCs passing through the spleen are forced to squeeze into the narrow slits between the endothelial cells, and stiffer cells which get stuck are killed and digested by macrophages. To investigate this important physiological process, we employ three different approaches to study RBCs passage through these small slits, including analytical theory, Dissipative Particle Dynamics (DPD) simulation and Multiscale Finite Element Method (MS-FEM). By applying the analytical theory, we estimate the critical limiting geometries RBCs can pass. By using the DPD method, we study the full fluid-structure interaction problem, and compute RBC deformation under different pressure gradients. By employing the MS-FEM approach, we model the lipid bilayer and the cytoskeleton as two distinct layers, and focus on the cytoskeleton deformation and the bilayer-skeleton interaction force at the molecular level. Finally the results of these three approaches are compared to each other and correlated to the experimental observations.

Holographic analysis on deformation and restoration of malaria-infected red blood cells by antimalarial drug

NASA Astrophysics Data System (ADS)

Byeon, Hyeokjun; Ha, Young-Ran; Lee, Sang Joon

2015-11-01

Malaria parasites induce morphological, biochemical, and mechanical changes in red blood cells (RBCs). Mechanical variations are closely related to the deformability of individual RBCs. The deformation of various RBCs, including healthy and malaria-infected RBCs (iRBCs), can be directly observed through quantitative phase imaging (QPI). The effects of chloroquine treatment on the mechanical property variation of iRBCs were investigated using time-resolved holographic QPI of single live cells on a millisecond time scale. The deformabilities of healthy RBCs, iRBCs, and drug-treated iRBCs were compared, and the effect of chloroquine on iRBC restoration was experimentally examined. The present results are beneficial to elucidate the dynamic characteristics of iRBCs and the effect of the antimalarial drug on iRBCs.

Holographic analysis on deformation and restoration of malaria-infected red blood cells by antimalarial drug.

PubMed

Byeon, Hyeokjun; Ha, Young-Ran; Lee, Sang Joon

2015-11-01

Malaria parasites induce morphological, biochemical, and mechanical changes in red blood cells (RBCs). Mechanical variations are closely related to the deformability of individual RBCs. The deformation of various RBCs, including healthy and malaria-infected RBCs (iRBCs), can be directly observed through quantitative phase imaging (QPI). The effects of chloroquine treatment on the mechanical property variation of iRBCs were investigated using time-resolved holographic QPI of single live cells on a millisecond time scale. The deformabilities of healthy RBCs, iRBCs, and drug-treated iRBCs were compared, and the effect of chloroquine on iRBC restoration was experimentally examined. The present results are beneficial to elucidate the dynamic characteristics of iRBCs and the effect of the antimalarial drug on iRBCs.

Three-dimensional refractive index tomograms and deformability of individual human red blood cells from cord blood of newborn infants and maternal blood.

PubMed

Park, HyunJoo; Ahn, Taegyu; Kim, Kyoohyun; Lee, Sangyun; Kook, Song-Yi; Lee, Dongheon; Suh, In Bum; Na, Sunghun; Park, YongKeun

2015-01-01

Red blood cells (RBCs) from the cord blood of newborn infants have distinctive functions in fetal and infant development. To systematically investigate the biophysical characteristics of individual cord RBCs in newborn infants, a comparative study was performed on RBCs from the cord blood of newborn infants and from adult mothers or nonpregnant women using optical holographic microtomography. Optical measurements of the distributions of the three-dimensional refractive indices and the dynamic membrane fluctuations of individual RBCs were used to investigate the morphological, biochemical, and mechanical properties of cord, maternal, and adult RBCs at the individual cell level. The volume and surface area of the cord RBCs were significantly larger than those of the RBCs from nonpregnant women, and the cord RBCs had more flattened shapes than that of the RBCs in adults. In addition, the hemoglobin (Hb) content in the cord RBCs from newborns was significantly higher. The Hb concentration in the cord RBCs was higher than that in the nonpregnant women or maternal RBCs, but they were within the physiological range of adults. Interestingly, the amplitudes of the dynamic membrane fluctuations in cord RBCs were comparable to those in nonpregnant women and maternal RBCs, suggesting that the deformability of cord RBCs is similar to that of healthy RBCs in adults.

Rubisco Accumulation Factor 1 from Thermosynechococcus elongatus participates in the final stages of ribulose-1,5-bisphosphate carboxylase/oxygenase assembly in Escherichia coli cells and in vitro.

PubMed

Kolesinski, Piotr; Belusiak, Iwona; Czarnocki-Cieciura, Mariusz; Szczepaniak, Andrzej

2014-09-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) biosynthesis is a multi-step process in which specific chaperones are involved. Recently, a novel polypeptide, Rubisco Accumulation Factor 1 (RAF1), has been identified as a protein that is necessary for proper assembly of this enzyme in maize cells (Zea mays). However, neither its specific function nor its mode of action have as yet been determined. The results presented here show that the prokaryotic homolog of RAF1 from Thermosynechococcus elongatus is expressed in cyanobacterial cells and interacts with a large Rubisco subunit (RbcL). Using a heterologous expression system, it was demonstrated that this protein promotes Rubisco assembly in Escherichia coli cells. Moreover, when co-expressed with RbcL alone, a stable RbcL-RAF1 complex is formed. Molecular mass determination for this Rubisco assembly intermediate by size-exclusion chromatography coupled with multi-angle light scattering indicates that it consists of an RbcL dimer and two RAF1 molecules. A purified RbcL-RAF1 complex dissociated upon addition of a small Rubisco subunit (RbcS), leading to formation of the active holoenzyme. Moreover, titration of the octameric (RbcL8) core of Rubisco with RAF1 results in disassembly of such a stucture and creation of an RbcL-RAF1 intermediate. The results presented here are the first attempt to elucidate the role of cyanobacterial Rubisco Accumulation Factor 1 in the Rubisco biosynthesis process. © 2014 FEBS.

A Small Decrease in Rubisco Content by Individual Suppression of RBCS Genes Leads to Improvement of Photosynthesis and Greater Biomass Production in Rice Under Conditions of Elevated CO2.

PubMed

Kanno, Keiichi; Suzuki, Yuji; Makino, Amane

2017-03-01

Rubisco limits photosynthesis at low CO2 concentrations ([CO2]), but does not limit it at elevated [CO2]. This means that the amount of Rubisco is excessive for photosynthesis at elevated [CO2]. Therefore, we examined whether a small decrease in Rubisco content by individual suppression of the RBCS multigene family leads to increases in photosynthesis and biomass production at elevated [CO2] in rice (Oryza sativa L.). Our previous studies indicated that the individual suppression of RBCS decreased Rubisco content in rice by 10-25%. Three lines of BC2F2 progeny were selected from transgenic plants with individual suppression of OsRBCS2, 3 and 5. Rubisco content in the selected lines was 71-90% that of wild-type plants. These three transgenic lines showed lower rates of CO2 assimilation at low [CO2] (28 Pa) but higher rates of CO2 assimilation at elevated [CO2] (120 Pa). Similarly, the biomass production and relative growth rate (RGR) of the two lines were also smaller at low [CO2] but greater than that of wild-type plants at elevated [CO2]. This greater RGR was caused by the higher net assimilation rate (NAR). When the nitrogen use efficiency (NUE) for the NAR was estimated by dividing the NAR by whole-plant leaf N content, the NUE for NAR at elevated [CO2] was higher in these two lines. Thus, a small decrease in Rubisco content leads to improvements of photosynthesis and greater biomass production in rice under conditions of elevated CO2. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Platelet Transfusion – the Art and Science of Compromise

PubMed Central

Cid, Joan; Harm, Sarah K.; Yazer, Mark H.

2013-01-01

Summary Many modern therapies depend on platelet (PLT) transfusion support. PLTs have a 4- to 7-day shelf life and are frequently in short supply. In order to optimize the inventory PLTs are often transfused to adults without regard for ABO compatibility. Hemolytic reactions are infrequent despite the presence of ‘high titer’ anti-A and anti-B antibodies in some of the units. Despite the low risk for hemolysis, some centers provide only ABO identical PLTs to their recipients; this practice might have other beneficial outcomes that remain to be proven. Strategies to mitigate the risk of hemolysis and the clinical and laboratory outcomes following ABO-matched and mismatched transfusions will be discussed. Although the PLTs themselves do not carry the D antigen, a small number of RBCs are also transfused with every PLT dose. The quantity of RBCs varies by the type of PLT preparation, and even a small quantity of D+ RBCs can alloimmunize a susceptible D− host. Thus PLT units are labeled as D+/–, and most transfusion services try to prevent the transfusion of D+ PLTs to D– females of childbearing age. A similar policy for patients with hematological diseases is controversial, and the elements and mechanisms of anti-D alloimmunization will be discussed. PMID:23922541

Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model.

PubMed

Shah, Sandeep N; Gelderman, Monique P; Lewis, Emily M A; Farrel, John; Wood, Francine; Strader, Michael Brad; Alayash, Abdu I; Vostal, Jaroslav G

2016-01-01

Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time, known as 'the storage lesion'. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product.

Subunit association of gamma-glutamyltranspeptidase of Escherichia coli K-12.

PubMed

Hashimoto, W; Suzuki, H; Nohara, S; Tachi, H; Yamamoto, K; Kumagai, H

1995-12-01

gamma-Glutamyltranspeptidase [EC 2.3.2.2] of Escherichia coli K-12 consists of one large subunit and one small subunit, which can be separated from each other by high-performance liquid chromatography. Using ion spray mass spectrometry, the masses of the large and the small subunit were determined to be 39,207 and 20,015, respectively. The large subunit exhibited no gamma-glutamyltranspeptidase activity and the small subunit had little enzymatic activity, but a mixture of the two subunits showed partial recovery of the enzymatic activity. The results of native-polyacrylamide gel electrophoresis suggested that they could partially recombine, and that the recombined dimer exhibited enzymatic activity. The gene of gamma-glutamyltranspeptidase encoded a signal peptide, and the large and small subunits in a single open reading frame in that order. Two kinds of plasmid were constructed encoding the signal peptide and either the large or the small subunit. A gamma-glutamyltranspeptidase-less mutant of E. coli K-12 was transformed with each plasmid or with both of them. The strain harboring the plasmid encoding each subunit produced a small amount of the corresponding subunit protein in the periplasmic space but exhibited no enzymatic activity. The strain transformed with both plasmids together exhibited the enzymatic activity, but its specific activity was approximately 3% of that of a strain harboring a plasmid encoding the intact structural gene. These results indicate that a portion of the separated large and small subunits can be reconstituted in vitro and exhibit the enzymatic activity, and that the expressed large and small subunits independently are able to associate in vivo and be folded into an active structure, though the specific activity of the associated subunits was much lower than that of native enzyme. This suggests that the synthesis of gamma-glutamyltranspeptidase in a single precursor polypeptide and subsequent processing are more effective to construct the intact structure of gamma-glutamyltranspeptidase than the association of the separated large and small subunits.

Mechanosensing Dynamics of Red blood Cells

NASA Astrophysics Data System (ADS)

Wan, Jiandi

2015-11-01

Mechanical stress-induced deformation of human red blood cells (RBCs) plays important physiopathological roles in oxygen delivery, blood rheology, transfusion, and malaria. Recent studies demonstrate that, in response to mechanical deformation, RBCs release adenosine-5'-triphosphate (ATP), suggesting the existence of mechanotransductive pathways in RBCs. Most importantly, the released ATP from RBCs regulates vascular tone and impaired release of ATP from RBCs has been linked to diseases such as type II diabetes and cystic fibrosis. To date, however, the mechanisms of mechanotransductive release of ATP from RBCs remain unclear. Given that RBCs experience shear stresses continuously during the circulation cycle and the released ATP plays a central role in vascular physiopathology, understanding the mechanotransductive release of ATP from RBCs will provide not only fundamental insights to the role of RBCs in vascular homeostasis but also novel therapeutic strategies for red cell dysfunction and vascular disease. This talk describes the main research in my group on integrating microfluidic-based approaches to study the mechanosensing dynamics of RBCs. Specifically, I will introduce a micro?uidic approach that can probe the dynamics of shear-induced ATP release from RBCs with millisecond resolution and provide quantitative understandings of the mechanosensitive ATP release processes in RBCs. Furthermore, I will also describe our recent findings about the roles of the Piezo1 channel, a newly discovered mechanosensitive cation channel in the mechanotransductive ATP release in RBCs. Last, possible functions of RBCs in the regulation of cerebral blood flow will be discussed.

Association of Plasmodium falciparum with Human Endothelial Cells in vitro

PubMed Central

Utter, Christopher; Serrano, Adelfa E.; Glod, John W.; Leibowitz, Michael J.

2017-01-01

Endothelial abnormalities play a critical role in the pathogenesis of malaria caused by the human pathogen, Plasmodium falciparum. In serious infections and especially in cerebral malaria, red blood cells infected with the parasite are sequestered in small venules in various organs, resulting in endothelial activation and vascular occlusion, which are believed to be largely responsible for the morbidity and mortality caused by this infection, especially in children. We demonstrate that after incubation with infected red blood cells (iRBCs), cultured human umbilical vein endothelial cells (HUVECs) contain parasite protein, genomic DNA, and RNA, as well as intracellular vacuoles with apparent parasite-derived material, but not engulfed or adherent iRBCs. The association of this material with the HUVECs is observed over 96 hours after removal of iRBCs. This phenomenon may occur in endothelial cells in vivo by the process of trogocytosis, in which transfer of material between cells depends on direct cell contact. This process may contribute to the endothelial activation and disruption involved in the pathogenesis of cerebral malaria. PMID:28656007

Vesicle dynamics in a confined Poiseuille flow: from steady state to chaos.

PubMed

Aouane, Othmane; Thiébaud, Marine; Benyoussef, Abdelilah; Wagner, Christian; Misbah, Chaouqi

2014-09-01

Red blood cells (RBCs) are the major component of blood, and the flow of blood is dictated by that of RBCs. We employ vesicles, which consist of closed bilayer membranes enclosing a fluid, as a model system to study the behavior of RBCs under a confined Poiseuille flow. We extensively explore two main parameters: (i) the degree of confinement of vesicles within the channel and (ii) the flow strength. Rich and complex dynamics for vesicles are revealed, ranging from steady-state shapes (in the form of parachute and slipper shapes) to chaotic dynamics of shape. Chaos occurs through a cascade of multiple periodic oscillations of the vesicle shape. We summarize our results in a phase diagram in the parameter plane (degree of confinement and flow strength). This finding highlights the level of complexity of a flowing vesicle in the small Reynolds number where the flow is laminar in the absence of vesicles and can be rendered turbulent due to elasticity of vesicles.

Propensity of red blood cells to undergo P2X7 receptor-mediated phosphatidylserine exposure does not alter during in vivo or ex vivo aging.

PubMed

Sophocleous, Reece A; Mullany, Phillip R F; Winter, Kelly M; Marks, Denese C; Sluyter, Ronald

2015-08-01

Phosphatidylserine (PS) exposure facilitates the removal of red blood cells (RBCs) from the circulation, potentially contributing to the loss of stored RBCs after transfusion, as well as senescent RBCs. Activation of the P2X7 receptor by extracellular adenosine 5'-triphosphate (ATP) can induce PS exposure on freshly isolated human RBCs, but whether this process occurs in stored RBCs or changes during RBC aging is unknown. RBCs were processed and stored according to Australian blood banking guidelines. PS exposure was determined by annexin V binding and flow cytometry. Efficacy of P2X antagonists was assessed by flow cytometric measurements of ATP-induced ethidium+ uptake in RPMI 8226 cells. Osmotic fragility was assessed by lysis in hypotonic saline. RBCs were fractionated by discontinuous density centrifugation. ATP (1 mmol/L) induced PS exposure on RBCs stored for less than 1 week. This process was near-completely inhibited by the P2X7 antagonists A438079 and AZ10606120 and the P2X1/P2X7 antagonist MRS2159 but not the P2X1 antagonist NF499. ATP-induced PS exposure on RBCs was not dependent on K+, Na+, or Cl- fluxes. ATP did not alter the osmotic fragility of stored RBCs. ATP-induced PS exposure was similar between RBCs of different densities. ATP-induced PS exposure was also similar between RBCs stored for less than 1 week or for 6 weeks. The propensity of RBCs to undergo P2X7-mediated PS exposure does not alter during in vivo and ex vivo aging. Thus, P2X7 activation is unlikely to be involved in the removal of senescent RBCs or stored RBCs after transfusion. © 2015 AABB.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jack Preiss

Conversion of the Potato tuber ADP-glucose Pyrophopshorylase Regulatory Subunit into a Catalytic Subunit. ADP-glucose synthesis, a rate-limiting reaction in starch synthesis, is catalyzed by ADP-glucose pyrophosphorylase (ADPGlc PPase). The enzyme in plants is allosterically activated by 3-phosphoglycerate (3PGA) and inhibited by inorganic phosphate (Pi) and is composed of two subunits as a heterotetramer, a2b2. Subunit a is the catalytic subunit and subunit b is designated as the regulatory subunit.The b subunit increases the affinty of the activator for the catalytic subunit. Recent results have shown that the subunits are derived from the same ancestor subunit as the regulatory subunit canmore » be converted to a catalytically subunit via mutation of just two amino acids. Lys44 and Thr54 in the large subunit from potato tuber were converted to the homologous catalytic subunit residues, Arg33 and Lys43. The activity of the large subunit mutants cannot be readily tested with a co-expressed wild-type small (catalytic) subunit because of the intrinsic activity of the latter. We co-expressed the regulatory-subunit mutants with SmallD145N, an inactive S subunit in which the catalytic Asp145 was mutated. The activity of the small (catalytic) subunit was reduced more than three orders of magnitude. Coexpression of the L subunit double mutant LargeK44R/T54K with SmallD145N generated an enzyme with considerable activity, 10% and 18% of the wildtype enzyme, in the ADP-glucose synthetic and pyrophosphorolytic direction, respectively. Replacement of those two residues in the small subunit by the homologous amino acids in the L subunits (mutations R33K and K43T) decreased the activity one and two orders of magnitude. The wild-type enzyme and SmallD145NLargeK44R/T54K had very similar kinetic properties indicating that the substrate site has been conserved. The fact that only two mutations in the L subunit restored enzyme activity is very strong evidence that the large subunit is derived from the catalytic ancestor. Previous results showed that Asp145 in the small subunit of the wild-type is essential for catalysis, whereas the homologous Asp160 in the Large WT subunit is not. However, in this study, mutation D160N or D160E in the LK44R/T54K subunit abolished the activity, which shows the ancestral essential role of this residue and confirms that the catalysis of SmallD145NLarge K44R/T54K occurs in the L(b) subunit. A phylogenetic tree of the ADP-Glc PPases present in photosynthetic eukaryotes also sheds information about the origin of the subunits. The tree showed that plant Small and Large subunits can be divided into two and four distinct groups, respectively. The two main groups of S subunits are from dicot and monocot plants, whereas Large subunit groups correlate better with their documented tissue expression. The first Large-subunit group is generally expressed in photosynthetic tissues and comprises Large subunits from dicots and monocots. Group II displays a broader expression pattern, whereas groups III and IV are expressed in storage organs (roots, stems, tubers, seeds). Subunits from group III are only from dicot plants, whereas group IV are seed-specific subunits from monocots. These last two groups stem from the same branch of the phylogenetic tree and split before monocot and dicot separation. Thus few as two mutations turned the L subunit from Solanum tuberosum catalytic, showing that L and S subunits share a common catalytic ancestor, rather than a non-catalytic one. The L subunit evolved to have a regulatory role, lost catalytic residues more than 130 million years ago before monocots and dicots diverged, and preserved, possibly as a byproduct, the active site domain.« less

[The modification of electrophysiology affected by ectopic synapse in ON-retinal bipolar cells of RCS rats].

PubMed

Zhang, Chen-xing; Yin, Zheng-qin; Weng, Chuan-huang; Zeng, Yu-xiao

2011-03-01

To study the influence of the ectopic synapse for electrophysiological characteristics modification in ON retinal bipolar cells (ON-RBCs) of RCS rat. Immunofluorescence of the retinal frozen section was taken in P60 d, P90 d of RCS rat (RCS) and control rat (CTR) with the anti-mGluR6 and anti-Synaptophysin, Lucifer Yellow staining solo ON-RBCs was taken in all the group. The whole cell recording was performed in the retinal slice of P60 d, P90 d in RCS and CTR. The modification of the passive membrane properties and the outward currents properties in RCS-ON-RBCs, CTR-ON-RBCs and CTR-OFF-RBCs were observed. RCS-ON-RBCs stretched out the ectopic neurite in different direction and the activity of synapse could be detected around the ectopic neurite. From Pn60d, passive membrane properties of RCS-ON-RBCs kept immature, The RMP in RCS-ON-RBCs and CTR-ON-RBCs were (-61.8 ± 3.07), (-50.44 ± 1.36) mV and (-63.1 ± 2.59), (-48.37 ± 3.69) mV when P60 d and P90 d, there ware significantly higher than CTR group (t = 2.191, 2.435, 5.817, 6.912;P < 0.05). The IR in RCS-ON-RBCs and CTR-ON-RBCs were (323.3 ± 42.6), (337.6 ± 71.3) MΩ and (321.2 ± 58.6), (340.3 ± 62.8) MΩ when P60 d and P90 d, there ware significantly higher than CTR group (t = 3.561, 1.987, 5.211, 4.034; P < 0.05). Outward currents were recorded when giving hyper- and depolarized voltage steps. In retinal degeneration, the amplitude of outward currents in RCS-ON-RBCs is significantly different with CTR-ON-RBCs (t = 5.561, 6.341; P < 0.05) or CTR-OFF-RBCs (t = 5.357, 6.997; P < 0.05). The ectopic neurite from RCS-ON-RBCs has the possibility for translating the signal. In retinitis pigmentosa, the modification of electrophysiology characteristics in RCS-ON-RBCs was significantly different with CTR-ON-RBCs and CTR-OFF-RBCs. Influence with the ectopic neurite is the possible cause.

Automated red blood cells extraction from holographic images using fully convolutional neural networks.

PubMed

Yi, Faliu; Moon, Inkyu; Javidi, Bahram

2017-10-01

In this paper, we present two models for automatically extracting red blood cells (RBCs) from RBCs holographic images based on a deep learning fully convolutional neural network (FCN) algorithm. The first model, called FCN-1, only uses the FCN algorithm to carry out RBCs prediction, whereas the second model, called FCN-2, combines the FCN approach with the marker-controlled watershed transform segmentation scheme to achieve RBCs extraction. Both models achieve good segmentation accuracy. In addition, the second model has much better performance in terms of cell separation than traditional segmentation methods. In the proposed methods, the RBCs phase images are first numerically reconstructed from RBCs holograms recorded with off-axis digital holographic microscopy. Then, some RBCs phase images are manually segmented and used as training data to fine-tune the FCN. Finally, each pixel in new input RBCs phase images is predicted into either foreground or background using the trained FCN models. The RBCs prediction result from the first model is the final segmentation result, whereas the result from the second model is used as the internal markers of the marker-controlled transform algorithm for further segmentation. Experimental results show that the given schemes can automatically extract RBCs from RBCs phase images and much better RBCs separation results are obtained when the FCN technique is combined with the marker-controlled watershed segmentation algorithm.

Automated red blood cells extraction from holographic images using fully convolutional neural networks

PubMed Central

Yi, Faliu; Moon, Inkyu; Javidi, Bahram

2017-01-01

In this paper, we present two models for automatically extracting red blood cells (RBCs) from RBCs holographic images based on a deep learning fully convolutional neural network (FCN) algorithm. The first model, called FCN-1, only uses the FCN algorithm to carry out RBCs prediction, whereas the second model, called FCN-2, combines the FCN approach with the marker-controlled watershed transform segmentation scheme to achieve RBCs extraction. Both models achieve good segmentation accuracy. In addition, the second model has much better performance in terms of cell separation than traditional segmentation methods. In the proposed methods, the RBCs phase images are first numerically reconstructed from RBCs holograms recorded with off-axis digital holographic microscopy. Then, some RBCs phase images are manually segmented and used as training data to fine-tune the FCN. Finally, each pixel in new input RBCs phase images is predicted into either foreground or background using the trained FCN models. The RBCs prediction result from the first model is the final segmentation result, whereas the result from the second model is used as the internal markers of the marker-controlled transform algorithm for further segmentation. Experimental results show that the given schemes can automatically extract RBCs from RBCs phase images and much better RBCs separation results are obtained when the FCN technique is combined with the marker-controlled watershed segmentation algorithm. PMID:29082078

In vitro measures of membrane changes reveal differences between red blood cells stored in SAGM and AS-1 additive solutions: a paired study

PubMed Central

Sparrow, Rosemary L; Sran, Amrita; Healey, Geraldine; Veale, Margaret F; Norris, Philip J

2014-01-01

Background Saline-Adenine-Glucose-Mannitol (SAGM) and a variant solution, AS-1 have been used for over 30 years to preserve red blood cells (RBCs). Reputedly these RBC components have similar quality, although no paired study has been reported. To determine whether differences exist, a paired study of SAGM-RBCs and AS-1-RBCs was conducted to identify membrane changes, including microparticle (MP) quantitation and in vitro RBC-endothelial cell (EC) interaction. Study Design and Methods Two whole blood packs were pooled-and-split and RBCs prepared (n=6 pairs). One pack was suspended in SAGM and one in AS-1. Samples were collected during 42 days of refrigerated storage. RBC shape/size, glycophorin A (GPA)+ and phosphatidylserine (PS)+ MPs were measured by flow cytometry. RBC adhesion to ECs was determined by an in vitro flow perfusion assay. Routine parameters (pH, hemolysis) were also measured. Results Compared to SAGM-RBCs, AS-1-RBCs had lower hemolysis (p<0.04), lower GPA+ MPs (p<0.03) and lower PS+ MPs (p<0.03) from day 14 onwards. AS-1-RBCs had higher (p<0.02) side scatter from day 28 onwards, compared to SAGM-RBCs. SAGM-RBCs were more adherent to ECs at day 28 of storage compared to AS-1 RBCs (p=0.04), but reversed at day 42 (p=0.02). No significant differences in forward scatter or pH were found. Conclusion SAGM-RBCs lose more membrane during storage. SAGM-RBCs had increased adherence to ECs at day 28 of storage, while AS-1-RBCs were more adherent at day 42. The effect of these differences on the function and survival of SAGM-RBCs and AS-1-RBCs following transfusion remains to be determined. PMID:23869602

ON-retinal bipolar cell survival in RCS rats.

PubMed

Zhang, Chen Xing; Yin, Zheng Qin; Chen, Li-Feng; Weng, Chuang-Huang; Zeng, Yu-Xiao

2010-11-01

In retinitis pigmentosa (RP), the slow and progressive death of inner retinal neurons is thought to be inevitable after the death of photoreceptors. However, even in the advanced stage of RP, all inner retinal neurons are not completely lost. The morphological and electrophysiological modifications in ON-retinal bipolar cells (ON-RBCs) of Royal College of Surgeons (RCS) rats (RCS-ON-RBCs) were investigated to elucidate the mechanisms of survival of RCS-ON-RBCs in RP. Control (CTR) and RCS rats were divided into age groups according to postnatal stage: postnatal day 21 (Pn21d), postnatal day 30 (Pn30d), postnatal day 60 (Pn60d), and postnatal day 90 (Pn90d). Lucifer yellow staining of single ON-RBCs and double-immunofluorescence of the retinal frozen sections were used to detect the morphological modifications and loss of RCS-ON-RBCs in different retinal regions. The whole-cell patch clamping technique was used to record the electrophysiological properties of ON-RBCs. There was a significant loss of RCS-ON-RBCs compared with CTR (p < 0.01) at Pn60d. Loss of the RCS-ON-RBCs differed by region. From Pn60d onwards, the loss was more severe in the peripheral retinal regions (p < 0.01). From Pn21d, the ectopic neurites from the RCS-ON-RBCs reached the outer and inner nuclear layers. At Pn60d, terminal branches of RCS-ON-RBCs axons vanished and ectopic neurites from the RCS-ON-RBCs became entwined. The resting membrane potential, input resistance and outward membrane current amplitude of RCS-ON-RBCs were significantly higher than those of the ON-RBCs of CTR rats at Pn60d (p < 0.05). Our results indicate that more RCS-ON-RBCs survived in the central retinal area near cone clusters, potentially as a result of ectopic neuritis. Meanwhile the surviving RCS-ON-RBCs remained immature and had no normal electrophysiological characteristics.

Red blood cells inhibit activation-induced cell death and oxidative stress in human peripheral blood T lymphocytes.

PubMed

Fonseca, A M; Porto, G; Uchida, K; Arosa, F A

2001-05-15

Red blood cells (RBCs) are known to perform one prominent function: to carry and deliver oxygen to the tissues. Earlier studies, however, suggested a role for RBCs in potentiating T-cell proliferation in vitro. Here it is shown that the presence of RBCs in cultures of stimulated human peripheral blood lymphocytes strengthens T-cell proliferation and survival. Analysis of phosphatidylserine externalization and DNA fragmentation showed that RBCs inhibit T-cell apoptosis. This inhibition correlated with a reduction in CD71 but not CD95 expression. RBCs enhanced T-cell proliferation and survival upon activation with phytohemagglutinin and with OKT3 antibodies. Studies aimed at characterizing the cellular and molecular basis of the protection afforded to T cells by RBCs showed that (1) optimal protection required intact RBCs and red cell/T-cell contact but not monocytes; (2) RBCs markedly reduced the level of intracellular reactive oxygen species; and (3) RBCs inhibited the formation of protein-bound acrolein, a peroxidation adduct in biologic systems. Overall, these data indicate that human RBCs protect T cells from activation-induced cell death, at least in part by reducing the pro-oxidant state, and suggest a role for RBCs as conceivable modulators of T-cell homeostasis.

Case report: massive postpartum transfusion of Jr(a+) red cells in the presence of anti-Jra.

PubMed

Yuan, S; Armour, R; Reid, A; Abdel-Rahman, K F; Rumsey, D M; Phillips, M; Nester, T

2005-01-01

Jr(a) is a high-prevalence antigen. The rare Jr(a-) individuals can form anti-Jr(a) after exposure to the Jr(a) antigen through transfusion or pregnancy. The clinical significance of anti-Jr(a) is not well established. This study reports a case of a 31-year-old woman with a previously identified anti-Jr(a) who required massive transfusion of RBCs after developing life-threatening postpartum disseminated intravascular coagulopathy. Despite the emergent transfusion of 15 units of Jr(a) untested RBCs, she did not develop laboratory or clinical evidence of acute hemolysis. The patient's anti-Jr(a) had a pretransfusion titer of 4 and a monocyte monolayer assay (MMA) reactivity of 68.5% (reactivity > 5% is considered capable of shortening the survival of incompatible RBCs). The titer increased fourfold to 64 and the MMA reactivity was 72.5% on Day 10 posttransfusion. Review of laboratory data showed evidence of a mild delayed hemolytic transfusion reaction by Day 10 posttransfusion. Despite rare reports of hemolytic transfusion reactions due to anti-Jr(a) in the literature, most cases, including this one, report that this antibody is clinically insignificant or causes only mild delayed hemolysis. Clinicians should be advised to balance the risks of withholding transfusion with the small chance of significant hemolysis after transfusion of Jr(a+) RBCs in the presence of anti-Jr(a).

Recovery of autologous sickle cells by hypotonic wash.

PubMed

Wilson, Emily; Kezeor, Kelly; Crosby, Monica

2018-01-01

It is important to isolate autologous red blood cells (RBCs) from transfused RBCs in samples from recently transfused patients to ensure that accurate serologic results are obtained. Typically, this isolation can be performed using methods that separate patient reticulocytes from transfused, older donor RBCs. Patients with sickle cell disease (SCD), however, characteristically have RBCs with altered membrane and morphological features, causing their RBCs to take on a sickle-shape appearance different from the biconcave disc-shape appearance of "normal" RBCs. These characteristics enable the use of hypotonic saline solution to lyse normal RBCs while allowing "sickle cells" to remain intact. Because many patients with SCD undergo frequent transfusions to treat their condition, the use of hypotonic saline solution provides a rapid method to obtain autologous RBCs for serologic testing from this patient population using standard laboratory equipment and supplies.

Accelerated removal of antibody-coated red blood cells from the circulation is accurately tracked by a biotin label

PubMed Central

Mock, Donald M.; Lankford, Gary L.; Matthews, Nell I.; Burmeister, Leon F.; Kahn, Daniel; Widness, John A.; Strauss, Ronald G.

2013-01-01

BACKGROUND Safe, accurate methods to reliably measure circulating red blood cell (RBC) kinetics are critical tools to investigate pathophysiology and therapy of anemia, including hemolytic anemias. This study documents the ability of a method using biotin-labeled RBCs (BioRBCs) to measure RBC survival (RCS) shortened by coating with a highly purified monomeric immunoglobulin G antibody to D antigen. STUDY DESIGN AND METHODS Autologous RBCs from 10 healthy D+ subjects were labeled with either biotin or 51Cr (reference method), coated (opsonized) either lightly (n = 4) or heavily (n = 6) with anti-D, and transfused. RCS was determined for BioRBCs and for 51Cr independently as assessed by three variables: 1) posttransfusion recovery at 24 hours (PTR24) for short-term RCS; 2) time to 50% decrease of the label (T50), and 3) mean potential life span (MPL) for long-term RCS. RESULTS BioRBCs tracked both normal and shortened RCS accurately relative to 51Cr. For lightly coated RBCs, mean PTR24, T50, and MPL results were not different between BioRBCs and 51Cr. For heavily coated RBCs, both short-term and long-term RCS were shortened by approximately 17 and 50%, respectively. Mean PTR24 by BioRBCs (84 ± 18%) was not different from 51Cr (81 ± 10%); mean T50 by BioRBCs (23 ± 17 days) was not different from 51Cr (22 ± 18 days). CONCLUSION RCS shortened by coating with anti-D can be accurately measured by BioRBCs. We speculate that BioRBCs will be useful for studying RCS in conditions involving accelerated removal of RBCs including allo- and autoimmune hemolytic anemias. PMID:22023312

Impaired ATP release from red blood cells promotes their adhesion to endothelial cells: A mechanism of hypoxemia after transfusion

PubMed Central

Zhu, Hongmei; Zennadi, Rahima; Xu, Bruce X.; Eu, Jerry P.; Torok, Jordan A.; Telen, Marilyn J.; McMahon, Timothy J.

2011-01-01

Objective Transfusion of red blood cells (RBCs) has been linked to disappointing clinical outcomes in the critically ill, but specific mechanisms of organ dysfunction after transfusion remain poorly understood. We tested the hypothesis that RBC storage impairs the ability of RBCs to release ATP and that impaired ATP-release was injurious in vivo, in part through increased RBC adhesion. Design Prospective, controlled, mechanistic study. Setting University research laboratory. Subjects Human and mouse blood donors; nude mouse transfusion recipients. Interventions Manipulation of ATP release, supplemental ATP, and antibodies to RBC and endothelial adhesion receptors were used in vitro and in vivo to probe the roles of released ATP and adhesion in responses to (transfused) RBCs. Measurements and main results The ability of stored RBCs to release ATP declined markedly within 14 days after collection, despite relatively stable levels of ATP within the RBCs. Inhibiting ATP release promoted the adhesion of stored RBCs to endothelial cells in vitro and RBC sequestration in the lungs of transfused mice in vivo. Unlike transfusion of fresh human RBCs, stored-RBC transfusion in mice decreased blood oxygenation and increased extravasation of RBCs into the lung’s alveolar airspaces. Similar findings were seen with transfusion of fresh RBCs treated with the ATP-release inhibitors glibenclamide and carbenoxolone. These findings were prevented by either co-infusion of an ATP analog or pre-transfusion incubation of the RBCs with an antibody against the erythrocyte adhesion receptor LW (Landsteiner-Wiener; ICAM-4). Conclusions The normal flow of RBCs in pulmonary microvessels depends in part on the release of anti-adhesive ATP from RBCs, and storage-induced deficiency in ATP release from transfused RBCs may promote or exacerbate microvascular pathophysiology in the lung, in part through increased RBC adhesion. PMID:21765360

Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

NASA Astrophysics Data System (ADS)

Wu, Jinxia; Hu, Zhangli; Wang, Chaogang; Li, Shuangfei; Lei, Anping

2008-08-01

To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein (GFP) was expressed in C. reinhardtii under the control of promoters: RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae: RBCS2 mediated strain Tran-I and HSP70A-RBCS2 mediated strain Tran-II. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-II was at least double of that in Tran-I. In addition, a threefold increase of GFP in Tran-II was induced by heat shock at 40°C. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.

Oxygen carrying capacity of salvaged blood in patients undergoing off-pump coronary artery bypass grafting surgery: a prospective observational study.

PubMed

Li, Xiu Liang; Dong, Peng; Tian, Ming; Ni, Jia Xiang; Smith, Fang Gao

2015-10-14

Intraoperative cell salvage (ICS), hereby referred to 'mechanical red cell salvage', has been widely used and proven to be an effective way to reduce or avoid the need for allogeneic red blood cells (RBCs)transfusion and its associated complications in surgeries involving major blood loss. However, little is known about the influence of this technique on the functional state of salvaged RBCs. Furthermore, there are no articles that describe the change of free hemoglobin (fHb) in salvage blood during storage, which is a key index of the quality control of salvaged blood. Therefore, in this study, the influence of ICS on the function of salvaged RBCs and the changes of salvaged RBCs during storage were studied with respect to the presence of oxyhemoglobin affinity (recorded as a P50 value) and the level of 2, 3-diphosphoglycerate (2, 3-DPG) and fHb by comparing salvaged RBCs with self-venous RBCs and 2-week-old packed RBCs. Fifteen patients undergoing off-pump coronary artery bypass grafting (OPCAB) surgery were enrolled. Blood was collected and processed using a Dideco Electa device. The level of P50, 2, 3-DPG and fHB from salvaged RBCs, venous RBCs and 2-week-old packed RBCs was measured. We also measured the changes of these indicators among salvaged RBCs at 4 h (storage at 21-24 °C) and at 24 h (storage at 1-6 °C). The P50 value of salvaged RBCs at 0 h (28.77 ± 0.27 mmHg) was significantly higher than the value of venous RBCs (27.07 ± 0.23 mmHg, p=0.000) and the value of the 2-week-old packed RBCs (16.26 ± 0.62 mmHg, p=0.000). P50 value did not change obviously at 4 h (p=0.121) and 24 h (p=0.384) compared with the value at 0 h. The 2, 3-DPG value of salvaged RBCs at 0 h (17.94 ± 6.91 μmol/g Hb) was significantly higher than the value of venous RBCs (12.73 ± 6.52 mmHg, p = 0.007) and the value of the 2-week-old packed RBCs (2.62 ± 3.13 mmHg, p=0.000). The level of 2, 3-DPG slightly decreased at 4 h (p=0.380) and 24 h (p=0.425) compared with the value at 0 h. Percentage of hemolysis of the salvaged blood at 0 h(0.51 ± 0.27 %) was significantly higher than the level of venous blood (0.07 ± 0.05 %, p=0.000) and the value of 2-week-old packed RBCs (0.07 ± 0.05 %, p=0.000), and reached 1.11 ± 0.42 % at 4 h (p=0.002) and 1.83 ± 0.77 % at 24 h (p=0.000). The oxygen transport function of salvaged RBCs at 0 h was not influenced by the cell salvage process and was better than that of the venous RBCs and 2-week-old packed RBCs. At the end of storage, the oxygen transport function of salvaged RBCs did not change obviously, but percentage of hemolysis significantly increased.

The sensing of poorly deformable red blood cells by the human spleen can be mimicked in vitro

PubMed Central

Deplaine, Guillaume; Safeukui, Innocent; Jeddi, Fakhri; Lacoste, François; Brousse, Valentine; Perrot, Sylvie; Biligui, Sylvestre; Guillotte, Micheline; Guitton, Corinne; Dokmak, Safi; Aussilhou, Béatrice; Sauvanet, Alain; Cazals Hatem, Dominique; Paye, François; Thellier, Marc; Mazier, Dominique; Milon, Geneviève; Mohandas, Narla; Mercereau-Puijalon, Odile; David, Peter H.

2011-01-01

Retention of poorly deformable red blood cells (RBCs) by the human spleen has been recognized as a critical determinant of pathogenesis in hereditary spherocytosis, malaria, and other RBC disorders. Using an ex vivo perfusion system, we had previously shown that retention of Plasmodium falciparum–infected RBCs (Pf-RBCs) occur in the splenic red pulp, upstream from the sinus wall. To experimentally replicate the mechanical sensing of RBCs by the splenic microcirculation, we designed a sorting device where a mixture of 5- to 25-μm-diameter microbeads mimics the geometry of narrow and short interendothelial splenic slits. Heated RBCs, Pf-RBCs, and RBCs from patients with hereditary spherocytosis were retained in the microbead layer, without hemolysis. The retention rates of Pf-RBCs were similar in microbeads and in isolated perfused human spleens. These in vitro results directly confirm the importance of the mechanical sensing of RBCs by the human spleen. In addition, rigid and deformable RBC subpopulations could be separated and characterized at the molecular level, and the device was used to deplete a stored RBC population from its subpopulation of rigid RBCs. This experimental approach may contribute to a better understanding of the role of the spleen in the pathogenesis of inherited and acquired RBC disorders. PMID:21163923

Anti-Uz found in mother's serum and child's eluate.

PubMed

Read, S M; Taylor, M M; Reid, M E; Popovsky, M A

1993-01-01

A saline-reactive antibody, anti-Uz, that reacted stronger with S+ than with S- red blood cells (RBCs) and failed to react with U- or ficin-treated RBCs has been previously reported. We describe an antibody of similar specificity in the postpartum serum of an untransfused woman and the eluate from her fourth child's cord RBCs. The mother's RBCs typed S-s+U+, He+(weak), and appeared to have normal glycophorin A and B content, as deter- mined by immunoblotting. The direct antiglobulin test (DAT) and the autocontrol were negative. Her serum reacted stronger with S + RBCs only in the antiglobulin phase, and failed to react with U- or ficin-treated RBCs. The antibody was adsorbed completely by S-s+U+ RBCs, proving that anti-S was not present. Monocyte monolayer assay results with S+s-U+ and S-s+U+ RBCs indicated that transfusion of incompatible blood would not result in significant hemolysis. The child's cord RBCs typed S-s+. The DAT was 3+ with anti-IgG, and an eluate prepared from these RBCs had the same reactivity as the maternal serum. The child showed no clinical signs of hemolytic disease of the newborn. In contrast to previous reports, these results suggest an immune form of anti-Uz.

The Effect of Polymeric Nanoparticles on Biocompatibility of Carrier Red Blood Cells

PubMed Central

Pan, Daniel; Vargas-Morales, Omayra; Zern, Blaine; Anselmo, Aaron C.; Gupta, Vivek; Zakrewsky, Michael; Mitragotri, Samir; Muzykantov, Vladimir

2016-01-01

Red blood cells (RBCs) can be used for vascular delivery of encapsulated or surface-bound drugs and carriers. Coupling to RBC prolongs circulation of nanoparticles (NP, 200 nm spheres, a conventional model of polymeric drug delivery carrier) enabling their transfer to the pulmonary vasculature without provoking overt RBC elimination. However, little is known about more subtle and potentially harmful effects of drugs and drug carriers on RBCs. Here we devised high-throughput in vitro assays to determine the sensitivity of loaded RBCs to osmotic stress and other damaging insults that they may encounter in vivo (e.g. mechanical, oxidative and complement insults). Sensitivity of these tests is inversely proportional to RBC concentration in suspension and our results suggest that mouse RBCs are more sensitive to damaging factors than human RBCs. Loading RBCs by NP at 1:50 ratio did not affect RBCs, while 10–50 fold higher NP load accentuated RBC damage by mechanical, osmotic and oxidative stress. This extensive loading of RBC by NP also leads to RBCs agglutination in buffer; however, addition of albumin diminished this effect. These results provide a template for analyses of the effects of diverse cargoes loaded on carrier RBCs and indicate that: i) RBCs can tolerate carriage of NP at doses providing loading of millions of nanoparticles per microliter of blood; ii) tests using protein-free buffers and mouse RBCs may overestimate adversity that may be encountered in humans. PMID:27003833

Effect of additive solutions on red blood cell (RBC) membrane properties of stored RBCs prepared from whole blood held for 24 hours at room temperature.

PubMed

Veale, Margaret F; Healey, Gerry; Sparrow, Rosemary L

2011-01-01

The quality of RBC components is influenced by collection, processing and storage conditions. Regulations require that whole blood (WB) units be refrigerated within 8 hours and processed into RBCs within 24 hours of collection. Overnight room temperature hold of WB has logistical advantages, but the effect on RBC quality has not been fully investigated. RBC additive solutions were compared for their ability to provide improved quality of RBCs prepared from WB held at room temperature for 24 hours. Leukocyte-reduced RBCs were prepared from WB held at 20°C on cooling plates for 24 hours prior to processing. RBCs were stored in additive solutions, SAG-M (control), Erythrosol-4, and PAGGSM, under standard blood banking conditions and sampled during 49 days of storage. Stored RBCs were evaluated for RBC shape and microparticle (MP) accumulation using flow cytometry. Osmotic fragility, adhesion of RBCs to endothelium under shear stress conditions (0.5 dyne/cm(2) ), and routine RBC quality parameters were assessed. RBCs stored in Erythrosol-4 and PAGGSM had decreased cell size, reduced osmotic fragility, and decreased accumulation of glycophorin A-positive MPs and annexin V-binding MPs compared with RBCs stored in SAG-M. RBCs stored in erythrosol-4 had increased adherence to endothelium at days 42 and 49 compared with RBCs stored in SAG-M or PAGGSM. RBCs stored in PAGGSM or Erythrosol-4 had improved retention of RBC membrane and osmotic resilience. The development of new additive solutions may offer improved quality of RBC components prepared from WB held overnight at room temperature. © 2010 American Association of Blood Banks.

The rat red blood cell proteome is altered by priming with 2-butoxyethanol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Palkar, Prajakta S.; Kakhniashvili, David G.; Goodman, Steven R.

2008-08-01

Administration of a low priming dose of 2-butoxyethanol (BE, 500 mg/kg, p.o.) 7 days prior to a larger LD{sub 90} dose (1500 mg BE/kg, p.o.) offers protection against the lethal dose-induced hemolysis and death in female Sprague Dawley rats because of prompt and efficient replacement of red blood cells (RBCs) with new resilient RBCs. The objective of the present work was to analyze the altered proteome of RBCs upon priming with BE in order to identify the potential anti-hemolytic survival proteins induced in the primed rat RBCs (P-RBCs) as opposed to vehicle-treated RBCs (V-RBCs). The RBCs from the two groupsmore » were fractionated into membrane and cytosolic fractions. The cytosolic fractions were further fractionated for proteomic analysis into 3 fractions. The fractions were labeled with Cy3 and Cy5 fluorescent dyes and subjected to 2-dimensional differential gel electrophoresis (DIGE) to analyze the protein profiles. Seven membrane and 8 cytosolic proteins were found to be significantly increased ({>=} 2.5 fold) in P-RBCs as compared to V-RBCs. The identified proteins can be classified into antioxidant, membrane skeleton, protein turnover, lipid raft, and energy metabolism components. Increased levels of the proteins from antioxidant and membrane skeleton groups were confirmed by Western blot analysis. The study provides the first report on protein profiling of rat RBCs as well as on alteration of the proteome upon exposure to a priming dose of hemotoxicant. Further studies are needed to prove the protective role of the identified proteins and will initiate the field of survival/protective/anti-hemolytic proteins in RBCs.« less

Disrupting malaria parasite AMA1-RON2 interaction with a small molecule prevents erythrocyte invasion.

PubMed

Srinivasan, Prakash; Yasgar, Adam; Luci, Diane K; Beatty, Wandy L; Hu, Xin; Andersen, John; Narum, David L; Moch, J Kathleen; Sun, Hongmao; Haynes, J David; Maloney, David J; Jadhav, Ajit; Simeonov, Anton; Miller, Louis H

2013-01-01

Plasmodium falciparum resistance to artemisinin derivatives, the first-line antimalarial drug, drives the search for new classes of chemotherapeutic agents. Current discovery is primarily directed against the intracellular forms of the parasite. However, late schizont-infected red blood cells (RBCs) may still rupture and cause disease by sequestration; consequently targeting invasion may reduce disease severity. Merozoite invasion of RBCs requires interaction between two parasite proteins AMA1 and RON2. Here we identify the first inhibitor of this interaction that also blocks merozoite invasion in genetically distinct parasites by screening a library of over 21,000 compounds. We demonstrate that this inhibition is mediated by the small molecule binding to AMA1 and blocking the formation of AMA1-RON complex. Electron microscopy confirms that the inhibitor prevents junction formation, a critical step in invasion that results from AMA1-RON2 binding. This study uncovers a strategy that will allow for highly effective combination therapies alongside existing antimalarial drugs.

Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells.

PubMed

Kurita, Ryo; Suda, Noriko; Sudo, Kazuhiro; Miharada, Kenichi; Hiroyama, Takashi; Miyoshi, Hiroyuki; Tani, Kenzaburo; Nakamura, Yukio

2013-01-01

Transfusion of red blood cells (RBCs) is a standard and indispensable therapy in current clinical practice. In vitro production of RBCs offers a potential means to overcome a shortage of transfusable RBCs in some clinical situations and also to provide a source of cells free from possible infection or contamination by microorganisms. Thus, in vitro production of RBCs may become a standard procedure in the future. We previously reported the successful establishment of immortalized mouse erythroid progenitor cell lines that were able to produce mature RBCs very efficiently. Here, we have developed a reliable protocol for establishing immortalized human erythroid progenitor cell lines that are able to produce enucleated RBCs. These immortalized cell lines produce functional hemoglobin and express erythroid-specific markers, and these markers are upregulated following induction of differentiation in vitro. Most importantly, these immortalized cell lines all produce enucleated RBCs after induction of differentiation in vitro, although the efficiency of producing enucleated RBCs remains to be improved further. To the best of our knowledge, this is the first demonstration of the feasibility of using immortalized human erythroid progenitor cell lines as an ex vivo source for production of enucleated RBCs.

SPH-DEM approach to numerically simulate the deformation of three-dimensional RBCs in non-uniform capillaries.

PubMed

Polwaththe-Gallage, Hasitha-Nayanajith; Saha, Suvash C; Sauret, Emilie; Flower, Robert; Senadeera, Wijitha; Gu, YuanTong

2016-12-28

Blood continuously flows through the blood vessels in the human body. When blood flows through the smallest blood vessels, red blood cells (RBCs) in the blood exhibit various types of motion and deformed shapes. Computational modelling techniques can be used to successfully predict the behaviour of the RBCs in capillaries. In this study, we report the application of a meshfree particle approach to model and predict the motion and deformation of three-dimensional RBCs in capillaries. An elastic spring network based on the discrete element method (DEM) is employed to model the three-dimensional RBC membrane. The haemoglobin in the RBC and the plasma in the blood are modelled as smoothed particle hydrodynamics (SPH) particles. For validation purposes, the behaviour of a single RBC in a simple shear flow is examined and compared against experimental results. Then simulations are carried out to predict the behaviour of RBCs in a capillary; (i) the motion of five identical RBCs in a uniform capillary, (ii) the motion of five identical RBCs with different bending stiffness (K b ) values in a stenosed capillary, (iii) the motion of three RBCs in a narrow capillary. Finally five identical RBCs are employed to determine the critical diameter of a stenosed capillary. Validation results showed a good agreement with less than 10% difference. From the above simulations, the following results are obtained; (i) RBCs exhibit different deformation behaviours due to the hydrodynamic interaction between them. (ii) Asymmetrical deformation behaviours of the RBCs are clearly observed when the bending stiffness (K b ) of the RBCs is changed. (iii) The model predicts the ability of the RBCs to squeeze through smaller blood vessels. Finally, from the simulations, the critical diameter of the stenosed section to stop the motion of blood flow is predicted. A three-dimensional spring network model based on DEM in combination with the SPH method is successfully used to model the motion and deformation of RBCs in capillaries. Simulation results reveal that the condition of blood flow stopping depends on the pressure gradient of the capillary and the severity of stenosis of the capillary. In addition, this model is capable of predicting the critical diameter which prevents motion of RBCs for different blood pressures.

Haemocompatibility of iron oxide nanoparticles synthesized for theranostic applications: a high-sensitivity microfluidic tool

NASA Astrophysics Data System (ADS)

Rodrigues, Raquel O.; Bañobre-López, Manuel; Gallo, Juan; Tavares, Pedro B.; Silva, Adrián M. T.; Lima, Rui; Gomes, Helder T.

2016-07-01

The poor heating efficiency of the most reported magnetic nanoparticles (MNPs), allied to the lack of comprehensive biocompatibility and haemodynamic studies, hampers the spread of multifunctional nanoparticles as the next generation of therapeutic bio-agents in medicine. The present work reports the synthesis and characterization, with special focus on biological/toxicological compatibility, of superparamagnetic nanoparticles with diameter around 18 nm, suitable for theranostic applications (i.e. simultaneous diagnosis and therapy of cancer). Envisioning more insights into the complex nanoparticle-red blood cells (RBCs) membrane interaction, the deformability of the human RBCs in contact with magnetic nanoparticles (MNPs) was assessed for the first time with a microfluidic extensional approach, and used as an indicator of haematological disorders in comparison with a conventional haematological test, i.e. the haemolysis analysis. Microfluidic results highlight the potential of this microfluidic tool over traditional haemolysis analysis, by detecting small increments in the rigidity of the blood cells, when traditional haemotoxicology analysis showed no significant alteration (haemolysis rates lower than 2 %). The detected rigidity has been predicted to be due to the wrapping of small MNPs by the bilayer membrane of the RBCs, which is directly related to MNPs size, shape and composition. The proposed microfluidic tool adds a new dimension into the field of nanomedicine, allowing to be applied as a high-sensitivity technique capable of bringing a better understanding of the biological impact of nanoparticles developed for clinical applications.

Proper cytoskeletal architecture beneath the plasma membrane of red blood cells requires Ttll4

PubMed Central

Ijaz, Faryal; Hatanaka, Yasue; Hatanaka, Takahiro; Tsutsumi, Koji; Iwaki, Takayuki; Umemura, Kazuo; Ikegami, Koji; Setou, Mitsutoshi

2017-01-01

Mammalian red blood cells (RBCs) circulate through blood vessels, including capillaries, for tens of days under high mechanical stress. RBCs tolerate this mechanical stress while maintaining their shape because of their elastic membrane skeleton. This membrane skeleton consists of spectrin-actin lattices arranged as quasi-hexagonal units beneath the plasma membrane. In this study, we found that the organization of the RBC cytoskeleton requires tubulin tyrosine ligase–like 4 (Ttll4). RBCs from Ttll4-knockout mice showed larger average diameters in smear test. Based on the rate of hemolysis, Ttll4-knockout RBCs showed greater vulnerability to phenylhydrazine-induced oxidative stress than did wild-type RBCs. Ultrastructural analyses revealed the macromolecular aggregation of cytoskeletal components in RBCs of Ttll4-knockout mice. Immunoprecipitation using the anti-glutamylation antibody GT335 revealed nucleosome assembly protein 1 (NAP1) to be the sole target of TTLL4 in the RBCs, and NAP1 glutamylation was completely lost in Ttll4-knockout RBCs. In wild-type RBCs, the amount of glutamylated NAP1 in the membrane was nearly double that in the cytosol. Furthermore, the absence of TTLL4-dependent glutamylation of NAP1 weakened the binding of NAP1 to the RBC membrane. Taken together, these data demonstrate that Ttll4 is required for proper cytoskeletal organization in RBCs. PMID:27974641

Identification of Rubisco rbcL and rbcS in Camellia oleifera and their potential as molecular markers for selection of high tea oil cultivars.

PubMed

Chen, Yongzhong; Wang, Baoming; Chen, Jianjun; Wang, Xiangnan; Wang, Rui; Peng, Shaofeng; Chen, Longsheng; Ma, Li; Luo, Jian

2015-01-01

Tea oil derived from seeds of Camellia oleifera Abel. is high-quality edible oil in China. This study isolated full-length cDNAs of Rubisco subunits rbcL and rbcS from C. oleifera. The rbcL has 1,522 bp with a 1,425 bp coding region, encoding 475 amino acids; and the rbcS has 615 bp containing a 528 bp coding region, encoding 176 amino acids. The expression level of the two genes, designated as Co-rbcL and Co-rbcS, was determined in three C. oleifera cultivars: Hengchong 89, Xianglin 1, and Xianglin 14 whose annual oil yields were 546.9, 591.4, and 657.7 kg ha(-1), respectively. The Co-rbcL expression in 'Xianglin 14' was significantly higher than 'Xianglin 1', and 'Xianglin 1' was greater than 'Hengchong 89'. The expression levels of Co-rbcS in 'Xianglin 1' and 'Xianglin 14' were similar but were significantly greater than in 'Hengchong 89'. The net photosynthetic rate of 'Xianglin 14' was significantly higher than 'Xianglin 1', and 'Xianglin 1' was higher than 'Hengchong 89'. Pearson's correlation analysis showed that seed yields and oil yields were highly correlated with the expression level of Co-rbcL at P < 0.001 level; and the expression of Co-rbcS was correlated with oil yield at P < 0.01 level. Net photosynthetic rate was also correlated with oil yields and seed yields at P < 0.001 and P < 0.01 levels, respectively. Our results suggest that Co-rbcS and Co-rbcL in particular could potentially be molecular markers for early selection of high oil yield cultivars. In combination with the measurement of net photosynthetic rates, the early identification of potential high oil production cultivars would significantly shorten plant breeding time and increase breeding efficiency.

Red blood cells in thrombosis.

PubMed

Byrnes, James R; Wolberg, Alisa S

2017-10-19

Red blood cells (RBCs) have historically been considered passive bystanders in thrombosis. However, clinical and epidemiological studies have associated quantitative and qualitative abnormalities in RBCs, including altered hematocrit, sickle cell disease, thalassemia, hemolytic anemias, and malaria, with both arterial and venous thrombosis. A growing body of mechanistic studies suggests that RBCs can promote thrombus formation and enhance thrombus stability. These findings suggest that RBCs may contribute to thrombosis pathophysiology and reveal potential strategies for therapeutically targeting RBCs to reduce thrombosis. © 2017 by The American Society of Hematology.

Anisotropic light scattering of individual sickle red blood cells.

PubMed

Kim, Youngchan; Higgins, John M; Dasari, Ramachandra R; Suresh, Subra; Park, YongKeun

2012-04-01

We present the anisotropic light scattering of individual red blood cells (RBCs) from a patient with sickle cell disease (SCD). To measure light scattering spectra along two independent axes of elongated-shaped sickle RBCs with arbitrary orientation, we introduce the anisotropic Fourier transform light scattering (aFTLS) technique and measured both the static and dynamic anisotropic light scattering. We observed strong anisotropy in light scattering patterns of elongated-shaped sickle RBCs along its major axes using static aFTLS. Dynamic aFTLS analysis reveals the significantly altered biophysical properties in individual sickle RBCs. These results provide evidence that effective viscosity and elasticity of sickle RBCs are significantly different from those of the healthy RBCs.

Highly conserved small subunit residues influence rubisco large subunit catalysis.

PubMed

Genkov, Todor; Spreitzer, Robert J

2009-10-30

The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

Quantitative microscopy and nanoscopy of sickle red blood cells performed by wide field digital interferometry

NASA Astrophysics Data System (ADS)

Shaked, Natan T.; Satterwhite, Lisa L.; Telen, Marilyn J.; Truskey, George A.; Wax, Adam

2011-03-01

We have applied wide-field digital interferometry (WFDI) to examine the morphology and dynamics of live red blood cells (RBCs) from individuals who suffer from sickle cell anemia (SCA), a genetic disorder that affects the structure and mechanical properties of RBCs. WFDI is a noncontact, label-free optical microscopy approach that can yield quantitative thickness profiles of RBCs and measurements of their membrane fluctuations at the nanometer scale reflecting their stiffness. We find that RBCs from individuals with SCA are significantly stiffer than those from a healthy control. Moreover, we show that the technique is sensitive enough to distinguish classes of RBCs in SCA, including sickle RBCs with apparently normal morphology, compared to the stiffer crescent-shaped sickle RBCs. We expect that this approach will be useful for diagnosis of SCA and for determining efficacy of therapeutic agents.

Efficacy of fresh packed red blood transfusion in organophosphate poisoning.

PubMed

Bao, Hang-Xing; Tong, Pei-Jian; Li, Cai-Xia; Du, Jing; Chen, Bing-Yu; Huang, Zhi-Hui; Wang, Ying

2017-03-01

The mortality rate caused by organophosphate (OP) poisoning is still high, even the standard treatment such as atropine and oxime improves a lot. To search for alternative therapies, this study was aimed to investigate the effects of packed red blood cell (RBC) transfusion in acute OP poisoning, and compare the therapeutic effects of RBCs at different storage times.Patients diagnosed with OP poisoning were included in this prospective study. Fresh RBCs (packed RBCs stored less than 10 days) and longer-storage RBCs (stored more than 10 days but less than 35 days) were randomly transfused or not into OP poisoning patients. Cholinesterase (ChE) levels in blood, atropine usage and durations, pralidoxime durations were measured.We found that both fresh and longer-storage RBCs (200-400 mL) significantly increased blood ChE levels 6 hours after transfusion, shortened the duration for ChE recovery and length of hospital stay, and reduced the usage of atropine and pralidoxime. In addition, fresh RBCs demonstrated stronger therapeutic effects than longer-storage RBCs.Packed RBCs might be an alternative approach in patients with OP poisoning, especially during early stages.

Refractive index tomograms and dynamic membrane fluctuations of red blood cells from patients with diabetes mellitus.

PubMed

Lee, SangYun; Park, HyunJoo; Kim, Kyoohyun; Sohn, YongHak; Jang, Seongsoo; Park, YongKeun

2017-04-21

In this paper, we present the optical characterisations of diabetic red blood cells (RBCs) in a non-invasive manner employing three-dimensional (3-D) quantitative phase imaging. By measuring 3-D refractive index tomograms and 2-D time-series phase images, the morphological (volume, surface area and sphericity), biochemical (haemoglobin concentration and content) and mechanical (membrane fluctuation) parameters were quantitatively retrieved at the individual cell level. With simultaneous measurements of individual cell properties, systematic correlative analyses on retrieved RBC parameters were also performed. Our measurements show there exist no statistically significant alterations in morphological and biochemical parameters of diabetic RBCs, compared to those of healthy (non-diabetic) RBCs. In contrast, membrane deformability of diabetic RBCs is significantly lower than that of healthy, non-diabetic RBCs. Interestingly, non-diabetic RBCs exhibit strong correlations between the elevated glycated haemoglobin in RBC cytoplasm and decreased cell deformability, whereas diabetic RBCs do not show correlations. Our observations strongly support the idea that slow and irreversible glycation of haemoglobin and membrane proteins of RBCs by hyperglycaemia significantly compromises RBC deformability in diabetic patients.

2,3-Diphosphoglycerate Concentrations in Autologous Salvaged Versus Stored Red Blood Cells and in Surgical Patients After Transfusion.

PubMed

Scott, Andrew V; Nagababu, Enika; Johnson, Daniel J; Kebaish, Khaled M; Lipsitz, Joshua A; Dwyer, Ian M; Zuckerberg, Gabriel S; Barodka, Viachaslau M; Berkowitz, Dan E; Frank, Steven M

2016-03-01

Stored red blood cells (RBCs) are deficient in 2,3-diphosphoglycerate (2,3-DPG), but it is unclear how autologous salvaged blood (ASB) compares with stored blood and how rapidly 2,3-DPG levels return to normal after transfusion. Therefore, we compared levels of 2,3-DPG in stored versus ASB RBCs and in patients' blood after transfusion. Twenty-four patients undergoing multilevel spine fusion surgery were enrolled. We measured 2,3-DPG and the oxyhemoglobin dissociation curve (P50) in samples taken from the ASB and stored blood bags before transfusion and in blood samples drawn from patients before and after transfusion. The mean storage duration for stored RBCs was 24 ± 8 days. Compared with fresh RBCs, stored RBCs had decreased 2,3-DPG levels (by approximately 90%; P < 0.0001) and a decreased P50 (by approximately 30%; P < 0.0001). However, ASB RBCs did not exhibit these changes. The mean 2,3-DPG concentration decreased by approximately 20% (P < 0.05) in postoperative blood sampled from patients who received 1 to 3 stored RBC units and by approximately 30% (P < 0.01) in those who received ≥4 stored RBC units. 2,3-DPG was unchanged in patients who received no stored blood or ASB alone. After surgery, 2,3-DPG levels recovered gradually over 3 postoperative days in patients who received stored RBCs. Stored RBCs, but not ASB RBCs, have decreased levels of 2,3-DPG and a left-shift in the oxyhemoglobin dissociation curve. Postoperatively, 2,3-DPG levels remain below preoperative baseline levels for up to 3 postoperative days in patients who receive stored RBCs but are unchanged in those who receive only ASB RBCs.

2,3-Diphosphoglycerate Concentrations in Autologous Salvaged Versus Stored Red Blood Cells and in Surgical Patients After Transfusion

PubMed Central

Scott, Andrew V.; Nagababu, Enika; Johnson, Daniel J.; Kebaish, Khaled M.; Lipsitz, Joshua A.; Dwyer, Ian M.; Zuckerberg, Gabriel S.; Barodka, Viachaslau M.; Berkowitz, Dan E.; Frank, Steven M.

2016-01-01

Background Stored red blood cells (RBCs) are deficient in 2,3-diphosphoglycerate (2,3-DPG), but it is unclear how autologous salvaged blood (ASB) compares to stored blood, and how rapidly 2,3-DPG levels return to normal after transfusion. Therefore, we compared levels of 2,3-DPG in stored versus ASB RBCs, and in patients’ blood following transfusion. Methods Twenty-four patients undergoing multilevel spine fusion surgery were enrolled. We measured 2,3-DPG and the oxyhemoglobin dissociation curve (P50) in samples taken from the ASB and stored blood-bags before transfusion, and in blood samples drawn from patients before and after transfusion. Results The mean storage duration for stored RBCs was 24 ± 8 days. Compared with fresh RBCs, stored RBCs had decreased 2,3-DPG levels (by ≈ 90%; P<0.0001), and a decreased P50 (by ≈ 30%; P<0.0001). However, ASB RBCs did not exhibit these changes. The mean 2,3-DPG concentration decreased by ≈ 20% (P<0.05) in postoperative blood sampled from patients who received 1 – 3 stored RBC units, and by ≈ 30% (P<0.01) in those who received ≥4 stored RBC units. 2,3-DPG was unchanged in patients who received no stored blood or ASB alone. After surgery, 2,3-DPG levels recovered gradually over three postoperative days in patients who received stored RBCs. Conclusions Stored RBCs, but not ASB RBCs, have decreased levels of 2,3-DPG and a left shift in the oxyhemoglobin dissociation curve. Postoperatively, 2,3-DPG levels remain below preoperative baseline levels for up to three postoperative days in patients who receive stored RBCs, but are unchanged in those who receive only ASB RBCs. PMID:26891388

The effects of red blood cell preparation method on in vitro markers of red blood cell aging and inflammatory response.

PubMed

Radwanski, Katherine; Garraud, Olivier; Cognasse, Fabrice; Hamzeh-Cognasse, Hind; Payrat, Jean-Marc; Min, Kyungyoon

2013-12-01

Studies are currently under way examining whether the age of stored red blood cells (RBCs) affects clinical outcome in transfusion recipients. The effects of storage duration on the RBC storage lesion are well documented, while fewer studies are available regarding the effect of RBC production method. In this study, we compared in vitro RBC quality variables and markers of inflammatory response in apheresis and whole blood (WB)-derived RBCs, specifically those prepared after an overnight room temperature hold (RTH) of WB. SAGM RBCs, prepared from WB after overnight RTH (n = 10), were compared to SAGM RBCs prepared using an apheresis device (Alyx, n = 10). As a control, SAGM RBCs were also prepared within 2 hours of WB collection (2-hr WB, n = 10). All RBCs were stored at 4°C for 42 days with weekly assay of in vitro variables, cytokines and/or chemokines, and neutrophil activation after incubation with RBC supernatant. RTH WB RBCs exhibited decreased levels of 2,3-diphosphoglycerate acid (2.3 μmol/g hemoglobin [Hb] ± 2.1 vs. 13.7 ± 1.3 μmol/g Hb) and morphology (160 ± 10 vs. 192 ± 5) on Day 1 and increased hemolysis (0.45 ± 0.21% vs. 0.31 ± 0.09%) and microparticles (6.1 ± 2.8/10(3) RBCs vs. 3.9 ± 1.1/10(3) RBCs) on Day 42 compared to apheresis RBCs. Gro-α and ENA-78 cytokine levels were significantly higher in RTH WB than Alyx RBCs during storage. CD11b expression was highest in neutrophils exposed to supernatant from RTH WB RBCs (p < 0.05). RBC preparation method has a meaningful effect on the RBC storage lesion, which should be taken into account in addition to length of storage. © 2013 American Association of Blood Banks.

Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea.

PubMed

Kasai, Yuki; Oshima, Kohei; Ikeda, Fukiko; Abe, Jun; Yoshimitsu, Yuya; Harayama, Shigeaki

2015-01-01

Microalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because it accumulates lipids to more than 30 % of dry cell weight under nitrogen-depleted conditions. It also grows rapidly under acidic conditions at which most protozoan grazers of microalgae may not be tolerant. The lipid productivity of this alga could be improved using genetic engineering techniques; however, genetically modified organisms are the subject of regulation by specific laws. Therefore, the aim of this study was to develop a self-cloning-based positive selection system for the breeding of P. ellipsoidea. In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system. The cDNA of the uridine monophosphate synthase gene (PeUMPS) of P. ellipsoidea was cloned downstream of the promoter of either a beta-tubulin gene (PeTUBULIN1) or the gene for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (PeRBCS) to construct the pUT1 or pUT2 plasmid, respectively. These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates. Use of Noble agar as the solidifying agent was essential to avoid the development of false-positive colonies. It took more than 6 weeks for the formation of colonies of pUT1 transformants, whereas pUT2 transformants formed colonies in 2 weeks. Real-time PCR revealed that there were more PeUMPS transcripts in pUT2 transformants than in pUT1 transformants. Uracil synthesis (Ura(+)) transformants were also obtained using a gene cassette consisting solely of PeUMPS flanked by the PeRBCS promoter and terminator. A self-cloning-based positive selection system for the genetic transformation of P. ellipsoidea was developed. Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation.

Effects of non-leukocyte-reduced and leukocyte-reduced packed red blood cell transfusions on oxygenation of rat spinotrapezius muscle

PubMed Central

Sundararajan, Sripriya; Dodhy, Sami C.; Pittman, Roland N.; Lewis, Stephen J.

2015-01-01

Leukoreduction of blood used for transfusion alleviates febrile transfusion reactions, graft versus host disease and alloimmunization to leukocyte antigen. However, the actual clinical benefit of leukoreduction in terms of microcirculatory tissue O2 delivery after packed red blood cell (pRBC) transfusion has not been investigated. As such, the aim of this study was to determine the effects of non-leukoreduced (NLR) and leukoreduced (LR) fresh pRBC transfusion on interstitial oxygenation in anesthetized male Sprague-Dawley rats. Interstitial fluid PO2 and arteriolar diameters in spinotrapezius muscle preparations were monitored before and after transfusion with NLR- or LR-pRBCs. The major findings were that (1) transfusion of NLR-pRBCs significantly decreased interstitial oxygenation whereas transfusion of LR-pRBCs did not, and (2) transfusion with LR-pRBCs elicited a substantially greater increase in arterial blood pressure (ABP) than did transfusion with NLR-pRBCs. These changes in PO2 and ABP were not associated with changes in the diameters of resistance arterioles in the spinotrapezius muscle. These data suggest that transfusion of fresh NLR-pRBCs may negatively affect tissue oxygenation via enhanced leukocyte influx and decreased O2 delivery. They also suggest that leukocytes diminish the capability of transfused pRBCs to increase cardiac output. As such, transfusion of LR-pRBCs may be less deleterious on tissue PO2 levels than NLR-pRBCs although a concomitantly greater increase in ABP may accompany transfusion of LR-pRBCs. PMID:24189119

Red blood cells for transfusion in patients with sepsis: respective roles of unit age and exposure to recipient plasma.

PubMed

Chadebech, Philippe; Bodivit, Gwellaouen; Razazi, Keyvan; de Vassoigne, Christophe; Pellé, Laurence; Burin-des-Roziers, Nicolas; Bocquet, Thibault; Bierling, Philippe; Djoudi, Rachid; Mekontso-Dessap, Armand; Pirenne, France

2017-08-01

Red blood cell (RBC) storage in blood banks is not exempt from cellular injury. Alterations not observed on RBCs freshly isolated from units can rapidly appear in circulation. The transfusion of old blood units, even if this is a controversial issue, could therefore have adverse effects on the recipient. We wanted to determine the respective effects of storage duration and recipient plasma on RBCs for transfusion into patients with severe sepsis. Eleven stored RBC units were sampled at various time points, approximately Days 3 to 8 (referred to as fresh RBCs) and Days 38 to 42 (old RBCs) and tested in coincubation experiments with plasma obtained from 13 patients with severe sepsis and 17 healthy donors as controls. RBCs were tested after 24 or 48 hours at 37°C for the detection of senescence markers (phosphatidylserine exposure, calcium influx, and reactive oxygen species detection and decrease in size) with or without exposure to plasma. We confirmed that a 42-day refrigerated storage of RBCs alone (without any incubation in plasma) had no significant effect on RBCs and no senescence marker detected. By contrast, ex vivo exposure to plasma samples altered both fresh and old RBCs, with a much larger effect for old RBCs, regardless of the plasma used (sepsis vs. control). We show that the main factor affecting the senescence of RBCs for transfusion into patients with severe sepsis is the age of the stored units rather than the clinical status of the recipient. © 2017 AABB.

Use of a flow-cell system to investigate virucidal dimethylmethylene blue phototreatment in two RBC additive solutions.

PubMed

Wagner, Stephen; Skripchenko, Andrey; Thompson-Montgomery, Dedeene

2002-09-01

Limited photoinactivation kinetics, use of low-volume 30 percent Hct RBCs, and hemolysis have restricted the practicality of the use of dimethylmethylene blue (DMMB) and light for RBC decontamination. A flow-cell system was developed to rapidly treat larger volumes of oxygenated 45 percent Hct RBCs with high-intensity red light. CPD-whole blood was WBC reduced, RBCs were diluted in additive solutions (either Adsol or Erythrosol), and suspensions were subsequently oxygenated by gas overlay. Intracellular or extracellular VSV and DMMB were sequentially added. VSV-infected RBC suspensions (45% Hct) were passed through 1-mm-thick flow cells and illuminated. Samples were titered for VSV, stored for up to 42 days, and assayed for Hb, supernatant potassium, ATP, and MCV. The use of oxygenated RBCs resulted in rapid and reproducible photoinactivaton of > or = 6.6 log extracellular and approximately 4.0 log intracellular VSV independent of additive solution. Phototreated Adsol RBCs exhibited more than 10 times greater hemolysis and 30 percent greater MCV during storage than identically treated Erythrosol RBCs. Phototreatment caused RBC potassium leakage from RBCs in both additive solutions. ATP levels were better preserved in Erythrosol than Adsol RBCs. A rapid, reproducible, and robust method for photoinactivating model virus in RBC suspensions was developed. Despite improved hemolysis and ATP levels in Erythrosol-phototreated RBCs, storage properties were not maintained for 42 days.

Surface decoration of red blood cells with maleimidophenyl-polyethylene glycol facilitated by thiolation with iminothiolane: an approach to mask A, B, and D antigens to generate universal red blood cells.

PubMed

Nacharaju, Parimala; Boctor, Fouad N; Manjula, Belur N; Acharya, Seetharama A

2005-03-01

The surface decoration of red blood cells (RBCs) by polyethylene glycol (PEG) chains has been an approach developed to camouflage the blood group antigens from their antibodies. A PEGylation protocol, however, that can mask the antigens appropriately to inhibit the agglutination of RBCs with the respective antibodies is not available so far. A new approach for PEGylation of RBC membrane proteins has been designed with thiolation-mediated maleimide chemistry. The accessibility of the surface lysine residues of membrane proteins to bulky PEG reagents was increased by linking an extension arm carrying a thiol group. RBCs have been PEGylated by thiolation-mediated chemistry with maleimidophenyl-PEG (Mal-Phe-PEG) reagents of different chain lengths. Mal-Phe-PEG-5000 chains alone masked the most important antigens of the Rh system (C, c, E, e, and D) from their antibodies. The masking of the A and B antigens needed a combination of Mal-Phe-PEG-5000 and Mal-Phe-PEG-20000 chains to inhibit the agglutination of RBCs completely with anti-A or anti-B. Thiolation-mediated PEGylation of RBCs with Mal-Phe-PEG-5000 and Mal-Phe-PEG-20000 converts Group A Rh(D)+ and B Rh(D)+ RBCs into RBCs with serologic behavior comparable to Group O Rh(D)- RBCs that are considered as universal RBCs for transfusion.

Factors affecting red blood cell storage age at the time of transfusion.

PubMed

Dzik, Walter H; Beckman, Neil; Murphy, Michael F; Delaney, Meghan; Flanagan, Peter; Fung, Mark; Germain, Marc; Haspel, Richard L; Lozano, Miguel; Sacher, Ronald; Szczepiorkowski, Zbigniew; Wendel, Silvano

2013-12-01

Clinical trials are investigating the potential benefit resulting from a reduced maximum storage interval for red blood cells (RBCs). The key drivers that determine RBC age at the time of issue vary among individual hospitals. Although progressive reduction in the maximum storage period of RBCs would be expected to result in smaller hospital inventories and reduced blood availability, the magnitude of the effect is unknown. Data on current hospital blood inventories were collected from 11 hospitals and three blood centers in five nations. A general predictive model for the age of RBCs at the time of issue was developed based on considerations of demand for RBCs in the hospital. Age of RBCs at issue is sensitive to the following factors: ABO group, storage age at the time of receipt by the hospital, the restock interval, inventory reserve, mean demand, and variation in demand. A simple model, based on hospital demand, may serve as the basis for examining factors affecting the storage age of RBCs in hospital inventories. The model suggests that the age of RBCs at the time of their issue to the patient depends on factors external to the hospital transfusion service. Any substantial change in the expiration date of stored RBCs will need to address the broad variation in demand for RBCs while attempting to balance considerations of availability and blood wastage. © 2013 American Association of Blood Banks.

Infectious pancreatic necrosis virus triggers antiviral immune response in rainbow trout red blood cells, despite not being infective

PubMed Central

Nombela, Ivan; Carrion, Aurora; Puente-Marin, Sara; Chico, Verónica; Mercado, Luis; Perez, Luis; Coll, Julio; Ortega-Villaizan, Maria del Mar

2017-01-01

Background: Some fish viruses, such as piscine orthoreovirus and infectious salmon anemia virus, target red blood cells (RBCs), replicate inside them and induce an immune response. However, the roles of RBCs in the context of infectious pancreatic necrosis virus (IPNV) infection  have not been studied yet. Methods: Ex vivo rainbow trout RBCs were obtained from peripheral blood, Ficoll purified and exposed to IPNV in order to analyze infectivity and immune response using RT-qPCR, immune fluorescence imaging, flow cytometry and western-blotting techniques. Results: IPNV could not infect RBCs; however, IPNV increased the expression of the INF1-related genes ifn-1, pkr and mx genes. Moreover, conditioned media from IPNV-exposed RBCs conferred protection against IPNV infection in CHSE-214 fish cell line. Conclusions: Despite not being infected, rainbow trout RBCs could respond to IPNV with increased expression of antiviral genes. Fish RBCs could be considered as mediators of the antiviral response and therefore targets of new strategies against fish viral infections. Further research is ongoing to completely understand the molecular mechanism that triggers this antiviral response in rainbow trout RBCs. PMID:29333244

Infectious pancreatic necrosis virus triggers antiviral immune response in rainbow trout red blood cells, despite not being infective.

PubMed

Nombela, Ivan; Carrion, Aurora; Puente-Marin, Sara; Chico, Verónica; Mercado, Luis; Perez, Luis; Coll, Julio; Ortega-Villaizan, Maria Del Mar

2017-01-01

Background : Some fish viruses, such as piscine orthoreovirus and infectious salmon anemia virus, target red blood cells (RBCs), replicate inside them and induce an immune response. However, the roles of RBCs in the context of infectious pancreatic necrosis virus (IPNV) infection  have not been studied yet. Methods : Ex vivo rainbow trout RBCs were obtained from peripheral blood, Ficoll purified and exposed to IPNV in order to analyze infectivity and immune response using RT-qPCR, immune fluorescence imaging, flow cytometry and western-blotting techniques. Results : IPNV could not infect RBCs; however, IPNV increased the expression of the INF1-related genes ifn-1 , pkr and mx genes. Moreover, conditioned media from IPNV-exposed RBCs conferred protection against IPNV infection in CHSE-214 fish cell line. Conclusions : Despite not being infected, rainbow trout RBCs could respond to IPNV with increased expression of antiviral genes. Fish RBCs could be considered as mediators of the antiviral response and therefore targets of new strategies against fish viral infections. Further research is ongoing to completely understand the molecular mechanism that triggers this antiviral response in rainbow trout RBCs.

Delivery of Iron-Sulfur Clusters to the Hydrogen-Oxidizing [NiFe]-Hydrogenases in Escherichia coli Requires the A-Type Carrier Proteins ErpA and IscA

PubMed Central

Pinske, Constanze; Sawers, R. Gary

2012-01-01

During anaerobic growth Escherichia coli synthesizes two membrane-associated hydrogen-oxidizing [NiFe]-hydrogenases, termed hydrogenase 1 and hydrogenase 2. Each enzyme comprises a catalytic subunit containing the [NiFe] cofactor, an electron-transferring small subunit with a particular complement of [Fe-S] (iron-sulfur) clusters and a membrane-anchor subunit. How the [Fe-S] clusters are delivered to the small subunit of these enzymes is unclear. A-type carrier (ATC) proteins of the Isc (iron-sulfur-cluster) and Suf (sulfur mobilization) [Fe-S] cluster biogenesis pathways are proposed to traffic pre-formed [Fe-S] clusters to apoprotein targets. Mutants that could not synthesize SufA had active hydrogenase 1 and hydrogenase 2 enzymes, thus demonstrating that the Suf machinery is not required for hydrogenase maturation. In contrast, mutants devoid of the IscA, ErpA or IscU proteins of the Isc machinery had no detectable hydrogenase 1 or 2 activities. Lack of activity of both enzymes correlated with the absence of the respective [Fe-S]-cluster-containing small subunit, which was apparently rapidly degraded. During biosynthesis the hydrogenase large subunits receive their [NiFe] cofactor from the Hyp maturation machinery. Subsequent to cofactor insertion a specific C-terminal processing step occurs before association of the large subunit with the small subunit. This processing step is independent of small subunit maturation. Using western blotting experiments it could be shown that although the amount of each hydrogenase large subunit was strongly reduced in the iscA and erpA mutants, some maturation of the large subunit still occurred. Moreover, in contrast to the situation in Isc-proficient strains, these processed large subunits were not membrane-associated. Taken together, our findings demonstrate that both IscA and ErpA are required for [Fe-S] cluster delivery to the small subunits of the hydrogen-oxidizing hydrogenases; however, delivery of the Fe atom to the active site might have different requirements. PMID:22363723

Photoinduced reduction of the medial FeS center in the hydrogenase small subunit HupS from Nostoc punctiforme.

PubMed

Raleiras, Patrícia; Hammarström, Leif; Lindblad, Peter; Styring, Stenbjörn; Magnuson, Ann

2015-07-01

The small subunit from the NiFe uptake hydrogenase, HupSL, in the cyanobacterium Nostoc punctiforme ATCC 29133, has been isolated in the absence of the large subunit (P. Raleiras, P. Kellers, P. Lindblad, S. Styring, A. Magnuson, J. Biol. Chem. 288 (2013) 18,345-18,352). Here, we have used flash photolysis to reduce the iron-sulfur clusters in the isolated small subunit, HupS. We used ascorbate as electron donor to the photogenerated excited state of Ru(II)-trisbipyridine (Ru(bpy)3), to generate Ru(I)(bpy)3 as reducing agent. Our results show that the isolated small subunit can be reduced by the Ru(I)(bpy)3 generated through flash photolysis. Copyright © 2015 Elsevier Inc. All rights reserved.

Cloning, Purification, and Characterization of a Heterodimeric β-Galactosidase from Lactobacillus kefiranofaciens ZW3.

PubMed

He, Xi; Han, Ning; Wang, Yan-Ping

2016-01-01

Lactobacillus kefiranofaciens ZW3 was obtained from kefir grains, which have high lactose hydrolytic activity. In this study, a heterodimeric LacLM-type β-galactosidase gene (lacLM) from ZW3 was isolated, which was composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp) encoding large and small subunits with calculated molecular masses of 73,620 and 35,682 Da, respectively. LacLM, LacL, and LacM were expressed in Escherichia coli BL21(DE3) and these recombinant proteins were purified and characterized. The results showed that, compared with the recombinant holoenzyme, the recombinant large subunit exhibits obviously lower thermostability and hydrolytic activity. Moreover, the optimal temperature and pH of the holoenzyme and large subunit are 60°C and 7.0, and 50°C and 8.0, respectively. However, the recombinant small subunit alone has no activity. Interestingly, the activity and thermostability of the large subunit were greatly improved after mixing it with the recombinant small subunit. Therefore, the results suggest that the small subunit might play an important role in maintaining the stability of the structure of the catalytic center located in the large subunit.

Enhanced immunization via dissolving microneedle array-based delivery system incorporating subunit vaccine and saponin adjuvant

PubMed Central

Zhao, Ji-Hui; Zhang, Qi-Bo; Liu, Bao; Piao, Xiang-Hua; Yan, Yu-Lu; Hu, Xiao-Ge; Zhou, Kuan; Zhang, Yong-Tai; Feng, Nian-Ping

2017-01-01

Purpose To enhance the immunogenicity of the model subunit vaccine, ovalbumin (OVA) was combined with platycodin (PD), a saponin adjuvant. To reduce the toxicity of PD, OVA, and adjuvant were loaded together into liposomes before being incorporated into a dissolving microneedle array. Methods OVA- and PD-loaded liposomes (OVA-PD-Lipos) were prepared using the film dispersion method. Their uptake behavior, toxicity to mouse bone marrow dendritic cells (BMDCs), and hemolytic activity to rabbit red blood cells (RBCs) were evaluated. The OVA-PD-Lipos were incorporated into a dissolving microneedle array. The chemical stability of OVA and the physical stability of OVA-PD-Lipos in microneedle arrays were investigated. The immune response of Institute of Cancer Research mice and potential skin irritation reaction of rabbits to OVA-PD-Lipos-MNs were evaluated. Results The uptake of OVA by mouse BMDCs was greatly enhanced when OVA was prepared as OVA-PD-Lipos, and in this form, the toxicity of PD was dramatically reduced. OVA was chemically stable as OVA-PD-Lipos, when OVA-PD-Lipos was incorporated into a dissolving microneedle array. Institute of Cancer Research mice treated with OVA-PD-Lipos-MNs showed a significantly enhanced immune response. PD combined with OVA elicited a balanced Th1 and Th2 humoral immune response in mice, with minimal irritation in rabbit skin. Conclusion The dissolving microneedle array-based system is a promising delivery vehicle for subunit vaccine and its adjuvant. PMID:28740383

The gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis contains a group I intron.

PubMed Central

De Wachter, R; Neefs, J M; Goris, A; Van de Peer, Y

1992-01-01

The nucleotide sequence of the gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis was determined. It revealed the presence of a group I intron with a length of 411 nucleotides. This is the third occurrence of such an intron discovered in a small subunit rRNA gene encoded by a eukaryotic nuclear genome. The other two occurrences are in Pneumocystis carinii, a fungus of uncertain taxonomic status, and Ankistrodesmus stipitatus, a green alga. The nucleotides of the conserved core structure of 101 group I intron sequences present in different genes and genome types were aligned and their evolutionary relatedness was examined. This revealed a cluster including all group I introns hitherto found in eukaryotic nuclear genes coding for small and large subunit rRNAs. A secondary structure model was designed for the area of the Ustilago maydis small ribosomal subunit RNA precursor where the intron is situated. It shows that the internal guide sequence pairing with the intron boundaries fits between two helices of the small subunit rRNA, and that minimal rearrangement of base pairs suffices to achieve the definitive secondary structure of the 18S rRNA upon splicing. PMID:1561081

Comparison of the effects of glycerol, dimethyl sulfoxide, and hydroxyethyl starch solutions for cryopreservation of avian red blood cells.

PubMed

Graham, Jennifer E; Meola, Dawn M; Kini, Nisha R; Hoffman, Andrew M

2015-06-01

To compare effectiveness of glycerol, dimethyl sulfoxide (DMSO), and hydroxyethyl starch (HES) solutions for cryopreservation of avian RBCs. RBCs from 12 healthy Ameraucana hens (Gallus gallus domesticus). RBCs were stored in 20% (wt/vol) glycerol, 10% (wt/vol) DMSO freezing medium, or various concentrations of HES solution (7.5%, 11.5%, and 20% [wt/vol]) and frozen for 2 months in liquid nitrogen. Cells were then thawed and evaluated by use of cell recovery and saline stability tests, cell staining (7-aminoactinomycin D and annexin V) and flow cytometry, and scanning electron microscopy. Percentage of RBCs recovered was highest for 20% glycerol solution (mean ± SE, 99.71 ± 0.04%) and did not differ significantly from the value for 7.5% HES solution (99.57 ± 0.04%). Mean saline stability of RBCs was highest for 10% DMSO (96.11 ± 0.25%) and did not differ significantly from the value for 20% HES solution (95.74 ± 0.25%). Percentages of cells with 7-aminoactinomycin D staining but without annexin V staining (indicating necrosis or late apoptosis) were lowest for 10% DMSO freezing medium (3%) and 20% glycerol solution (1%) and highest for all HES concentrations (60% to 80%). Scanning electron microscopy revealed severe membrane changes in RBCs cryopreserved in 20% HES solution, compared with membrane appearance in freshly harvested RBCs and RBCs cryopreserved in 10% DMSO freezing medium. Cryopreservation of avian RBCs with HES solution, regardless of HES concentration, resulted in greater degrees of apoptosis and cell death than did cryopreservation with other media. Transfusion with RBCs cryopreserved in HES solution may result in posttransfusion hemolysis in birds.

[Optimization of trehalose loading in red blood cells before freeze-drying].

PubMed

Zhuang, Yuan; Liu, Jing-Han; Ouyang, Xi-Lin; Chen, Lin-Feng; Che, Ji

2007-04-01

The key points for better protection of trehalose in freeze-drying red blood cells (RBCs) are to resolve non-osmosis of trehalose to red blood cells and to make cytoplasmic trehalose to reach effective concentration. This study was aimed to investigate the regularity of loading RBCs with trehalose, screen out optimal loading condition and evaluate the effect of trehalose on physico-chemical parameters of RBCs during the period of loading. The cytoplasmic trehalose concentration in red blood cells, free hemoglobin and ATP level were determined at different incubation temperatures (4, 22 and 37 degrees C), different trehaolse concentrations (0, 200, 400, 600, 800 and 1000 mmol/L) and different incubation times (2, 4, 6, 8 and 10 hours), the cytoplasmic trehalose, free hemoglobin (FHb), hemoglobin (Hb) and mean corpuscular volume (MCV) in fresh RBCs and RBCs stored for 72 hours at 4 degrees C were compared, when loading condition was ensured. The results showed that with increase of incubation temperature, time and extracellular trehalose concentration, the loading of trehalose in RBCs also increased. Under the optimal loading condition, cytoplasmic trehalose concentration and free hemoglobin level of fresh RBCs and RBCs stored for 72 hours at 4 degrees C were 65.505 +/- 6.314 mmol/L, 66.2 +/- 5.002 mmol/L and 6.567 +/- 2.568 g/L, 16.168 +/- 3.922 g/L respectively. It is concluded that the most optimal condition of loading trehalose is that fresh RBCs incubate in 800 mmol/L trehalose solution for 8 hours at 37 degrees C. This condition can result in a efficient cytoplasmic trehalose concentration. The study provides an important basis for long-term preservation of RBCs.

Shape matters: the effect of red blood cell shape on perfusion of an artificial microvascular network.

PubMed

Piety, Nathaniel Z; Reinhart, Walter H; Pourreau, Patrick H; Abidi, Rajaa; Shevkoplyas, Sergey S

2016-04-01

The shape of human red blood cells (RBCs) deteriorates progressively throughout hypothermic storage, with echinocytosis being the most prevalent pathway of this morphologic lesion. As a result, each unit of stored blood contains a heterogeneous mixture of cells in various stages of echinocytosis and normal discocytes. Here we studied how the change in shape of RBCs following along the path of the echinocytic transformation affects perfusion of an artificial microvascular network (AMVN). Blood samples were obtained from healthy consenting volunteers. RBCs were leukoreduced, resuspended in saline, and treated with various concentrations of sodium salicylate to induce shape changes approximating the stages of echinocytosis experienced by RBCs during hypothermic storage (e.g., discocyte, echinocyte I, echinocyte II, echinocyte III, spheroechinocyte, and spherocyte). The AMVN perfusion rate was measured for 40% hematocrit suspensions of RBCs with different shapes. The AMVN perfusion rates for RBCs with discocyte and echinocyte I shapes were similar, but there was a significant decline in the AMVN perfusion rate between RBCs with shapes approximating each subsequent stage of echinocytosis. The difference in AMVN perfusion between discocytes and spherocytes (the last stage of the echinocytic transformation) was 34%. The change in shape of RBCs from normal discocytes progressively through various stages of echinocytosis to spherocytes produced a substantial decline in the ability of these cells to perfuse an AMVN. Echinocytosis induced by hypothermic storage could therefore be responsible for a similarly substantial impairment of deformability previously observed for stored RBCs. © 2015 AABB.

Glycomics-based analysis of chicken red blood cells provides insight into the selectivity of the viral agglutination assay.

PubMed

Aich, Udayanath; Beckley, Nia; Shriver, Zachary; Raman, Rahul; Viswanathan, Karthik; Hobbie, Sven; Sasisekharan, Ram

2011-05-01

Agglutination of red blood cells (RBCs), including chicken RBCs (cRBCs), has been used extensively to estimate viral titer, to screen glycan-receptor binding preference, and to assess the protective response of vaccines. Although this assay enjoys widespread use, some virus strains do not agglutinate RBCs. To address these underlying issues and to increase the usefulness of cRBCs as tools for studying viruses, such as influenza, we analyzed the cell surface N-glycans of cRBCs. On the basis of the results obtained from complementary analytical strategies, including MS, 1D and 2D-NMR spectroscopy, exoglycosidase digestions, and HPLC profiling, we report the major glycan structures present on cRBCs. By comparing the glycan structures of cBRCs with those of representative human upper respiratory cells, we offer a possible explanation for the fact that certain influenza strains do not agglutinate cRBCs, using specific human-adapted influenza hemagglutinins as examples. Finally, recent understanding of the role of various glycan structures in high affinity binding to influenza hemagglutinins provides context to our findings. These results illustrate that the field of glycomics can provide important information with respect to the experimental systems used to characterize, detect and study viruses. © 2011 The Authors Journal compilation © 2011 FEBS.

Extravascular red blood cells and hemoglobin promote tumor growth and therapeutic resistance as endogenous danger signals.

PubMed

Yin, Tao; He, Sisi; Liu, Xiaoling; Jiang, Wei; Ye, Tinghong; Lin, Ziqiang; Sang, Yaxiong; Su, Chao; Wan, Yang; Shen, Guobo; Ma, Xuelei; Yu, Min; Guo, Fuchun; Liu, Yanyang; Li, Ling; Hu, Qiancheng; Wang, Yongsheng; Wei, Yuquan

2015-01-01

Hemorrhage is a common clinical manifestation in patients with cancer. Intratumor hemorrhage has been demonstrated to be a poor prognostic factor for cancer patients. In this study, we investigated the role of RBCs and hemoglobin (Hb) in the process of tumor progression and therapeutical response. RBCs and Hb potently promoted tumor cell proliferation and syngenic tumor growth. RBCs and Hb activated the reactive oxygen species-NF-κB pathway in both tumor cells and macrophages. RBCs and Hb also induced chemoresistance mediated, in part, by upregulating ABCB1 gene expression. Tumor growth induced by RBCs was accompanied by an inflammatory signature, increased tumor vasculature, and influx of M2 macrophages. In both the peritoneal cavity and tumor microenvironment, extravascular RBCs rapidly recruited monocyte-macrophages into the lesion sites. In addition, RBCs and Hb increased several nucleotide-binding oligomerization domain-like receptors' expression and induced IL-1β release. Our results provide novel insights into the protumor function of RBCs and Hb as endogenous danger signals, which can promote tumor cell proliferation, macrophage recruitment, and polarization. Hemorrhage may represent a useful prognostic factor for cancer patients because of its role in tumor promotion and chemoresistance. Copyright © 2014 by The American Association of Immunologists, Inc.

Microconfined flow behavior of red blood cells.

PubMed

Tomaiuolo, Giovanna; Lanotte, Luca; D'Apolito, Rosa; Cassinese, Antonio; Guido, Stefano

2016-01-01

Red blood cells (RBCs) perform essential functions in human body, such as gas exchange between blood and tissues, thanks to their ability to deform and flow in the microvascular network. The high RBC deformability is mainly due to the viscoelastic properties of the cell membrane. Since an impaired RBC deformability could be found in some diseases, such as malaria, sickle cell anemia, diabetes and hereditary disorders, there is the need to provide further insight into measurement of RBC deformability in a physiologically relevant flow field. Here, RBCs deformability has been studied in terms of the minimum apparent plasma-layer thickness by using high-speed video microscopy of RBCs flowing in cylindrical glass capillaries. An in vitro systematic microfluidic investigation of RBCs in micro-confined conditions has been performed, resulting in the determination of the RBCs time recovery constant, RBC volume and surface area and RBC membrane shear elastic modulus and surface viscosity. It has been noticed that the deformability of RBCs induces cells aggregation during flow in microcapillaries, allowing the formation of clusters of cells. Overall, our results provide a novel technique to estimate RBC deformability and also RBCs collective behavior, which can be used for the analysis of pathological RBCs, for which reliable quantitative methods are still lacking. Copyright © 2015 IPEM. Published by Elsevier Ltd. All rights reserved.

Activation of interspecies-hybrid Rubisco enzymes to assess different models for the Rubisco-Rubisco activase interaction.

PubMed

Wachter, Rebekka M; Salvucci, Michael E; Carmo-Silva, A Elizabete; Barta, Csengele; Genkov, Todor; Spreitzer, Robert J

2013-11-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is prone to inactivation from non-productive binding of sugar-phosphates. Reactivation of Rubisco requires conformational remodeling by a specific chaperone, Rubisco activase. Rubisco activase from tobacco and other plants in the family Solanaceae is an inefficient activator of Rubisco from non-Solanaceae plants and from the green alga Chlamydomonas reinhardtii. To determine if the Rubisco small subunit plays a role in the interaction with Rubisco activase, a hybrid Rubisco (SSNT) composed of tobacco small subunits and Chlamydomonas large subunits was constructed. The SSNT hybrid, like other hybrid Rubiscos containing plant small subunits, supported photoautotrophic growth in Chlamydomonas, but growth in air was much slower than for cells containing wild-type Rubisco. The kinetic properties of the SSNT hybrid Rubisco were similar to the wild-type enzyme, indicating that the poor growth in air was probably caused by disruption of pyrenoid formation and the consequent impairment of the CO2concentrating mechanism. Recombinant Rubisco activase from Arabidopsis activated the SSNT hybrid Rubisco and hybrid Rubiscos containing spinach and Arabidopsis small subunits at rates similar to the rates with wild-type Rubisco. However, none of the hybrid Rubiscos was activated by tobacco Rubisco activase. That replacement of Chlamydomonas small subunits with plant small subunits does not affect the species-specific interaction between Rubisco and Rubisco activase suggests that the association is not dominated by the small subunits that surround the Rubisco central solvent channel. Therefore, the geometry of a side-on binding mode is more consistent with the data than a top-on or ring-stacking binding mode.

Characterization of the Genes Encoding the Cytosolic and Plastidial Forms of ADP-Glucose Pyrophosphorylase in Wheat Endosperm1

PubMed Central

Burton, Rachel A.; Johnson, Philip E.; Beckles, Diane M.; Fincher, Geoffrey B.; Jenner, Helen L.; Naldrett, Mike J.; Denyer, Kay

2002-01-01

In most species, the synthesis of ADP-glucose (Glc) by the enzyme ADP-Glc pyrophosphorylase (AGPase) occurs entirely within the plastids in all tissues so far examined. However, in the endosperm of many, if not all grasses, a second form of AGPase synthesizes ADP-Glc outside the plastid, presumably in the cytosol. In this paper, we show that in the endosperm of wheat (Triticum aestivum), the cytosolic form accounts for most of the AGPase activity. Using a combination of molecular and biochemical approaches to identify the cytosolic and plastidial protein components of wheat endosperm AGPase we show that the large and small subunits of the cytosolic enzyme are encoded by genes previously thought to encode plastidial subunits, and that a gene, Ta.AGP.S.1, which encodes the small subunit of the cytosolic form of AGPase, also gives rise to a second transcript by the use of an alternate first exon. This second transcript encodes an AGPase small subunit with a transit peptide. However, we could not find a plastidial small subunit protein corresponding to this transcript. The protein sequence of the purified plastidial small subunit does not match precisely to that encoded by Ta.AGP.S.1 or to the predicted sequences of any other known gene from wheat or barley (Hordeum vulgare). Instead, the protein sequence is most similar to those of the plastidial small subunits from chickpea (Cicer arietinum) and maize (Zea mays) and rice (Oryza sativa) seeds. These data suggest that the gene encoding the major plastidial small subunit of AGPase in wheat endosperm has yet to be identified. PMID:12428011

Pf155/RESA protein influences the dynamic microcirculatory behavior of ring-stage Plasmodium falciparum infected red blood cells

PubMed Central

Diez-Silva, Monica; Park, YongKeun; Huang, Sha; Bow, Hansen; Mercereau-Puijalon, Odile; Deplaine, Guillaume; Lavazec, Catherine; Perrot, Sylvie; Bonnefoy, Serge; Feld, Michael S.; Han, Jongyoon; Dao, Ming; Suresh, Subra

2012-01-01

Proteins exported by Plasmodium falciparum to the red blood cell (RBC) membrane modify the structural properties of the parasitized RBC (Pf-RBC). Although quasi-static single cell assays show reduced ring-stage Pf-RBCs deformability, the parameters influencing their microcirculatory behavior remain unexplored. Here, we study the dynamic properties of ring-stage Pf-RBCs and the role of the parasite protein Pf155/Ring-Infected Erythrocyte Surface Antigen (RESA). Diffraction phase microscopy revealed RESA-driven decreased Pf-RBCs membrane fluctuations. Microfluidic experiments showed a RESA-dependent reduction in the Pf-RBCs transit velocity, which was potentiated at febrile temperature. In a microspheres filtration system, incubation at febrile temperature impaired traversal of RESA-expressing Pf-RBCs. These results show that RESA influences ring-stage Pf-RBCs microcirculation, an effect that is fever-enhanced. This is the first identification of a parasite factor influencing the dynamic circulation of young asexual Pf-RBCs in physiologically relevant conditions, offering novel possibilities for interventions to reduce parasite survival and pathogenesis in its human host. PMID:22937223

Pf155/RESA protein influences the dynamic microcirculatory behavior of ring-stage Plasmodium falciparum infected red blood cells

NASA Astrophysics Data System (ADS)

Diez-Silva, Monica; Park, Yongkeun; Huang, Sha; Bow, Hansen; Mercereau-Puijalon, Odile; Deplaine, Guillaume; Lavazec, Catherine; Perrot, Sylvie; Bonnefoy, Serge; Feld, Michael S.; Han, Jongyoon; Dao, Ming; Suresh, Subra

2012-08-01

Proteins exported by Plasmodium falciparum to the red blood cell (RBC) membrane modify the structural properties of the parasitized RBC (Pf-RBC). Although quasi-static single cell assays show reduced ring-stage Pf-RBCs deformability, the parameters influencing their microcirculatory behavior remain unexplored. Here, we study the dynamic properties of ring-stage Pf-RBCs and the role of the parasite protein Pf155/Ring-Infected Erythrocyte Surface Antigen (RESA). Diffraction phase microscopy revealed RESA-driven decreased Pf-RBCs membrane fluctuations. Microfluidic experiments showed a RESA-dependent reduction in the Pf-RBCs transit velocity, which was potentiated at febrile temperature. In a microspheres filtration system, incubation at febrile temperature impaired traversal of RESA-expressing Pf-RBCs. These results show that RESA influences ring-stage Pf-RBCs microcirculation, an effect that is fever-enhanced. This is the first identification of a parasite factor influencing the dynamic circulation of young asexual Pf-RBCs in physiologically relevant conditions, offering novel possibilities for interventions to reduce parasite survival and pathogenesis in its human host.

Probing the In Vitro Cytotoxicity of the Veterinary Drug Oxytetracycline

PubMed Central

Chi, Zhenxing; Liu, Rutao; You, Hong; Ma, Shanshan; Cui, Hao; Zhang, Qiang

2014-01-01

The study investigated the effect of oxytetracycline (OTC) on the anti-oxidative defense system, the structure (hemolysis rate and morphology) and function (ATP enzyme activity) of human red blood cells (hRBCs) to investigate the possible toxic mechanism of OTC to hRBCs. The experimental results indicate that OTC can cause a decline in the function of the antioxidant defense system of hRBCs, resulting in oxidative stress. OTC can bring about morphological changes to hRBCs, and further leads to hemolysis, when the concentration of OTC is over 8×10−5 M (about 164 µg/ml). At a low OTC concentration, below 4×10−5 M (82 µg/ml), OTC can enhance the activity of ATP enzyme of hRBCs, known as hormesis. However, at a high concentration, above 4×10−5 M (about 82 µg/ml), the ATP enzymatic activity was inhibited, affecting the function of hRBCs. The estalished mechanism of toxicity of OTC to hRBCs can facilitate a deeper understanding of the toxicity of OTC in vivo. PMID:25019386

A mesoscopic simulation on distributions of red blood cells in a bifurcating channel

NASA Astrophysics Data System (ADS)

Inoue, Yasuhiro; Takagi, Shu; Matsumoto, Yoichiro

2004-11-01

Transports of red blood cells (RBCs) or particles in bifurcated channels have been attracting renewed interest since the advent of concepts of MEMS for sorting, analyzing, and removing cells or particles from sample medium. In this talk, we present a result on a transport of red blood cells (RBCs) in a bifurcating channel studied by using a mesoscale simulation technique of immiscible droplets, where RBCs have been modeled as immiscible droplets. The distribution of RBCs is represented by the fractional RBC flux into two daughters as a function of volumetric flow ratio between the daughters. The data obtained in our simulations are examined with a theoretical prediction, in which, we assume an exponential distribution for positions of RBCs in the mother channel. The theoretical predictions show a good agreement with simulation results. A non-uniform distribution of RBCs in the mother channel affects disproportional separation of RBC flux at a bifurcation.

A definition of the domains Archaea, Bacteria and Eucarya in terms of small subunit ribosomal RNA characteristics

NASA Technical Reports Server (NTRS)

Winker, S.; Woese, C. R.

1991-01-01

The number of small subunit rRNA sequences is now great enough that the three domains Archaea, Bacteria and Eucarya (Woese et al., 1990) can be reliably defined in terms of their sequence "signatures". Approximately 50 homologous positions (or nucleotide pairs) in the small subunit rRNA characterize and distinguish among the three. In addition, the three can be recognized by a variety of nonhomologous rRNA characters, either individual positions and/or higher-order structural features. The Crenarchaeota and the Euryarchaeota, the two archaeal kingdoms, can also be defined and distinguished by their characteristic compositions at approximately fifteen positions in the small subunit rRNA molecule.

Single-photon emission computed tomography enhanced Tc-99m-pertechnetate disodium-labelled red blood cell scintigraphy in the localization of small intestine bleeding: a single-centre twelve-year study.

PubMed

Dolezal, Jiri; Vizda, Jaroslav; Kopacova, Marcela

2011-01-01

To present our experience with the detection of bleeding in the small intestine by means of scintigraphy with in vivo-labelled red blood cells (RBCs) in the period of 1998-2009. A 12-year prospective study was accomplished with 40 patients (23 men, 17 women, aged 12-91, mean 56 years) who had lower gastrointestinal bleeding (obscure-overt bleeding) and underwent scintigraphy with in vivo-labelled RBCs by means of technetium 99m. The scintigraphy was usually performed after other diagnostic tests had failed to locate the bleeding. A total of 26 patients had a positive scintigraphy with in vivo-labelled RBCs and 14 patients had negative scintigraphy. The final diagnosis was confirmed in 20 of 26 patients with a positive scintigraphy by push enteroscopy (6/20), intraoperative enteroscopy (7/20), surgery (4/20), duodenoscopy (1/20), double-balloon enteroscopy (1/20) and X-ray angiography (1/20). The correct location of the bleeding site was identified by RBC scintigraphy in 15 of 20 (75%) patients with the confirmed source. The locations of the bleeding site identified by scintigraphy and enteroscopy (push, intraoperative) and surgical investigations were highly correlated in patients with a positive scintigraphy within the first 3 h. Eleven of the 20 correctly localized studies and none of the incorrectly localized studies were positive in the dynamic phase of imaging. In 5 patients (all erroneously localized), scintigraphy was positive only at a period longer than 18 h. RBC scintigraphy is an effective imaging modality in localizing lower gastrointestinal bleeding in patients for whom other diagnostic tests have failed to locate the bleeding. RBC scintigraphy can be successful in the detection of bleeding sites in the small intestine. Copyright © 2011 S. Karger AG, Basel.

Small things considered: the small accessory subunits of RNA polymerase in Gram-positive bacteria

PubMed Central

Weiss, Andy; Shaw, Lindsey N.

2015-01-01

The DNA-dependent RNA polymerase core enzyme in Gram-positive bacteria consists of seven subunits. Whilst four of them (α2ββ′) are essential, three smaller subunits, δ, ε and ω (∼9–21.5 kDa), are considered accessory. Both δ and ω have been viewed as integral components of RNAP for several decades; however, ε has only recently been described. Functionally these three small subunits carry out a variety of tasks, imparting important, supportive effects on the transcriptional process of Gram-positive bacteria. While ω is thought to have a wide range of roles, reaching from maintaining structural integrity of RNAP to σ factor recruitment, the only suggested function for ε thus far is in protecting cells from phage infection. The third subunit, δ, has been shown to have distinct influences in maintaining transcriptional specificity, and thus has a key role in cellular fitness. Collectively, all three accessory subunits, although dispensable under laboratory conditions, are often thought to be crucial for proper RNAP function. Herein we provide an overview of the available literature on each subunit, summarizing landmark findings that have deepened our understanding of these proteins and their function, and outline future challenges in understanding the role of these small subunits in the transcriptional process. PMID:25878038

Shape-Shifted Red Blood Cells: A Novel Red Blood Cell Stage?

PubMed Central

Chico, Verónica; Puente-Marin, Sara; Ciordia, Sergio; Mena, María Carmen; Carracedo, Begoña; Mercado, Luis; Coll, Julio

2018-01-01

Primitive nucleated erythroid cells in the bloodstream have long been suggested to be more similar to nucleated red cells of fish, amphibians, and birds than the red cells of fetal and adult mammals. Rainbow trout Ficoll-purified red blood cells (RBCs) cultured in vitro undergo morphological changes, especially when exposed to stress, and enter a new cell stage that we have coined shape-shifted RBCs (shRBCs). We have characterized these shRBCs using transmission electron microscopy (TEM) micrographs, Wright–Giemsa staining, cell marker immunostaining, and transcriptomic and proteomic evaluation. shRBCs showed reduced density of the cytoplasm, hemoglobin loss, decondensed chromatin in the nucleus, and striking expression of the B lymphocyte molecular marker IgM. In addition, shRBCs shared some features of mammalian primitive pyrenocytes (extruded nucleus surrounded by a thin rim of cytoplasm and phosphatidylserine (PS) exposure on cell surface). These shRBCs were transiently observed in heat-stressed rainbow trout bloodstream for three days. Functional network analysis of combined transcriptomic and proteomic studies resulted in the identification of proteins involved in pathways related to the regulation of cell morphogenesis involved in differentiation, cellular response to stress, and immune system process. In addition, shRBCs increased interleukin 8 (IL8), interleukin 1 β (IL1β), interferon ɣ (IFNɣ), and natural killer enhancing factor (NKEF) protein production in response to viral hemorrhagic septicemia virus (VHSV). In conclusion, shRBCs may represent a novel cell stage that participates in roles related to immune response mediation, homeostasis, and the differentiation and development of blood cells. PMID:29671811

Salidroside as a Novel Protective Agent to Improve Red Blood Cell Cryopreservation.

PubMed

Alotaibi, Noha A S; Slater, Nigel K H; Rahmoune, Hassan

2016-01-01

Glycerol and trehalose have been widely examined as protective agents in the cryopreservation of red blood cells (RBCs). However, the effectiveness of these reagents alone on cell viability is moderate. Here, the addition of salidroside attenuated oxidative damage of sheep RBCs prior to and post cryostorage. The supplementation of salidroside to the cryopreservation media containing 10% glycerol improved RBC survival by approximately 61.1±4.8% vs 37.9±4.6%. A smaller effect was seen in RBCs cryopreserved in 300 mM trehalose where the addition of salidroside improved survival by 7.6±0.3%. Furthermore, the addition of salidroside to cold storage solution demonstrated a significant reduction of haemolysis after 4 days for RBCs loaded with either glycerol or trehalose, compared to cells incubated without salidroside. RBCs survival was 2-fold greater following freezing in trehalose, compared with glycerol. After 10 days, salidroside enabled a lower haemolysis of 16.7±1.3% compared to 29.0±8.4% for cells incubated without salidroside. However, salidroside had no effect on RBCs which had been frozen in glycerol as the resulting haemolysis rate by day 10 was approximately 60%. Salidroside increased glutathione reductase activity and decreased lactate dehydrogenase activity. Furthermore, it led to reduced carbonylation of proteins in both glycerol and trehalose loaded cells. Finally, no effect on lipid peroxidation was found in the glycerol loaded RBCs although this was reduced in RBCs loaded with trehalose and salidroside. The present findings confirm the potential use of salidroside as a novel protective agent in cryopreservation and refrigerated storage of sheep RBCs.

Riboflavin-ultraviolet light pathogen reduction treatment does not impact the immunogenicity of murine red blood cells.

PubMed

Tormey, Christopher A; Santhanakrishnan, Manjula; Smith, Nicole H; Liu, Jingchun; Marschner, Susanne; Goodrich, Raymond P; Hendrickson, Jeanne E

2016-04-01

Ultraviolet (UV) illumination/pathogen reduction effectively inactivates white blood cells (WBCs) in whole blood. Given that cotransfused WBCs may impact recipient immune responses, we hypothesized that pathogen reduction of whole blood may alter responses to RBC antigens. Transgenic mice expressing a model (HOD) antigen, authentic human (hGPA or KEL) antigens, or natural fluorescence (uGFP) on their RBCs were utilized as blood donors. Recipients were transfused with fresh whole blood to which riboflavin had been added or fresh whole blood treated by UV illumination/pathogen reduction treatment after the addition of riboflavin. Posttransfusion RBC recovery, survival, and alloimmunization were measured by flow cytometry. UV illumination/pathogen reduction treatment did not alter RBC antigen expression, and recipients of treated syngeneic RBCs had persistently negative direct antiglobulin tests. Greater than 75% of treated and untreated syngeneic RBCs were recovered 24 hours posttransfusion in all experiments, although alterations in the long-term posttransfusion survival of treated RBCs were observed. Treated and untreated KEL RBCs induced similar recipient alloimmune responses, with all recipients making anti-KEL glycoprotein immunoglobulins (p > 0.05). Alloimmune responses to treated HOD or hGPA RBCs were no different from untreated RBCs (p > 0.05). Pathogen inactivation treatment of fresh whole murine blood with riboflavin and UV illumination does not impact the rate or magnitude of RBC alloimmunization to three distinct RBC antigens. Further, UV illumination/pathogen reduction appears safe from an immunohematologic standpoint, with no immunogenic neoantigens detected on treated murine RBCs. Future studies with fresh and stored human RBCs are warranted to confirm these findings. © 2015 AABB.

Shape-Shifted Red Blood Cells: A Novel Red Blood Cell Stage?

PubMed

Chico, Verónica; Puente-Marin, Sara; Nombela, Iván; Ciordia, Sergio; Mena, María Carmen; Carracedo, Begoña; Villena, Alberto; Mercado, Luis; Coll, Julio; Ortega-Villaizan, María Del Mar

2018-04-19

Primitive nucleated erythroid cells in the bloodstream have long been suggested to be more similar to nucleated red cells of fish, amphibians, and birds than the red cells of fetal and adult mammals. Rainbow trout Ficoll-purified red blood cells (RBCs) cultured in vitro undergo morphological changes, especially when exposed to stress, and enter a new cell stage that we have coined shape-shifted RBCs (shRBCs). We have characterized these shRBCs using transmission electron microscopy (TEM) micrographs, Wright⁻Giemsa staining, cell marker immunostaining, and transcriptomic and proteomic evaluation. shRBCs showed reduced density of the cytoplasm, hemoglobin loss, decondensed chromatin in the nucleus, and striking expression of the B lymphocyte molecular marker IgM. In addition, shRBCs shared some features of mammalian primitive pyrenocytes (extruded nucleus surrounded by a thin rim of cytoplasm and phosphatidylserine (PS) exposure on cell surface). These shRBCs were transiently observed in heat-stressed rainbow trout bloodstream for three days. Functional network analysis of combined transcriptomic and proteomic studies resulted in the identification of proteins involved in pathways related to the regulation of cell morphogenesis involved in differentiation, cellular response to stress, and immune system process. In addition, shRBCs increased interleukin 8 (IL8), interleukin 1 β (IL1β), interferon ɣ (IFNɣ), and natural killer enhancing factor (NKEF) protein production in response to viral hemorrhagic septicemia virus (VHSV). In conclusion, shRBCs may represent a novel cell stage that participates in roles related to immune response mediation, homeostasis, and the differentiation and development of blood cells.

Molecular cloning of human protein 4.2: a major component of the erythrocyte membrane.

PubMed Central

Sung, L A; Chien, S; Chang, L S; Lambert, K; Bliss, S A; Bouhassira, E E; Nagel, R L; Schwartz, R S; Rybicki, A C

1990-01-01

Protein 4.2 (P4.2) comprises approximately 5% of the protein mass of human erythrocyte (RBC) membranes. Anemia occurs in patients with RBCs deficient in P4.2, suggesting a role for this protein in maintaining RBC stability and integrity. We now report the molecular cloning and characterization of human RBC P4.2 cDNAs. By immunoscreening a human reticulocyte cDNA library and by using the polymerase chain reaction, two cDNA sequences of 2.4 and 2.5 kilobases (kb) were obtained. These cDNAs differ only by a 90-base-pair insert in the longer isoform located three codons downstream from the putative initiation site. The 2.4- and 2.5-kb cDNAs predict proteins of approximately 77 and approximately 80 kDa, respectively, and the authenticity was confirmed by sequence identity with 46 amino acids of three cyanogen bromide-cleaved peptides of P4.2. Northern blot analysis detected a major 2.4-kb RNA species in reticulocytes. Isolation of two P4.2 cDNAs implies existence of specific regulation of P4.2 expression in human RBCs. Human RBC P4.2 has significant homology with human factor XIII subunit a and guinea pig liver transglutaminase. Sequence alignment of P4.2 with these two transglutaminases, however, revealed that P4.2 lacks the critical cysteine residue required for the enzymatic crosslinking of substrates. Images PMID:1689063

Photosynthetic characteristics in genetically modified sense-RbcS silver birch lines.

PubMed

Kontunen-Soppela, Sari; Sillanpää, Maarit; Tuhkanen, Eeva-Maria; Sutinen, Sirkka; Kangasjärvi, Jaakko; Vapaavuori, Elina; Häggman, Hely

2010-07-01

Transgenic silver birch lines carrying extra copies of endogenous small subunit of Rubisco (RbcS)-gene under 35S CaMV promoter were used to study the carbon use efficiency of silver birch (Betula pendula Roth). A five week greenhouse experiment was carried out with four transgenic lines, R3.2, R7.2, E5 and E25, and their corresponding wild types (wt). The first fully developed leaves were used for analyses. Three of the produced lines, R3.2, E5 and E25, differed from the wt lines. Line R3.2 showed an altered growth rhythm; its chlorophyll content, Rubisco amount and activity as well as photosynthetic characteristics were reduced at the beginning of the experiment, which resulted in decreased biomass and growth. In lines E25 and E5, the biomass accumulation was shifted to roots, and in line E25, the total biomass was also reduced. In line E25, the differences were particularly marked in the dry mass, indicating a difference in water use, seen as increased transpiration. Introduction of sense RbcS decreased the Rubisco amount in birch leaves to 80% of wt at times during the tree development, but the lower amount of Rubisco was usually not seen in photosynthesis. The accumulation and distribution of biomass within the plants was altered. 2010 Elsevier GmbH. All rights reserved.

Changes in growth, carbon and nitrogen enzyme activity and mRNA accumulation in the halophilic microalga Dunaliella viridis in response to NaCl stress

NASA Astrophysics Data System (ADS)

Wang, Dongmei; Wang, Weiwei; Xu, Nianjun; Sun, Xue

2016-12-01

Many species of microalga Dunaliella exhibit a remarkable tolerance to salinity and are therefore ideal for probing the effects of salinity. In this work, we assessed the effects of NaCl stress on the growth, activity and mRNA level of carbon and nitrogen metabolism enzymes of D. viridis. The alga could grow over a salinity range of 0.44 mol L-1 to 3.00 mol L-1 NaCl, but the most rapid growth was observed at 1.00 mol L-1 NaCl, followed by 2.00 mol L-1 NaCl. Paralleling these growth patterns, the highest initial and total Rubisco activities were detected in the presence of 1.00 mol L-1 NaCl, decreasing to 37.33% and 26.39% of those values, respectively, in the presence of 3.00 mol L-1 NaCl, respectively. However, the highest extracellular carbonic anhydrase (CA) activity was measured in the presence of 2.00 mol L-1 NaCl, followed by 1.00 mol L-1 NaCl. Different from the two carbon enzymes, nitrate reductase (NR) activity showed a slight change under different NaCl concentrations. At the transcriptional level, the mRNAs of Rubisco large subunit ( rbcL), and small subunit ( rbcS), attained their highest abundances in the presence of 1.00 and 2.00 mol L-1 NaCl, respectively. The CA mRNA accumulation was induced from 0.44 mol L-1 to 3.00 mol L-1 NaCl, but the NR mRNA showed the decreasing tendency with the increasing salinity. In conclusion, the growth and carbon fixation enzyme of Rubisco displayed similar tendency in response to NaCl stress, CA was proved be salt-inducible within a certain salinity range and NR showed the least effect by NaCl in D. viridis.

Ultracold Polar Molecules

DTIC Science & Technology

2016-04-01

large positive scattering lengths make clouds difficult to overlap (but 85RbCs looked promising), but 85RbCs is difficult because 85Rb is hard to...produce at high densities. We revealed broad Feshbach resonances that we hope will allow production of higher-density 85Rb clouds . We are now...and this makes clouds of 87Rb and Cs hard to overlap. We therefore investi- gated 85RbCs, using the potentials we had developed for 85RbCs, and showed

Age Decline in the Activity of the Ca2+-sensitive K+ Channel of Human Red Blood Cells

PubMed Central

Tiffert, Teresa; Daw, Nuala; Etzion, Zipora; Bookchin, Robert M.; Lew, Virgilio L.

2007-01-01

The Ca2+-sensitive K+ channel of human red blood cells (RBCs) (Gardos channel, hIK1, hSK4) was implicated in the progressive densification of RBCs during normal senescence and in the mechanism of sickle cell dehydration. Saturating RBC Ca2+ loads were shown before to induce rapid and homogeneous dehydration, suggesting that Gardos channel capacity was uniform among the RBCs, regardless of age. Using glycated hemoglobin as a reliable RBC age marker, we investigated the age–activity relation of Gardos channels by measuring the mean age of RBC subpopulations exceeding a set high density boundary during dehydration. When K+ permeabilization was induced with valinomycin, the oldest and densest cells, which started nearest to the set density boundary, crossed it first, reflecting conservation of the normal age–density distribution pattern during dehydration. However, when Ca2+ loads were used to induce maximal K+ fluxes via Gardos channels in all RBCs (F max), the youngest RBCs passed the boundary first, ahead of the older RBCs, indicating that Gardos channel F max was highest in those young RBCs, and that the previously observed appearance of uniform dehydration concealed a substantial degree of age scrambling during the dehydration process. Further analysis of the Gardos channel age–activity relation revealed a monotonic decline in F max with cell age, with a broad quasi-Gaussian F max distribution among the RBCs. PMID:17470662

Age decline in the activity of the Ca2+-sensitive K+ channel of human red blood cells.

PubMed

Tiffert, Teresa; Daw, Nuala; Etzion, Zipora; Bookchin, Robert M; Lew, Virgilio L

2007-05-01

The Ca(2+)-sensitive K(+) channel of human red blood cells (RBCs) (Gardos channel, hIK1, hSK4) was implicated in the progressive densification of RBCs during normal senescence and in the mechanism of sickle cell dehydration. Saturating RBC Ca(2+) loads were shown before to induce rapid and homogeneous dehydration, suggesting that Gardos channel capacity was uniform among the RBCs, regardless of age. Using glycated hemoglobin as a reliable RBC age marker, we investigated the age-activity relation of Gardos channels by measuring the mean age of RBC subpopulations exceeding a set high density boundary during dehydration. When K(+) permeabilization was induced with valinomycin, the oldest and densest cells, which started nearest to the set density boundary, crossed it first, reflecting conservation of the normal age-density distribution pattern during dehydration. However, when Ca(2+) loads were used to induce maximal K(+) fluxes via Gardos channels in all RBCs (F(max)), the youngest RBCs passed the boundary first, ahead of the older RBCs, indicating that Gardos channel F(max) was highest in those young RBCs, and that the previously observed appearance of uniform dehydration concealed a substantial degree of age scrambling during the dehydration process. Further analysis of the Gardos channel age-activity relation revealed a monotonic decline in F(max) with cell age, with a broad quasi-Gaussian F(max) distribution among the RBCs.

Mechanical characterization of human red blood cells under different osmotic conditions by robotic manipulation with optical tweezers.

PubMed

Tan, Youhua; Sun, Dong; Wang, Jinzhi; Huang, Wenhao

2010-07-01

The physiological functions of human red blood cells (RBCs) play a crucial role to human health and are greatly influenced by their mechanical properties. Any alteration of the cell mechanics may cause human diseases. The osmotic condition is an important factor to the physiological environment, but its effect on RBCs has been little studied. To investigate this effect, robotic manipulation technology with optical tweezers is utilized in this paper to characterize the mechanical properties of RBCs in different osmotic conditions. The effectiveness of this technology is demonstrated first in the manipulation of microbeads. Then the optical tweezers are used to stretch RBCs to acquire the force-deformation relationships. To extract cell properties from the experimental data, a mechanical model is developed for RBCs in hypotonic conditions by extending our previous work , and the finite element model is utilized for RBCs in isotonic and hypertonic conditions. Through comparing the modeling results to the experimental data, the shear moduli of RBCs in different osmotic solutions are characterized, which shows that the cell stiffness increases with elevated osmolality. Furthermore, the property variation and potential biomedical significance of this study are discussed. In conclusion, this study indicates that the osmotic stress has a significant effect on the cell properties of human RBCs, which may provide insight into the pathology analysis and therapy of some human diseases.

IB-LBM simulation on blood cell sorting with a micro-fence structure.

PubMed

Wei, Qiang; Xu, Yuan-Qing; Tian, Fang-bao; Gao, Tian-xin; Tang, Xiao-ying; Zu, Wen-Hong

2014-01-01

A size-based blood cell sorting model with a micro-fence structure is proposed in the frame of immersed boundary and lattice Boltzmann method (IB-LBM). The fluid dynamics is obtained by solving the discrete lattice Boltzmann equation, and the cells motion and deformation are handled by the immersed boundary method. A micro-fence consists of two parallel slope post rows which are adopted to separate red blood cells (RBCs) from white blood cells (WBCs), in which the cells to be separated are transported one after another by the flow into the passageway between the two post rows. Effected by the cross flow, RBCs are schemed to get through the pores of the nether post row since they are smaller and more deformable compared with WBCs. WBCs are required to move along the nether post row till they get out the micro-fence. Simulation results indicate that for a fix width of pores, the slope angle of the post row plays an important role in cell sorting. The cells mixture can not be separated properly in a small slope angle, while obvious blockages by WBCs will take place to disturb the continuous cell sorting in a big slope angle. As an optimal result, an adaptive slope angle is found to sort RBCs form WBCs correctly and continuously.

Ceramic Hollow Fibre Constructs for Continuous Perfusion and Cell Harvest from 3D Hematopoietic Organoids

PubMed Central

Tahlawi, Asma; Li, Kang

2018-01-01

Tissue vasculature efficiently distributes nutrients, removes metabolites, and possesses selective cellular permeability for tissue growth and function. Engineered tissue models have been limited by small volumes, low cell densities, and invasive cell extraction due to ineffective nutrient diffusion and cell-biomaterial attachment. Herein, we describe the fabrication and testing of ceramic hollow fibre membranes (HFs) able to separate red blood cells (RBCs) and mononuclear cells (MNCs) and be incorporated into 3D tissue models to improve nutrient and metabolite exchange. These HFs filtered RBCs from human umbilical cord blood (CB) suspensions of 20% RBCs to produce 90% RBC filtrate suspensions. When incorporated within 5 mL of 3D collagen-coated polyurethane porous scaffold, medium-perfused HFs maintained nontoxic glucose, lactate, pH levels, and higher cell densities over 21 days of culture in comparison to nonperfused 0.125 mL scaffolds. This hollow fibre bioreactor (HFBR) required a smaller per-cell medium requirement and operated at cell densities > 10-fold higher than current 2D methods whilst allowing for continuous cell harvest through HFs. Herein, we propose HFs to improve 3D cell culture nutrient and metabolite diffusion, increase culture volume and cell density, and continuously harvest products for translational cell therapy biomanufacturing protocols. PMID:29760729

Early resuscitation with polymerized bovine hemoglobin reverses acidosis, but not peripheral tissue oxygenation, in a severe hamster shock model.

PubMed

Wettstein, Reto; Tsai, Amy G; Harder, Yves; Erni, Dominique; Intaglietta, Marcos

2006-11-01

Awake hamsters equipped with the dorsal window chamber preparation were subjected to hemorrhage of 50% of the estimated blood volume. Initial resuscitation (25% of estimated blood volume) with polymerized bovine hemoglobin (PBH) or 10% hydroxyethyl starch (HES) occurred in concert with an equivolumetric bleeding to simulate the early, prehospital setting (exchange transfusion). Resuscitation (25% of estimated blood volume) without bleeding was performed with PBH, HES, or autologous red blood cells (HES-RBCs). Peripheral microcirculation, tissue oxygenation, and systemic hemodynamic and blood gas parameters were assessed. After exchange transfusion, base deficit was -8.6 +/- 3.7 mmol/L (PBH) and -5.1 +/- 5.3 mmol/L (HES) (not significant). Functional capillary density was 17% +/- 6% of baseline (PBH) and 31% +/- 11% (HES) (P < 0.05) and arteriolar diameter 73% +/- 3% of baseline (PBH) and 90% + 5% (HES) (P < 0.01). At the end, hemoglobin levels were 3.7 +/- 0.3 g/dL with HES, 8.2 +/- 0.6 g/dL with PBH, and 10.4 +/- 0.8 g/dL with HES-RBCs (P < 0.01 HES vs. PBH and HES-RBCs, P < 0.05 PBH vs. HES-RBCs). Base excess was restored to baseline with PBH and HES-RBCs, but not with HES (P < 0.05). Functional capillary density was 46% +/- 5% of baseline (PBH), 62% + 20% (HES-RBCs), and 36% +/- 19% (HES) (P < 0.01 HES-RBCs vs. HES). Peripheral oxygen delivery and consumption was highest with HES-RBCs, followed by PBH (P < 0.05 HES-RBCs vs. PBH, P < 0.01 HES-RBCs and PBH vs. HES). In conclusion, the PBH led to a correction of base deficit comparable to blood transfusion. However, oxygenation of the peripheral tissue was inferior with PBH. This was attributed to its negative impact on the peripheral microcirculation caused by arteriolar vasoconstriction.

Polymer-mediated immunocamouflage of red blood cells: effects of polymer size on antigenic and immunogenic recognition of allogeneic donor blood cells.

PubMed

Wang, DunCheng; Kyluik, Dana L; Murad, Kari L; Toyofuku, Wendy M; Scott, Mark D

2011-07-01

Developing a practical means of reducing alloimmunization in chronically transfused patients would be of significant clinical benefit. Immunocamouflaging red blood cells (RBCs) by membrane grafting of methoxypoly(ethylene glycol) (mPEG) may reduce the risk of allo-immunization. The results of this study showed that antibody recognition of non-ABO antigens was significantly reduced in an mPEG-dose- and polymer size-dependent manner, with higher molecular weight mPEGs providing better immunoprotection. Furthermore, in vivo immunogenicity was significantly reduced in mice serially transfused with mPEG-modified xenogeneic (sheep; sRBCs), allogeneic (C57Bl/6), or syngeneic (Balb/c) RBCs. Following a primary transfusion of sRBCs, mice receiving mPEG-sRBCs showed a >90% reduction in anti-sRBC IgG antibody levels. After two transfusions, mice receiving mPEG-sRBCs showed reductions of >80% in anti-sRBC IgG levels. Importantly, mPEG-modified autologous cells did not induce neoantigens or an immune (IgG or IgM) response. These data suggest that the global immunocamouflage of RBCs by polymer grafting may provide a safe and cost-effective means of reducing the risk of alloimmunization.

Piezo1 regulates mechanotransductive release of ATP from human RBCs.

PubMed

Cinar, Eyup; Zhou, Sitong; DeCourcey, James; Wang, Yixuan; Waugh, Richard E; Wan, Jiandi

2015-09-22

Piezo proteins (Piezo1 and Piezo2) are recently identified mechanically activated cation channels in eukaryotic cells and associated with physiological responses to touch, pressure, and stretch. In particular, human RBCs express Piezo1 on their membranes, and mutations of Piezo1 have been linked to hereditary xerocytosis. To date, however, physiological functions of Piezo1 on normal RBCs remain poorly understood. Here, we show that Piezo1 regulates mechanotransductive release of ATP from human RBCs by controlling the shear-induced calcium (Ca(2+)) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca(2+) influx decrease significantly. Remarkably, a critical extracellular Ca(2+) concentration is required to trigger significant ATP release, but membrane-associated ATP pools in RBCs also contribute to the release of ATP. Our results show how Piezo1 channels are likely to function in normal RBCs and suggest a previously unidentified mechanotransductive pathway in ATP release. Thus, we anticipate that the study will impact broadly on the research of red cells, cellular mechanosensing, and clinical studies related to red cell disorders and vascular disease.

Hemoglobin encapsulation in vesicles retards NO and CO binding and O2 release when perfused through narrow gas-permeable tubes.

PubMed

Sakai, Hiromi; Okuda, Naoto; Sato, Atsushi; Yamaue, Tatsuya; Takeoka, Shinji; Tsuchida, Eishun

2010-03-01

Intravenous administration of cell-free Hb induces vasoconstriction and circulatory disorders, presumably because of the intrinsic affinities to endogenous nitric oxide (NO) and carbon monoxide (CO) as vasorelaxation factors and because of the facilitated O(2) release that might induce autoregulatory vasoconstriction. We examined these gas reactions when Hb-containing solutions of four kinds were perfused through artificial narrow tubes at a practical Hb concentration (10 g/dl). Purified Hb solution, polymerized bovine Hb (Poly(B)Hb), encapsulated Hb [Hb-vesicles (HbV), 279 nm], and red blood cells (RBCs) were perfused through a gas-permeable narrow tube (25 microm inner diameter) at 1 mm/s centerline velocity. The level of reactions was determined microscopically based on the visible-light absorption spectrum of Hb. When the tube was immersed in NO and CO atmospheres, both NO binding and CO binding of deoxygenated Hb (deoxy-Hb) and Poly(B)Hb in the tube was faster than those of HbV and RBCs, and HbV and RBCs showed almost identical binding rates. When the tube was immersed in a N(2) atmosphere, oxygenated Hb and Poly(B)Hb showed much faster O(2) release than did HbV and RBCs. Poly(B)Hb showed a faster reaction than Hb because of the lower O(2) affinity of Poly(B)Hb than Hb. The diffusion process of the particles was simulated using Navier-Stokes and Maxwell-Stefan equations. Results clarified that small Hb (6 nm) diffuses laterally and mixes rapidly. However, the large-dimension HbV shows no such rapid diffusion. The purely physicochemical differences in diffusivity of the particles and the resulting reactivity with gas molecules are one factor inducing biological vasoconstriction of Hb-based oxygen carriers.

Subunit Conformations and Assembly States of a DNA Translocating Motor: The Terminase of Bacteriophage P22

PubMed Central

Němeček, Daniel; Gilcrease, Eddie B.; Kang, Sebyung; Prevelige, Peter E.; Casjens, Sherwood; Thomas, George J.

2007-01-01

Bacteriophage P22, a podovirus infecting strains of Salmonella typhimurium, packages a 42 kbp genome using a headful mechanism. DNA translocation is accomplished by the phage terminase, a powerful molecular motor consisting of large and small subunits. Although many of the structural proteins of the P22 virion have been well characterized, little is known about the terminase subunits and their molecular mechanism of DNA translocation. We report here structural and assembly properties of ectopically expressed and highly purified terminase large and small subunits. The large subunit (gp2), which contains the nuclease and ATPase activities of terminase, exists as a stable monomer with an α/β fold. The small subunit (gp3), which recognizes DNA for packaging and may regulate gp2 activity, exhibits a highly α-helical secondary structure and self-associates to form a stable oligomeric ring in solution. For wildtype gp3, the ring contains nine subunits, as demonstrated by hydrodynamic measurements, electron microscopy and native mass spectrometry. We have also characterized a gp3 mutant (Ala 112 → Thr) that forms a ten subunit ring, despite a subunit fold indistinguishable from wildtype. Both the nonameric and decameric gp3 rings exhibit nonspecific DNA binding activity, and gp2 is able to bind strongly to the DNA/gp3 complex but not to DNA alone. We propose a scheme for the roles of P22 terminase large and small subunits in the recruitment and packaging of viral DNA and discuss the model in relation to proposals for terminase-driven DNA translocation in other phages. PMID:17945256

Biocompatible coupling of therapeutic fusion proteins to human erythrocytes

PubMed Central

Villa, Carlos H.; Pan, Daniel C.; Johnston, Ian H.; Greineder, Colin F.; Walsh, Landis R.; Hood, Elizabeth D.; Cines, Douglas B.; Poncz, Mortimer; Siegel, Don L.

2018-01-01

Carriage of drugs by red blood cells (RBCs) modulates pharmacokinetics, pharmacodynamics, and immunogenicity. However, optimal targets for attaching therapeutics to human RBCs and adverse effects have not been studied. We engineered nonhuman-primate single-chain antibody fragments (scFvs) directed to human RBCs and fused scFvs with human thrombomodulin (hTM) as a representative biotherapeutic cargo (hTM-scFv). Binding fusions to RBCs on band 3/glycophorin A (GPA; Wright b [Wrb] epitope) and RhCE (Rh17/Hr0 epitope) similarly endowed RBCs with hTM activity, but differed in their effects on RBC physiology. scFv and hTM-scFv targeted to band 3/GPA increased membrane rigidity and sensitized RBCs to hemolysis induced by mechanical stress, while reducing sensitivity to hypo-osmotic hemolysis. Similar properties were seen for other ligands bound to GPA and band 3 on human and murine RBCs. In contrast, binding of scFv or hTM-scFv to RhCE did not alter deformability or sensitivity to mechanical and osmotic stress at similar copy numbers bound per RBCs. Contrasting responses were also seen for immunoglobulin G antibodies against band 3, GPA, and RhCE. RBC-bound hTM-scFv generated activated protein C (APC) in the presence of thrombin, but RhCE-targeted hTM-scFv demonstrated greater APC generation per bound copy. Both Wrb- and RhCE-targeted fusion proteins inhibited fibrin deposition induced by tumor necrosis factor-α in an endothelialized microfluidic model using human whole blood. RhCE-bound hTM-scFv more effectively reduced platelet and leukocyte adhesion, whereas anti-Wrb scFv appeared to promote platelet adhesion. These data provide a translational framework for the development of engineered affinity ligands to safely couple therapeutics to human RBCs. PMID:29365311

Transfusion of fresher vs older red blood cells in hospitalized patients: a systematic review and meta-analysis.

PubMed

Alexander, Paul E; Barty, Rebecca; Fei, Yutong; Vandvik, Per Olav; Pai, Menaka; Siemieniuk, Reed A C; Heddle, Nancy M; Blumberg, Neil; McLeod, Shelley L; Liu, Jianping; Eikelboom, John W; Guyatt, Gordon H

2016-01-28

The impact of transfusing fresher vs older red blood cells (RBCs) on patient-important outcomes remains controversial. Two recently published large trials have provided new evidence. We summarized results of randomized trials evaluating the impact of the age of transfused RBCs. We searched MEDLINE, EMBASE, CINAHL, the Cochrane Database for Systematic Reviews, and Cochrane CENTRAL for randomized controlled trials enrolling patients who were transfused fresher vs older RBCs and reported outcomes of death, adverse events, and infection. Independently and in duplicate, reviewers determined eligibility, risk of bias, and abstracted data. We conducted random effects meta-analyses and rated certainty (quality or confidence) of evidence using the GRADE approach. Of 12 trials that enrolled 5229 participants, 6 compared fresher RBCs with older RBCs and 6 compared fresher RBCs with current standard practice. There was little or no impact of fresher vs older RBCs on mortality (relative risk [RR], 1.04; 95% confidence interval [CI], 0.94-1.14; P = .45; I(2) = 0%, moderate certainty evidence) or on adverse events (RR, 1.02; 95% CI, 0.91-1.14; P = .74; I(2) = 0%, low certainty evidence). Fresher RBCs appeared to increase the risk of nosocomial infection (RR, 1.09; 95% CI, 1.00-1.18; P = .04; I(2) = 0%, risk difference 4.3%, low certainty evidence). Current evidence provides moderate certainty that use of fresher RBCs does not influence mortality, and low certainty that it does not influence adverse events but could possibly increase infection rates. The existing evidence provides no support for changing practices toward fresher RBC transfusion. © 2016 by The American Society of Hematology.

Quality of red blood cells washed using a second wash sequence on an automated cell processor.

PubMed

Hansen, Adele L; Turner, Tracey R; Kurach, Jayme D R; Acker, Jason P

2015-10-01

Washed red blood cells (RBCs) are indicated for immunoglobulin (Ig)A-deficient recipients when RBCs from IgA-deficient donors are not available. Canadian Blood Services recently began using the automated ACP 215 cell processor (Haemonetics Corporation) for RBC washing, and its suitability to produce IgA-deficient RBCs was investigated. RBCs produced from whole blood donations by the buffy coat (BC) and whole blood filtration (WBF) methods were washed using the ACP 215 or the COBE 2991 cell processors and IgA and total protein levels were assessed. A double-wash procedure using the ACP 215 was developed, tested, and validated by assessing hemolysis, hematocrit, recovery, and other in vitro quality variables in RBCs stored after washing, with and without irradiation. A single wash using the ACP 215 did not meet Canadian Standards Association recommendations for washing with more than 2 L of solution and could not consistently reduce IgA to levels suitable for IgA-deficient recipients (24/26 BC RBCs and 0/9 WBF RBCs had IgA levels < 0.05 mg/dL). Using a second wash sequence, all BC and WBF units were washed with more than 2 L and had levels of IgA of less than 0.05 mg/dL. During 7 days' postwash storage, with and without irradiation, double-washed RBCs met quality control criteria, except for the failure of one RBC unit for inadequate (69%) postwash recovery. Using the ACP 215, a double-wash procedure for the production of components for IgA-deficient recipients from either BC or WBF RBCs was developed and validated. © 2015 AABB.

Storage duration and white blood cell content of red blood cell (RBC) products increases adhesion of stored RBCs to endothelium under flow conditions.

PubMed

Anniss, Angela M; Sparrow, Rosemary L

2006-09-01

Adherence of red blood cells (RBCs) to vascular endothelium impairs blood flow and decreases oxygen delivery. Although RBCs may be stored for up to 42 days before transfusion under current blood banking guidelines, little is known of how changes to RBCs during storage may affect their adherence properties. The influence of RBC product storage time and white blood cell (WBC) burden on the adherence of RBCs for transfusion to vascular endothelium under conditions of continuous flow was investigated in this study. RBC samples were collected from nonleukoreduced (S-RBC), buffy coat-poor (BCP-RBC), and leukofiltered (LF-RBC) products at fixed time points during storage. Samples were perfused, at controlled shear stress and temperature, across a confluent endothelial cell (EC) monolayer with a parallel-flow chamber mounted to an inverted microscope. RBC-EC interactions were recorded with a digital camera attached to the microscope. The number of RBCs adhering to the EC layer increased significantly with storage time in all RBC products; however, WBC reduction delayed this increase. LF-RBCs were also significantly less adherent than S-RBC or BCP-RBC products on Day 1 of storage (p < 0.05). The strength of RBC attachment to vascular endothelium was significantly stronger in S-RBC products compared to BCP-RBC and LF-RBC products. Our findings indicate that product storage time and WBC burden increase the number and strength of adhesion of RBCs to vascular endothelium. These results may lead to greater understanding of the interaction of transfused RBCs with recipient endothelium and the biologic consequences of this adherence.

Quantification by magnetic resonance imaging and liver consequences of post-transfusional iron overload alone in long term survivors after allogeneic hematopoietic stem cell transplantation (HSCT).

PubMed

Rose, Christian; Ernst, Olivier; Hecquet, Bernard; Maboudou, Patrice; Renom, Pascale; Noel, Marie Pierre; Yakoub-Agha, Ibrahim; Bauters, Francis; Jouet, Jean Pierre

2007-06-01

We quantified and studied the impact of post transfusional iron overload alone in post allogeneic HSCT. Median number of RBCs was 18. Ferritin was 532 mg/L. Liver iron content (LIC) was 117 mmoles/gdw. Correlation RBCs and ferritin was (r=0.81); RBCs and LIC was (r=0.84). The high ferritin group differed from normal ferritin group in terms of RBCs transfused (p<10(-3)), ALT (p<0.009). But occurrence of liver dysfunction was not significant. Magnitude of iron overload correlates closely to the number of RBCs and is quantified by MRI. Impact on liver dysfunction is moderate in absence of co-morbidity.

Numerical Simulations of the Motion and Deformation of Three RBCs during Poiseuille Flow through a Constricted Vessel Using IB-LBM.

PubMed

Wang, Rongyang; Wei, Yikun; Wu, Chuanyu; Sun, Liang; Zheng, Wenguang

2018-01-01

The immersed boundary-lattice Boltzmann method (IB-LBM) was used to examine the motion and deformation of three elastic red blood cells (RBCs) during Poiseuille flow through constricted microchannels. The objective was to determine the effects of the degree of constriction and the Reynolds (Re) number of the flow on the physical characteristics of the RBCs. It was found that, with decreasing constriction ratio, the RBCs experienced greater forced deformation as they squeezed through the constriction area compared to at other parts of the microchannel. It was also observed that a longer time was required for the RBCs to squeeze through a narrower constriction. The RBCs subsequently regained a stable shape and gradually migrated toward the centerline of the flow beyond the constriction area. However, a sick RBC was observed to be incapable of passing through a constricted vessel with a constriction ratio ≤1/3 for Re numbers below 0.40.

Numerical Simulations of the Motion and Deformation of Three RBCs during Poiseuille Flow through a Constricted Vessel Using IB-LBM

PubMed Central

Sun, Liang; Zheng, Wenguang

2018-01-01

The immersed boundary-lattice Boltzmann method (IB-LBM) was used to examine the motion and deformation of three elastic red blood cells (RBCs) during Poiseuille flow through constricted microchannels. The objective was to determine the effects of the degree of constriction and the Reynolds (Re) number of the flow on the physical characteristics of the RBCs. It was found that, with decreasing constriction ratio, the RBCs experienced greater forced deformation as they squeezed through the constriction area compared to at other parts of the microchannel. It was also observed that a longer time was required for the RBCs to squeeze through a narrower constriction. The RBCs subsequently regained a stable shape and gradually migrated toward the centerline of the flow beyond the constriction area. However, a sick RBC was observed to be incapable of passing through a constricted vessel with a constriction ratio ≤1/3 for Re numbers below 0.40. PMID:29681999

Cryopreservation of Genotyped ABO Subgroup RBCs for Quality Assurance of ABO Grouping Reagents.

PubMed

Kim, Sinyoung; Song, Sungwook; Kim, Hyun Ok

2018-03-01

Quality assurance of newly developed or manufactured blood grouping reagents with reagent red blood cells (RBCs) is crucial in the process of product approval by governmental agency. However, RBCs with rare blood group are not easily available in the fresh state. We investigated the feasibility of cryopreserved and genotyped ABO subgroup RBC reagents for quality assurance purpose. We obtained RBCs from 10 volunteers with ABO subgroup phenotypes. The ABO genotypes and alleles were analyzed by the sequencing of ABO exon 6 and 7. Using the 40% wt/vol high-glycerol method, RBC units were cryopreserved as reagent RBCs into separate cryovials at -80°C. The potency titrations were performed before and after cryopreservation for 6 months and 2 years to evaluate the stability of ABO antigens. ABO genotypes of cryopreserved RBCs were cis-AB01/O01 (n=2), cis-AB01/B101 (n=1), Aw10/B101 (n=2), A201/A201 (n=1), A205/B101 (n=1), A102/B112 (n=1), and A101/B306 (n=1). These ABO subgroup alleles are exclusively present in East-Asian population except for the known ABO*A201 allele. The potency titers of cryopreserved RBCs were not significantly different between pre-freezing and post-freezing. The national performance evaluation of ABO blood grouping reagents could be performed with cryopreserved and genotyped reagent RBCs. © 2018 by the Association of Clinical Scientists, Inc.

An acute hemolytic transfusion reaction due to anti-IH in a patient with sickle cell disease.

PubMed

Campbell, S A; Shirey, R S; King, K E; Ness, P M

2000-07-01

A hemolytic transfusion reaction (HTR) due to anti-IH is reported in a patient with sickle cell disease (SCD). An 18-year-old woman with SCD and a complete phenotype on file had been identified as group B-positive with negative antibody-screening tests and had received 1 unit of packed RBCs. Ten days later, she was readmitted in painful crisis with a Hb of 4.2 g per dL. Antibody-screening tests and panel cells were positive at all test phases with a negative autocontrol, which suggested alloantibodies. Phenotypically matched group O RBCs were issued emergently. After the transfusion of 100 mL, the patient had an HTR with chills, fever, and tachycardia and laboratory findings of hemoglobinemia, hemoglobinuria, and negative DATs. A high-titer, IgM anti-IH with a high thermal amplitude (reactive with group O, but not group B RBCs at 37 degrees C) was identified. Autologous RBCs appeared to have normal I antigen expression, but less H antigen than pooled group B RBCs. She was given group B RBCs, uneventfully, by use of a blood warmer. This is a rare case of anti-IH as the cause of a HTR, as a serologic problem that may be seen in SCD, and as an autoantibody that may mimic an alloantibody. Ironically, this HTR resulted from the effort to provide phenotypically matched RBCs, which necessitated the selection of group O RBCs.

Identification of Rubisco rbcL and rbcS in Camellia oleifera and their potential as molecular markers for selection of high tea oil cultivars

PubMed Central

Chen, Yongzhong; Wang, Baoming; Chen, Jianjun; Wang, Xiangnan; Wang, Rui; Peng, Shaofeng; Chen, Longsheng; Ma, Li; Luo, Jian

2015-01-01

Tea oil derived from seeds of Camellia oleifera Abel. is high-quality edible oil in China. This study isolated full-length cDNAs of Rubisco subunits rbcL and rbcS from C. oleifera. The rbcL has 1,522 bp with a 1,425 bp coding region, encoding 475 amino acids; and the rbcS has 615 bp containing a 528 bp coding region, encoding 176 amino acids. The expression level of the two genes, designated as Co-rbcL and Co-rbcS, was determined in three C. oleifera cultivars: Hengchong 89, Xianglin 1, and Xianglin 14 whose annual oil yields were 546.9, 591.4, and 657.7 kg ha-1, respectively. The Co-rbcL expression in ‘Xianglin 14’ was significantly higher than ‘Xianglin 1’, and ‘Xianglin 1’ was greater than ‘Hengchong 89’. The expression levels of Co-rbcS in ‘Xianglin 1’ and ‘Xianglin 14’ were similar but were significantly greater than in ‘Hengchong 89’. The net photosynthetic rate of ‘Xianglin 14’ was significantly higher than ‘Xianglin 1’, and ‘Xianglin 1’ was higher than ‘Hengchong 89’. Pearson’s correlation analysis showed that seed yields and oil yields were highly correlated with the expression level of Co-rbcL at P < 0.001 level; and the expression of Co-rbcS was correlated with oil yield at P < 0.01 level. Net photosynthetic rate was also correlated with oil yields and seed yields at P < 0.001 and P < 0.01 levels, respectively. Our results suggest that Co-rbcS and Co-rbcL in particular could potentially be molecular markers for early selection of high oil yield cultivars. In combination with the measurement of net photosynthetic rates, the early identification of potential high oil production cultivars would significantly shorten plant breeding time and increase breeding efficiency. PMID:25873921

Elevated red blood cell 2,3-diphosphoglycerate levels in black blood donors.

PubMed

Wallas, C H

1978-01-01

Mean levels of 2,3-diphosphoglycerate (DPG) were significantly increased in erythrocytes (RBC) from 43 nonanemic black blood donors (4.80 +/- 0.06 micromoles/l RBC) compared with 22 white donors 4.47 +/- 0.08 micromoles/l RBCs from eight of the 12 black donors with DPG levels greater than 5 micromoles/l RBC. Although a potentially hemolytic disorder could be defined in four (AS hemoglobin, beta-Thalassemia minor, G6PD deficiency), reticulocyte counts were normal. However, when RBCs from the subgroup were compared to RBCs from an additional 25 unselected white donors, the following suggested an abnormally large population of young RBCs in the subgroup: 1) normal or elevated RBC-ATP with normal serum phosphate level; 2) significantly increased activities of RBC age-dependent enzymes hexokinase (p less than 0.02), pyruvate kinase (p less than 0.05), and glutamicoxaloacetic transaminase (p less than 0.01), with normal activity of phosphoglycerate kinase, an age-independent enzyme; 3) decreased dense (older) RBCs as determined by sedimentation in phthalate esters. Since DPG is increased in young RBCs and falls as the RBC ages, loss of older relatively DPG depleted RBCs due to shortened survival could account for the elevated DPG levels seen in the subgroup.

Changing the 30-min Rule in Canada: The Effect of Room Temperature on Bacterial Growth in Red Blood Cells.

PubMed

Ramirez-Arcos, Sandra; Kou, Yuntong; Ducas, Éric; Thibault, Louis

2016-11-01

To maintain product quality and safety, the '30-min rule' requires the discard of red blood cells (RBCs) that are exposed to uncontrolled temperatures for more than 30 min. Recent studies suggest this rule may safely be extended to a 60-min rule. A pool-and-split design study (N = 4) was run in parallel at Canadian Blood Services (SAGM RBCs) and Héma-Québec (AS-3 RBCs). RBCs were spiked with ∼1 colony-forming unit/ml of mesophilic and psychrophilic bacteria. Control units remained in storage at 1-6 °C for 42 days. Test 30 (T30) and T60 units were exposed to room temperature (RT) six times during storage, each time for 30 and 60 min, respectively. Bacterial proliferation was monitored. Mesophilic bacteria do not proliferate in RBCs. The growth of psychrophilic bacteria is not significantly different in RBCs exposed for 30 or 60 min to RT (p < 0.05). The study findings were the final evidence to support extension from a 30-min rule to a 60-min rule in Canada.

Application of α-N-acetylgalactosaminidase and α-galactosidase in AB to O red blood cells conversion.

PubMed

Gao, Hongwei; Li, Subo; Tan, Yingxia; Ji, Shouping; Wang, Yingli; Bao, Guoqiang; Xu, Lijuan; Gong, Feng

2013-02-01

Enzymatical conversion of A or B RBCs into group O RBCs (ECORBCs) was achieved by using α-N-acetylgalactosaminidase and α-galactosidase, respectively. Now, we initiated AB to O-RBC conversion by using these two enzymes together. But α-N-acetylgalactosaminidase and α-galactosidase's preserving and their reaction buffer were quite different. The aim of this study is to confirm an available system for converting AB to O RBCs, especially to study the maximal permission amount of PCS which was brought to the system-accompanied enzyme addition. Enzyme activity was detected by using GalNAc-pNp or Gal-pNp as substrates. The efficiency of the conversion of A or B antigen was evaluated by routine method and measured by fluorescence-activated cell sorting analysis. The optimal buffer component and the doses of α-N-acetylgalactosaminidase and α-galactosidase were confirmed according to A and B antigen epitope removal efficiency. The activity of α-N-acetylgalactosaminidase and α-galactosidase was not decreased drastically when they were kept in PCS Buffer in 4°C. The optimal reaction buffer composed of glycine 250 mM and NaCl 3 mM, pH 6.8 and PCS less than 10%(v/v). For converting A(1)B to O RBCs completely, the doses of α-N-acetylgalactosaminidase and α-galactosidase were confirmed as 0.015 mg/ml packed RBCs(pRBCs) for A(1) antigen epitopes and 0.005 mg/ml pRBCs for B epitopes. Approximately 0.004 mg α-N-acetylgalactosaminidase and 0.005 mg α-galactosidase were required to convert 1 ml pRBCs. Our studies indicated that α-N-acetylgalactosaminidase and α-galactosidase were stable in PCS buffer and a modified protocol which was propitious to converting AB to O RBCs was provided.

Deformability measurement of red blood cells using a microfluidic channel array and an air cavity in a driving syringe with high throughput and precise detection of subpopulations.

PubMed

Kang, Yang Jun; Ha, Young-Ran; Lee, Sang-Joon

2016-01-07

Red blood cell (RBC) deformability has been considered a potential biomarker for monitoring pathological disorders. High throughput and detection of subpopulations in RBCs are essential in the measurement of RBC deformability. In this paper, we propose a new method to measure RBC deformability by evaluating temporal variations in the average velocity of blood flow and image intensity of successively clogged RBCs in the microfluidic channel array for specific time durations. In addition, to effectively detect differences in subpopulations of RBCs, an air compliance effect is employed by adding an air cavity into a disposable syringe. The syringe was equally filled with a blood sample (V(blood) = 0.3 mL, hematocrit = 50%) and air (V(air) = 0.3 mL). Owing to the air compliance effect, blood flow in the microfluidic device behaved transiently depending on the fluidic resistance in the microfluidic device. Based on the transient behaviors of blood flows, the deformability of RBCs is quantified by evaluating three representative parameters, namely, minimum value of the average velocity of blood flow, clogging index, and delivered blood volume. The proposed method was applied to measure the deformability of blood samples consisting of homogeneous RBCs fixed with four different concentrations of glutaraldehyde solution (0%-0.23%). The proposed method was also employed to evaluate the deformability of blood samples partially mixed with normal RBCs and hardened RBCs. Thereafter, the deformability of RBCs infected by human malaria parasite Plasmodium falciparum was measured. As a result, the three parameters significantly varied, depending on the degree of deformability. In addition, the deformability measurement of blood samples was successfully completed in a short time (∼10 min). Therefore, the proposed method has significant potential in deformability measurement of blood samples containing hematological diseases with high throughput and precise detection of subpopulations in RBCs.

Artificially evolved Synechococcus PCC6301 Rubisco variants exhibit improvements in folding and catalytic efficiency.

PubMed

Greene, Dina N; Whitney, Spencer M; Matsumura, Ichiro

2007-06-15

The photosynthetic CO2-fixing enzyme, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), is responsible for most of the world's biomass, but is a slow non-specific catalyst. We seek to identify and overcome the chemical and biological constraints that limit the evolutionary potential of Rubisco in Nature. Recently, the horizontal transfer of Calvin cycle genes (rbcL, rbcS and prkA) from cyanobacteria (Synechococcus PCC6301) to gamma-proteobacteria (Escherichia coli) was emulated in the laboratory. Three unique Rubisco variants containing single (M259T) and double (M259T/A8S, M259T/F342S) amino acid substitutions in the L (large) subunit were identified after three rounds of random mutagenesis and selection in E. coli. Here we show that the M259T mutation did not increase steady-state levels of rbcL mRNA or L protein. It instead improved the yield of properly folded L subunit in E. coli 4-9-fold by decreasing its natural propensity to misfold in vivo and/or by enhancing its interaction with the GroES-GroEL chaperonins. The addition of osmolites to the growth media enhanced productive folding of the M259T L subunit relative to the wild-type L subunit, while overexpression of the trigger factor and DnaK/DnaJ/GrpE chaperones impeded Rubisco assembly. The evolved enzymes showed improvement in their kinetic properties with the M259T variant showing a 12% increase in carboxylation turnover rate (k(c)cat), a 15% improvement in its K(M) for CO2 and no change in its K(M) for ribulose-1,5-bisphosphate or its CO2/O2 selectivity. The results of the present study show that the directed evolution of the Synechococcus Rubisco in E. coli can elicit improvements in folding and catalytic efficiency.

Dietary supplementation with docosahexanoic acid (DHA) increases red blood cell membrane flexibility in mice with sickle cell disease.

PubMed

Wandersee, Nancy J; Maciaszek, Jamie L; Giger, Katie M; Hanson, Madelyn S; Zheng, Suilan; Guo, YiHe; Mickelson, Barbara; Hillery, Cheryl A; Lykotrafitis, George; Low, Philip S; Hogg, Neil

2015-02-01

Humans and mice with sickle cell disease (SCD) have rigid red blood cells (RBCs). Omega-3 fatty acids, such as docosahexanoic acid (DHA), may influence RBC deformability via incorporation into the RBC membrane. In this study, sickle cell (SS) mice were fed natural ingredient rodent diets supplemented with 3% DHA (DHA diet) or a control diet matched in total fat (CTRL diet). After 8weeks of feeding, we examined the RBCs for: 1) stiffness, as measured by atomic force microscopy; 2) deformability, as measured by ektacytometry; and 3) percent irreversibly sickled RBCs on peripheral blood smears. Using atomic force microscopy, it is found that stiffness is increased and deformability decreased in RBCs from SS mice fed CTRL diet compared to wild-type mice. In contrast, RBCs from SS mice fed DHA diet had markedly decreased stiffness and increased deformability compared to RBCs from SS mice fed CTRL diet. Furthermore, examination of peripheral blood smears revealed less irreversibly sickled RBCs in SS mice fed DHA diet as compared to CTRL diet. In summary, our findings indicate that DHA supplementation improves RBC flexibility and reduces irreversibly sickled cells by 40% in SS mice. These results point to potential therapeutic benefits of dietary omega-3 fatty acids in SCD. Copyright © 2014 Elsevier Inc. All rights reserved.

A case for stocking O D+ red blood cells in emergency room trauma bays.

PubMed

Meyer, Erin; Uhl, Lynne

2015-04-01

AABB Standard 5.27 requires transfusion services to have a process for urgent release of blood before completion of compatibility testing. Our institution endorses a policy for the emergency release of group O, D+ red blood cells (RBC; O+ RBC) to males and females at least 50 years of age. Our emergency department (ED) stocks 4 O- RBC units. To determine if O+ RBCs can replace ED O- RBCs, we performed a retrospective review. Patients admitted to the ED between January 2001 and August 2011 and transfused emergency-release O- RBCs were identified. Data were collected on sex, age, length of stay, clinical status, ABO/Rh, RBC transfusions, and RBC antibody screen results. A total of 498 ED O- RBC units were transfused to 268 patients (168 male, 100 female). A total of 322 units were transfused to males and 114 to females at least 50 years of age. Thirty-nine (14%) were D- with 18 receiving O+ RBCs. A total of 109 had follow-up antibody screens; one D- patient developed alloanti-D. The findings support the placement of O+ RBCs in the ED. The majority of ED O- RBCs (88%) went to patients who qualified for O+ RBCs; a minority (1.5%) of patients were D- females less than 50 years of age. The rate of alloimmunization was low. © 2014 AABB.

Machine learning-based in-line holographic sensing of unstained malaria-infected red blood cells.

PubMed

Go, Taesik; Kim, Jun H; Byeon, Hyeokjun; Lee, Sang J

2018-04-19

Accurate and immediate diagnosis of malaria is important for medication of the infectious disease. Conventional methods for diagnosing malaria are time consuming and rely on the skill of experts. Therefore, an automatic and simple diagnostic modality is essential for healthcare in developing countries that lack the expertise of trained microscopists. In the present study, a new automatic sensing method using digital in-line holographic microscopy (DIHM) combined with machine learning algorithms was proposed to sensitively detect unstained malaria-infected red blood cells (iRBCs). To identify the RBC characteristics, 13 descriptors were extracted from segmented holograms of individual RBCs. Among the 13 descriptors, 10 features were highly statistically different between healthy RBCs (hRBCs) and iRBCs. Six machine learning algorithms were applied to effectively combine the dominant features and to greatly improve the diagnostic capacity of the present method. Among the classification models trained by the 6 tested algorithms, the model trained by the support vector machine (SVM) showed the best accuracy in separating hRBCs and iRBCs for training (n = 280, 96.78%) and testing sets (n = 120, 97.50%). This DIHM-based artificial intelligence methodology is simple and does not require blood staining. Thus, it will be beneficial and valuable in the diagnosis of malaria. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterizations of individual human red blood cells from patients with diabetes mellitus (Conference Presentation)

NASA Astrophysics Data System (ADS)

Lee, SangYun; Jang, Seongsoo; Park, HyunJoo; Park, YongKeun

2016-03-01

We systematically measure the morphological, biochemical, and biomechanical properties of individual human red blood cells (RBCs) from patients with diabetes mellitus using quantitative phase imaging technique to characterize the diabetic red cells with respect to those of the healthy. The 3-D refractive index tomograms and 2-D dynamic membrane fluctuation maps of individual RBCs are reconstructed from a set of the retrieved complex optical fields at various laser incidence angles using the Common-path diffraction optical tomography, from which volume, surface area, sphericity, hemoglobin (Hb) concentration, Hb content, and membrane fluctuation are obtained simultaneously. The correlative relations among the retrieved red cell indices of diabetic and healthy RBCs are also investigated with capabilities of individual cell measurement. As expected, there are no significant alterations in morphologies (cellular volumes, surface area, and sphericity) between diabetic and healthy RBCs. However, despite the minute mean corpuscular Hb differences in cell blood count datasheet, the measured Hb concentrations and Hb contents of diabetic RBCs are statistically higher than those of healthy RBCs, which might be related to the glycation of Hb molecules by hyperglycemia. Meanwhile, the membrane fluctuations of diabetic RBCs are clearly diminished compared to healthy red cells, implying the significantly decreased RBC deformability. In particular, it seems that the membrane fluctuations have mild negative relationships with the reported HbA1c levels.

Additive solution-7 reduces the red blood cell cold storage lesion.

PubMed

Cancelas, Jose A; Dumont, Larry J; Maes, Lou A; Rugg, Neeta; Herschel, Louise; Whitley, Pamela H; Szczepiokowski, Zbigniew M; Siegel, Alan H; Hess, John R; Zia, Majid

2015-03-01

Transfusion of long-stored red blood cells (RBCs) is associated with decreased in vivo RBC recovery, delivery of RBC breakdown products, and increased morbidity and mortality. Reducing the burden of this RBC "storage lesion" is a major challenge in transfusion medicine. Additive solution-7 (AS-7) is a new RBC storage solution designed to improve RBC metabolism by providing phosphate and increasing buffering capacity. Storage quality in AS-7 was measured in a prospective, randomized, three-center trial using units of whole blood from healthy human subjects whose RBCs were stored for up to 56 days in AS-7 (n = 120) or for 42 days in the control solution AS-1 (n = 60). Hemolysis and shedding of protein-containing microvesicles were significantly reduced in RBCs stored in AS-7 for 42 and 56 days compared with RBCs stored in AS-1. Autologous in vivo recoveries of RBCs stored in AS-7 was 88 ± 5% at 42 days (n = 27) and 82 ± 3% at 56 days (n = 27), exceeding recoveries of RBCs stored in currently used solutions. Increasing the phosphate, pH range, and buffer capacity of a RBC storage system allowed RBCs to be stored better and longer than currently approved storage systems. AS-7 ameliorates the long-term storage lesion resulting in significantly increased viability in vitro and in vivo. © 2014 AABB.

Novel single-cell functional analysis of red blood cells using laser tweezers Raman spectroscopy: application for sickle cell disease.

PubMed

Liu, Rui; Mao, Ziliang; Matthews, Dennis L; Li, Chin-Shang; Chan, James W; Satake, Noriko

2013-07-01

Laser tweezers Raman spectroscopy was used to characterize the oxygenation response of single normal adult, sickle, and cord blood red blood cells (RBCs) to an applied mechanical force. Individual cells were subjected to different forces by varying the laser power of a single-beam optical trap, and the intensities of several oxygenation-specific Raman spectral peaks were monitored to determine the oxygenation state of the cells. For all three cell types, an increase in laser power (or mechanical force) induced a greater deoxygenation of the cell. However, sickle RBCs deoxygenated more readily than normal RBCs when subjected to the same optical forces. Conversely, cord blood RBCs were able to maintain their oxygenation better than normal RBCs. These results suggest that differences in the chemical or mechanical properties of fetal, normal, and sickle cells affect the degree to which applied mechanical forces can deoxygenate the cell. Populations of normal, sickle, and cord RBCs were identified and discriminated based on this mechanochemical phenomenon. This study demonstrates the potential application of laser tweezers Raman spectroscopy as a single-cell, label-free analytical tool to characterize the functional (e.g., mechanical deformability, oxygen binding) properties of normal and diseased RBCs. Copyright © 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Is Increased Intracellular Calcium in Red Blood Cells a Common Component in the Molecular Mechanism Causing Anemia?

PubMed Central

Hertz, Laura; Huisjes, Rick; Llaudet-Planas, Esther; Petkova-Kirova, Polina; Makhro, Asya; Danielczok, Jens G.; Egee, Stephane; del Mar Mañú-Pereira, Maria; van Wijk, Richard; Vives Corrons, Joan-Lluis; Bogdanova, Anna; Kaestner, Lars

2017-01-01

For many hereditary disorders, although the underlying genetic mutation may be known, the molecular mechanism leading to hemolytic anemia is still unclear and needs further investigation. Previous studies revealed an increased intracellular Ca2+ in red blood cells (RBCs) from patients with sickle cell disease, thalassemia, or Gardos channelopathy. Therefore we analyzed RBCs' Ca2+ content from 35 patients with different types of anemia (16 patients with hereditary spherocytosis, 11 patients with hereditary xerocytosis, 5 patients with enzymopathies, and 3 patients with hemolytic anemia of unknown cause). Intracellular Ca2+ in RBCs was measured by fluorescence microscopy using the fluorescent Ca2+ indicator Fluo-4 and subsequent single cell analysis. We found that in RBCs from patients with hereditary spherocytosis and hereditary xerocytosis the intracellular Ca2+ levels were significantly increased compared to healthy control samples. For enzymopathies and hemolytic anemia of unknown cause the intracellular Ca2+ levels in RBCs were not significantly different. These results lead us to the hypothesis that increased Ca2+ levels in RBCs are a shared component in the mechanism causing an accelerated clearance of RBCs from the blood stream in channelopathies such as hereditary xerocytosis and in diseases involving defects of cytoskeletal components like hereditary spherocytosis. Future drug developments should benefit from targeting Ca2+ entry mediating molecular players leading to better therapies for patients. PMID:28932200

Measurement of whole blood of different mammalian species in the oscillating shear field: influence of erythrocyte aggregation

NASA Astrophysics Data System (ADS)

Windberger, U.; Pöschl, Ch; Peters, S.; Huber, J.; van den Hoven, R.

2017-01-01

This is the first systematic analysis of mammalian blood of species with a high (horse), medium (man), and low (sheep) erythrocyte (RBC) aggregability by small amplitude oscillation technique. Amplitude and frequency sweep tests (linear viscoelastic mode) were performed with blood from healthy adult volunteers, horses, and sheep in CSS-mode. Blood samples were hematocrit (HCT) adjusted (40%, 50%, 60%) and tested at 7°C, 22°C, and 37°C. Generally, storage modulus (G´) increased with HCT and decreased with temperature in each species, but the gradient of this increase was species-specific. The lower dependency of G´ on the equine HCT value could be a benefit during physical performance when high numbers of RBCs are released from the spleen. In sheep, an HCT-threshold had to be overcome before the desired quasi-static condition of the blood sample could be achieved, suggesting that the contact between RBCs, and between RBCs and plasma molecules must be very low. The frequencies for tests under linear viscoelastic condition were in a narrow range around the physiologic heart rate of the species. In horse, time-dependent influences concurred at frequencies lower than 3 rad.s-1probably due to sedimentation of RBC aggregates. In conclusion, blood is a fragile suspension that shows its best stability around the resting heart rate of the species.

Whole blood of mammalian species in the oscillating shear field: influence of erythrocyte aggregation

NASA Astrophysics Data System (ADS)

Windberger, U.; Pöschl, Ch; Peters, S.; Huber, J.; van den Hoven, R.

2017-02-01

This is the rheologicalanalysis of mammalian blood of species with a high (horse), medium (man), and low (sheep) erythrocyte (RBC) aggregability by small amplitude oscillation technique. Amplitude and frequency sweep tests in linear mode were performed with blood from healthy adult volunteers, horses, and sheep in CSS-mode. Blood samples were hematocrit (HCT) adjusted (40%, 50%, 60%) and tested at 7°C, 22°C, and 37°C. Storage modulus (G‧) increased with HCT and decreased with temperature in each species, but the gradient of this increase was species-specific. The lower dependency of G‧ on the equine HCT value could be a benefit during physical performance when high numbers of RBCs are released from the spleen in the horse. In sheep, a HCT-threshold had to be overcome before elasticity of the blood sample could be measured, suggesting that the cohesive forces between RBCs, and between RBCs and plasma molecules must be very low. The frequencies for tests under quasi-staticcondition were in a narrow range around the physiologic heart rate of the species. In horse, time-dependent influences concurred at frequencies lower than 3 rad.s-1 probably due to sedimentation of RBC aggregates. In conclusion, elasticity of blood depends not only on the amount of blood cells, but also on their mechanical and functional properties.

Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tai, Lin-Ru; Chou, Chang-Wei; Wu, Jing-Ying

2013-11-15

Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20{sub NLS} mutant gene and examined polysome profile of cells that had been transfected with the S20{sub NLS} gene. As a result, we observed the formation of recombinant 40S carried S20{sub NLS} but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletionmore » and restoration, we were able to restrain the nuclear-resided S20{sub NLS} in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20{sub NLS} in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20{sub NLS} is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20{sub NLS}. • Cytoplasm-retained S20{sub NLS} is crucial for creating a functional small subunit.« less

In vitro biofilm formation on resin-based composites after different finishing and polishing procedures.

PubMed

Cazzaniga, Gloria; Ottobelli, Marco; Ionescu, Andrei C; Paolone, Gaetano; Gherlone, Enrico; Ferracane, Jack L; Brambilla, Eugenio

2017-12-01

To evaluate the influence of surface treatments of different resin-based composites (RBCs) on S. mutans biofilm formation. 4 RBCs (microhybrid, nanohybrid, nanofilled, bulk-filled) and 6 finishing-polishing (F/P) procedures (open-air light-curing, light-curing against Mylar strip, aluminum oxide discs, one-step rubber point, diamond bur, multi-blade carbide bur) were evaluated. Surface roughness (SR) (n=5/group), gloss (n=5/group), scanning electron microscopy morphological analysis (SEM), energy-dispersive X-ray spectrometry (EDS) (n=3/group), and S. mutans biofilm formation (n=16/group) were assessed. EDS analysis was repeated after the biofilm assay. A morphological evaluation of S. mutans biofilm was also performed using confocal laser-scanning microscopy (CLSM) (n=2/group). The data were analyzed using Wilcoxon (SR, gloss) and two-way ANOVA with Tukey as post-hoc tests (EDS, biofilm formation). F/P procedures as well as RBCs significantly influenced SR and gloss. While F/P procedures did not significantly influence S. mutans biofilm formation, a significant influence of RBCs on the same parameter was found. Different RBCs showed different surface elemental composition. Both F/P procedures and S. mutans biofilm formation significantly modified this parameter. The tested F/P procedures significantly influenced RBCs surface properties but did not significantly affect S. mutans biofilm formation. The significant influence of the different RBCs tested on S. mutans biofilm formation suggests that material characteristics and composition play a greater role than SR. F/P procedures of RBCs may unexpectedly play a minor role compared to that of the restoration material itself in bacterial colonization. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of diverse defense mechanisms in rainbow trout red blood cells in response to halted replication of VHS virus.

PubMed

Nombela, Ivan; Puente-Marin, Sara; Chico, Veronica; Villena, Alberto J; Carracedo, Begoña; Ciordia, Sergio; Mena, Maria Carmen; Mercado, Luis; Perez, Luis; Coll, Julio; Estepa, Amparo; Ortega-Villaizan, Maria Del Mar

2017-01-01

Background: It has been described that fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them and are their main target cell. The immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues has not yet been explored and is the objective of our study. Methods: Rainbow trout RBCs were obtained from peripheral blood, ficoll purified and exposed to Viral Haemorrhagic Septicaemia virus (VHSV). Immune response was evaluated by means of RT-qPCR, flow cytometry, immunofluorescence and isobaric tag for relative and absolute quantification (iTRAQ) protein profiling. Results: VHSV N gene transcripts incremented early postexposure and were drastically decreased after 6 hours postexposure (hpe). The expression of type I interferon ( ifn1 ) gene was significantly downregulated at early postexposure (3 hpe), together with a gradual downregulation of interferon-inducible mx and pkr genes until 72 hpe. Type I IFN protein was downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs, previously exposed to UV-inactivated VHSV, and TSS (stromal cell line from spleen) revealed IFN crosstalk between both cell types. On the other hand, anti-microbial peptide β-defensin 1 and neutrophil chemotactic factor interleukin 8 were slightly upregulated in VHSV-exposed RBCs. iTRAQ profiling revealed that VHSV exposure can induce a global protein downregulation in rainbow trout RBCs, mainly related to RNA stability and proteasome pathways. Antioxidant/antiviral response is also suggested to be involved in the response of rainbow trout RBCs to VHSV. Conclusions: A variety of mechanisms are proposed to be implicated in the antiviral response of rainbow trout RBCs against VHSV halted infection. Ongoing research is focused on understanding the mechanisms in detail.

Comparison of stored red blood cell washing techniques for priming extracorporeal circuits.

PubMed

Sasaki, Jun; Tirotta, Christopher; Lim, Hyunsoo; Kubes, Kathleen; Salvaggio, Jane; Hannan, Robert; Burke, Redmond; Ojito, Jorge

2018-03-01

The aim of this study was to compare three different blood washing techniques and describe the differences for the composition of the washed red blood cells (RBC). Stored RBCs less than 5 days old were washed using three different techniques. 1) Washing with normal saline with the COBE Model 2991 blood processor in the blood bank (BB-S). 2) Washing with normal saline with the Continuous AutoTransfusion System (C.A.T.S) in the operating room (OR-S). 3) Washing with Plasma-Lyte with the C.A.T.S in the operating room (OR-PL). Then, we compared the values for hemoglobin (Hb), hematocrit (Hct), blood volume, RBC volume, lactate, glucose, sodium and potassium of the three different groups. Forty-five units of RBCs were washed and analyzed (15 for each technique). The OR-S RBCs, when compared to the BB-S RBCs, had lower hemoglobin (g/dL) (22.8 vs 24.1, p=0.006), lower hematocrit (%) (67 vs 71, p=0.006), higher RBC volume (ml) (161 vs 130, p<0.001), higher glucose (mg/dL) (185 vs 46, p<0.001) and lower sodium (mmol/L) (153 vs 158, p<0.001). When compared to the OR-S RBCs, the OR-PL RBCs showed higher potassium (mmol/L) (5.3 vs 2, p<0.001) and lower sodium (mmol/L) (129 vs 153, p<0.001). RBCs washed with an autotransfusion device had a higher RBC volume and more physiological levels of glucose and sodium when compared with the blood processor in the blood bank. It can be an alternative option to use RBCs washed with an autotransfusion device for priming the extracorporeal circuits utilized in patients undergoing cardiac surgery.

Light transmittance and micro-mechanical properties of bulk fill vs. conventional resin based composites.

PubMed

Bucuta, Stefan; Ilie, Nicoleta

2014-11-01

The aim of this study was to quantify the blue light that passes through different incremental thicknesses of bulk fill in comparison to conventional resin-based composites (RBCs) and to relate it to the induced mechanical properties. Seven bulk fill, five nanohybrid and two flowable RBCs were analysed. Specimens (n = 5) of three incremental thicknesses (2, 4 and 6 mm) were cured from the top for 20 s, while at the bottom, a spectrometer monitored in real time the transmitted irradiance. Micro-mechanical properties (Vickers hardness, HV, and indentation modulus, E) were measured at the top and bottom after 24 h of storage in distilled water at 37 °C. Electron microscope images were taken for assessing the filler distribution and size. Bulk fill RBCs (except SonicFill) were more translucent than conventional RBCs. Low-viscosity bulk fill materials showed the lowest mechanical properties. HV depends highly on the following parameters: material (ηp (2) = 0.952), incremental thickness (0.826), filler volume (0.747), filler weight (0.746) and transmitted irradiance (0.491). The bottom-to-top HV ratio (HVbt) was higher than 80 % in all materials in 2- and 4-mm increments (except for Premise), whereas in 6-mm increments, this is valid only in four bulk fill materials (Venus Bulk Fill, SDR, x-tra fil, Tetric EvoCeram Bulk Fill). The depth of cure is dependent on the RBC's translucency. Low-viscosity bulk fill RBCs have lower mechanical properties than all other types of analysed materials. All bulk fill RBCs (except SonicFill) are more translucent for blue light than conventional RBCs. Although bulk fill RBCs are generally more translucent, the practitioner has to follow the manufacturer's recommendations on curing technique and maximum incremental thickness.

Identification of cytosolic phosphodiesterases in the erythrocyte: A possible role for PDE5

PubMed Central

Adderley, Shaquria P.; Thuet, Kelly M.; Sridharan, Meera; Bowles, Elizabeth A.; Stephenson, Alan H.; Ellsworth, Mary L.; Sprague, Randy S.

2011-01-01

Summary Background Within erythrocytes (RBCs), cAMP levels are regulated by phosphodiesterases (PDEs). Increases in cAMP and ATP release associated with activation of β-adrenergic receptors (βARs) and prostacyclin receptors (IPRs) are regulated by PDEs 2, 4 and PDE 3, respectively. Here we establish the presence of cytosolic PDEs in RBCs and determine a role for PDE5 in regulating levels of cGMP. Material/Methods Purified cytosolic proteins were obtained from isolated human RBCs and western analysis was performed using antibodies against PDEs 3A, 4 and 5. Rabbit RBCs were incubated with dbcGMP, a cGMP analog, to determine the effect of cGMP on cAMP levels. To determine if cGMP affects receptor-mediated increases in cAMP, rabbit RBCs were incubated with dbcGMP prior to addition of isoproterenol (ISO), a βAR receptor agonist. To demonstrate that endogenous cGMP produces the same effect, rabbit and human RBCs were incubated with SpNONOate (SpNO), a nitric oxide donor, and YC1, a direct activator of soluble guanylyl cyclase (sGC), in the absence and presence of a selective PDE5 inhibitor, zaprinast (ZAP). Results Western analysis identified PDEs 3A, 4D and 5A. dbcGMP produced a concentration dependent increase in cAMP and ISO-induced increases in cAMP were potentiated by dbcGMP. In addition, incubation with YC1 and SpNO in the presence of ZAP potentiated βAR-induced increases in cAMP. Conclusions PDEs 2, 3A and 5 are present in the cytosol of human RBCs. PDE5 activity in RBCs regulates cGMP levels. Increases in intracellular cGMP augment cAMP levels. These studies suggest a novel role for PDE5 in erythrocytes. PMID:21525805

Macrophages clear refrigerator storage-damaged RBCs and subsequently secrete cytokines in vivo, but not in vitro, in a murine model

PubMed Central

Wojczyk, Boguslaw S.; Kim, Nina; Bandyopadhyay, Sheila; Francis, Richard O.; Zimring, James C.; Hod, Eldad A.; Spitalnik, Steven L.

2014-01-01

BACKGROUND In mice, refrigerator-stored red blood cells (RBCs) are cleared by extravascular hemolysis and induce cytokine production. To enhance understanding of this phenomenon, we sought to model it in vitro. STUDY DESIGN AND METHODS Ingestion of refrigerator-stored murine RBCs and subsequent cytokine production were studied using J774A.1 mouse macrophage cells and primary murine splenic macrophages. Wild-type and Ccl2-GFP-reporter mice were used for RBC clearance in vivo. RESULTS Although J774A.1 cells and primary macrophages preferentially ingested refrigerator-stored RBCs in vitro, as compared to freshly-isolated RBCs, neither produced increased cytokines following erythrophagocytosis. In contrast, phagocytosis of refrigerator-stored RBCs in vivo induced increases in circulating monocyte chemoattractant protein-1 (MCP-1) and keratinocyte chemoattractant (KC), and correspondingly increased mRNA levels in mouse spleen and liver. In the spleen, these were predominantly expressed by CD11b+ cells. Using Ccl2-GFP-reporter mice, the predominant splenic population responsible for MCP-1 mRNA production were tissue-resident macrophages (i.e., CD45+, CD11b+, F4/80+, Ly6c+, CD11clow cells). CONCLUSION J774A.1 cells and primary macrophages selectively ingested refrigerator-stored RBCs by phagocytosis. Although cytokine expression was not enhanced, this approach could be used to identify the relevant receptor-ligand combination(s). In contrast, cytokine levels increased following phagocytosis of refrigerator-stored RBCs in vivo. These were primarily cleared in the liver and spleen, which demonstrated increased MCP-1 and KC mRNA expression. Finally, in mouse spleen, tissue-resident macrophages were predominantly involved in MCP-1 mRNA production. The differences between cytokine production in vitro and in vivo are not yet well understood. PMID:25041478

Evaluation of canine red blood cell quality after processing with an automated cell salvage device.

PubMed

Hofbauer, Nina; Windberger, Ursula; Schwendenwein, Ilse; Tichy, Alexander; Eberspächer, Eva

2016-05-01

To evaluate the properties of RBC concentrate harvested after processing fresh whole blood units from healthy dogs with an automated cell salvage device. Prospective, in vitro, experimental study. University teaching hospital. Sixteen healthy, privately owned dogs of various breeds. Fresh canine whole blood collected in bags with citrate phosphate dextrose adenine solution was processed with an automated cell salvage device and analyzed in vitro. Laboratory values determined before (baseline, from a catheter sample) and after processing RBCs (procRBCs) included a complete blood count, selected blood chemistry analytes, erythrocyte osmotic resistance, whole blood viscosity, RBC aggregation, and RBC deformability. Total recovery of RBCs was 80% ± 12%. Hematocrit of the procRBCs yielded by the device was 77% ± 3.7% (mean ± standard deviation). Gross morphology of the RBCs remained unchanged. The mean corpuscular volume, erythrocyte osmotic resistance, RBC deformability, RBC aggregation, and the activity of lactate dehydrogenase showed minor but statistically significant changes from baseline. No differences in the concentrations of free hemoglobin were observed. Whole blood viscosity was less in the procRBCs. Seventy-seven percent (mean) of the platelets were washed out, while a mean of 57% of the leukocytes remained in the procRBCs. Although processing canine blood with this automated cell salvage device leads to slight changes in some properties of RBCs, most of these changes are comparable to changes seen in human blood after processing. Present data indicate that the use of this cell salvage device does not induce changes in canine RBC concentrate that would preclude its use for transfusion. © Veterinary Emergency and Critical Care Society 2016.

Introduction of a closed-system cell processor for red blood cell washing: postimplementation monitoring of safety and efficacy.

PubMed

Acker, Jason P; Hansen, Adele L; Yi, Qi-Long; Sondi, Nayana; Cserti-Gazdewich, Christine; Pendergrast, Jacob; Hannach, Barbara

2016-01-01

After introduction of a closed-system cell processor, the effect of this product change on safety, efficacy, and utilization of washed red blood cells (RBCs) was assessed. This study was a pre-/postimplementation observational study. Efficacy data were collected from sequentially transfused washed RBCs received as prophylactic therapy by β-thalassemia patients during a 3-month period before and after implementation of the Haemonetics ACP 215 closed-system processor. Before implementation, an open system (TerumoBCT COBE 2991) was used to wash RBCs. The primary endpoint for efficacy was a change in hemoglobin (Hb) concentration corrected for the duration between transfusions. The primary endpoint for safety was the frequency of adverse transfusion reactions (ATRs) in all washed RBCs provided by Canadian Blood Services to the transfusion service for 12 months before and after implementation. Data were analyzed from more than 300 RBCs transfused to 31 recipients before implementation and 29 recipients after implementation. The number of units transfused per episode reduced significantly after implementation, from a mean of 3.5 units to a mean of 3.1 units (p < 0.005). The corrected change in Hb concentration was not significantly different before and after implementation. ATRs occurred in 0.15% of transfusions both before and after implementation. Safety and efficacy of washed RBCs were not affected after introduction of a closed-system cell processor. The ACP 215 allowed for an extended expiry time, improving inventory management and overall utilization of washed RBCs. Transfusion of fewer RBCs per episode reduced exposure of recipients to allogeneic blood products while maintaining efficacy. © 2015 AABB.

Randomized study of washing 40- to 42-day-stored red blood cells.

PubMed

Bennett-Guerrero, Elliott; Kirby, Brett S; Zhu, Hongmei; Herman, Annadele E; Bandarenko, Nicholas; McMahon, Timothy J

2014-10-01

Pretransfusion washing of red blood cells (RBCs) stored for a longer duration may have theoretical advantages but few data exist to support this practice. In many hospital settings, use of a point-of-care cell washer could conceivably be used to quickly wash allogeneic RBCs before transfusion. The purpose of this preliminary study was to compare a point-of-care device with a common blood bank device for washing longer-stored RBCs. Forty RBC units stored for 40 to 42 days were randomized to washing with the COBE 2991 device (Terumo BCT; FDA-cleared for washing stored RBCs) or the Cell Saver Elite (Haemonetics; FDA-cleared point-of-care device for processing and washing fresh autologous shed whole blood). Supernatant and unit RBCs from unwashed (baseline) and washed blood were assayed for potassium, lactate, intracellular ATP, percentage of RBC recovery, cell-free hemoglobin, RBC microparticles, and RBCs were examined for susceptibility to hemolysis by physical stress. Both devices recovered a high percentage of RBCs and efficiently removed extracelluar potassium. Washing with the Elite resulted in significant increases in cell-free Hb, percent hemolysis, and RBC microparticle production, whereas washing with the COBE 2991 did not (fold Δ = 2.1 vs. 1.0, 4.6 vs. 1.2, 2.0 vs. 1.1, respectively; p < 0.05). Hemolysis induced by physical stress was not altered by washing. Although point-of-care washing of longer-stored RBCs is appealing, these preliminary data suggest that transfusion of washed, longer-stored units could result in potentially greater exposure to plasma free Hb. More data are needed before this practice can be routinely recommended. © 2014 AABB.

Duration of storage influences the hemoglobin rising effect of red blood cells in patients undergoing major abdominal surgery.

PubMed

Hunsicker, Oliver; Hessler, Katarina; Krannich, Alexander; Boemke, Willehad; Braicu, Ioana; Sehouli, Jalid; Meyer, Oliver; Pruß, Axel; Spies, Claudia; Feldheiser, Aarne

2018-04-17

After transfusion of senescent red blood cells (RBCs) a considerable fraction is rapidly cleared from the recipients' circulation. Thus, transfusion of senescent RBCs may be less effective in terms of increasing hemoglobin concentration (cHb) after transfusion. Data were retrospectively obtained in patients who underwent major abdominal surgery between 2006 and 2012. Patients were eligible if they received RBCs during surgery and had at least two arterial blood gas analyses performed. The primary endpoint was the increase of recipients' cHb related to the transfusion of 1 unit of RBCs with respect to different storage periods. Four storage periods were defined according to the distribution of RBC storage of the study population. General estimating equation was used for calculation of the primary endpoint and to adjust for confounding variables. A total of 598 arterial blood gas samples from 120 patients, receiving 429 RBC units, were analyzed. Mean (±SD) RBC storage was 21 (±9) days. RBC storage duration and the increase in recipients' cHb were inversely and gradually related; that is, the older the RBCs, the lower the increase in the recipients' cHb after transfusion (storage < 12 days, ΔcHb per unit RBCs +0.82 [95% confidence interval, 0.42-1.21] g/dL, p < 0.01; storage 12-20 days, +0.66 [0.46-0.86] g/dL, p < 0.01; storage 21-29 days, +0.56 [0.33-0.79] g/dL, p < 0.01; storage ≥30 days, +0.39 [0.07 to 0.71] g/dL, p = 0.02). Transfusion of senescent RBCs increased cHb less effectively than transfusion of fresher RBCs. © 2018 AABB.

INRA, a new high-frequency antigen in the INDIAN (IN023) blood group system.

PubMed

Joshi, Sanmukh R; Sheladiya, Ankita; Mendapara-Dobariya, Kinjal V

2017-01-01

The INDIAN blood group system comprises 4 antigens sensitive to enzymes and 2-aminoethyl isothiouronium bromide (AET). The patient's antibody was investigated for its specificity to the high-frequency antigens (HFA) of this system. Low ionic strength solution (LISS)-tube/LISS-indirect antiglobulin test (IAT) methods were used. The patient's red blood cells (RBCs) were tested with antisera to HFA. Her antibody was tested with RBCs lacking the HFA. Furthermore, it was tested with RBCs as untreated or treated with enzyme or AET. The genetic sequence was studied for mutation in CD44 gene that encodes INDIAN antigens. The patient was grouped A1B, RhD+, antibody screening test positive, direct antiglobulin test negative. A negative autocontrol test had suggested to the alloantibody being present. Antibody had agglutinated RBCs in LISS-tube at RT and by LISS-IAT at 37°C. The RBCs of the 11-cell panel, those lacking HFA and from 50 random donors, were agglutinated by her antibody indicating its specificity to the HFA, though the RBCs of Lu (a-b-)/In (Lu) type showed a weaker reaction. The patient's RBCs were agglutinated by antisera to a number of the enzyme-sensitive HFA, including those of INDIAN blood groups. The antibody showed reduced reactivity with the RBCs treated with papain, chymotrypsin, and AET but resistant to trypsin and dithiothreitol. The patient's genetic sequence revealed a novel homozygous mutation 449G>A in exon 5 of CD44 . The antibody to enzyme sensitive HFA was tested for serological and molecular genetics studies and found to be directed to the novel HFA, named as INRA of the INDIAN blood group system and was assigned a numerical symbol IN: 005 by the International Society of Blood Transfusion (ISBT).

Differential targeting of Gbetagamma-subunit signaling with small molecules.

PubMed

Bonacci, Tabetha M; Mathews, Jennifer L; Yuan, Chujun; Lehmann, David M; Malik, Sundeep; Wu, Dianqing; Font, Jose L; Bidlack, Jean M; Smrcka, Alan V

2006-04-21

G protein betagamma subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gbetagamma subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gbetagamma subunit functions. Several compounds bound to Gbetagamma subunits with affinities from 0.1 to 60 muM and selectively modulated functional Gbetagamma-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor-dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein-coupled receptor signaling that may represent an important therapeutic strategy.

Development of a High-Throughput Magnetic Separation Device for Malaria-infected Erythrocytes

PubMed Central

Martin, A. Blue; Wu, Wei-Tao; Kameneva, Marina V.; Antaki, James F.

2017-01-01

This study describes a non-dilutive high-gradient magnetic separation (HGMS) device intended to continuously remove malaria-infected red blood cells (iRBCs) from the circulation. A mesoscale prototype device with disposable photo-etched ferromagnetic grid and reusable permanent magnet was designed with a computationally-optimized magnetic force. The prototype device was evaluated in-vitro using a non-pathogenic analog for malaria-infected blood, comprised of 24% healthy RBCs, 6% human methemoglobin RBCs (metRBCs), and 70% phosphate buffer solution (PBS). The device provided a 27.0 ± 2.2% reduction of metRBCs in a single pass at a flow rate of 77 μL min−1. This represents a clearance rate over 380 times greater throughput than microfluidic devices reported previously. These positive results encourage development of a clinical scale system that would economize time and donor blood for treating severe malaria. PMID:28924724

Development of a High-Throughput Magnetic Separation Device for Malaria-Infected Erythrocytes.

PubMed

Blue Martin, A; Wu, Wei-Tao; Kameneva, Marina V; Antaki, James F

2017-12-01

This study describes a non-dilutive high-gradient magnetic separation (HGMS) device intended to continuously remove malaria-infected red blood cells (iRBCs) from the circulation. A mesoscale prototype device with disposable photo-etched ferromagnetic grid and reusable permanent magnet was designed with a computationally-optimized magnetic force. The prototype device was evaluated in vitro using a non-pathogenic analog for malaria-infected blood, comprised of 24% healthy RBCs, 6% human methemoglobin RBCs (metRBCs), and 70% phosphate buffer solution (PBS). The device provided a 27.0 ± 2.2% reduction of metRBCs in a single pass at a flow rate of 77 μL min -1 . This represents a clearance rate over 380 times greater throughput than microfluidic devices reported previously. These positive results encourage development of a clinical scale system that would economize time and donor blood for treating severe malaria.

Comparison of red blood cells from gastric cancer patients and healthy persons using FTIR spectroscopy

NASA Astrophysics Data System (ADS)

Liu, Hui; Su, Qinglong; Sheng, Daping; Zheng, Wei; Wang, Xin

2017-02-01

In this paper, FTIR spectroscopy was used to compare gastric cancer patients' red blood cells (RBCs) with healthy persons' RBCs. IR spectra were acquired with high resolution. The A1653/A1543 (the protein secondary structures), A1543/A2958 (the relative content of proteins and lipids), A1106/A1166 (the structure and content changes of sugars) and A1543/A1106 (the relative content of proteins and sugars) ratios of gastric cancer patients' RBCs were significantly different from those of healthy persons' RBCs. Curve fitting results showed that the protein secondary structures and sugars' structures had differences between gastric cancer patients' and healthy persons' RBCs. Additionally, FTIR spectroscopy could obtain 95% sensitivity, 70% specificity, 84.2% accuracy and 80.9% positive predictive value in combination with canconical discriminant analysis. The above results indicate FTIR spectroscopy may be useful for diagnosing gastric cancer.

'Gardos Channelopathy': a variant of hereditary Stomatocytosis with complex molecular regulation.

PubMed

Fermo, Elisa; Bogdanova, Anna; Petkova-Kirova, Polina; Zaninoni, Anna; Marcello, Anna Paola; Makhro, Asya; Hänggi, Pascal; Hertz, Laura; Danielczok, Jens; Vercellati, Cristina; Mirra, Nadia; Zanella, Alberto; Cortelezzi, Agostino; Barcellini, Wilma; Kaestner, Lars; Bianchi, Paola

2017-05-11

The Gardos channel is a Ca 2+ sensitive, K + selective channel present in several tissues including RBCs, where it is involved in cell volume regulation. Recently, mutations at two different aminoacid residues in KCNN4 have been reported in patients with hereditary xerocytosis. We identified by whole exome sequencing a new family with two members affected by chronic hemolytic anemia carrying mutation R352H in the KCNN4 gene. No additional mutations in genes encoding for RBCs cytoskeletal, membrane or channel proteins were detected. We performed functional studies on patients' RBCs to evaluate the effects of R352H mutation on the cellular properties and eventually on the clinical phenotype. Gardos channel hyperactivation was demonstrated in circulating erythrocytes and erythroblasts differentiated ex-vivo from peripheral CD34+ cells. Pathological alterations in the function of multiple ion transport systems were observed, suggesting the presence of compensatory effects ultimately preventing cellular dehydration in patient's RBCs; moreover, flow cytometry and confocal fluorescence live-cell imaging showed Ca 2+ overload in the RBCs of both patients and hypersensitivity of Ca 2+ uptake by RBCs to swelling. Altogether these findings suggest that the 'Gardos channelopathy' is a complex pathology, to some extent different from the common hereditary xerocytosis.

Effect of pronase on high-incidence blood group antigens and the prevalence of antibodies to pronase-treated erythrocytes.

PubMed

Reid, M E; Greeen, C A; Hoffer, J; Øyen, R

1996-01-01

Pronase is a useful and relatively nonspecific protease that cleaves many red blood cell (RBC) membrane proteins that carry blood group antigens. Unexpected findings in tests using pronase-treated RBCs during the investigation of a patient's blood sample led us to test which high-incidence blood group antigens were sensitive and which were resistant to pronase treatment, and to determine the prevalence of antipronase in the serum of blood donors. Our results show that antigens in the Cromer and Lutheran blood group systems and the JMH antigen were sensitive to pronase treatment of RBCs. Antigens in the Dombrock blood group system and Sc1 were either sensitive to or markedly weakened by pronase treatment of RBCs. The following high-incidence antigens were resistant to treatment of RBCs with pronase: AnWj, Ata, Coa, Co3, Dib, EnaFR, Era, Fy3, Jk3, Jra, k, Kpb, Jsb, K14, Lan, Oka, Rh17, U, Vel, and Wrb. Over half of the serum samples from normal blood donors contained antibodies to pronase-treated RBCs. When testing human serum against pronase-treated RBCs, it is essential either to use an autocontrol or to perform the testing with an eluate.

Rouleaux red blood cells splitting in microscopic thin blood smear images via local maxima, circles drawing, and mapping with original RBCs.

PubMed

Rehman, Amjad; Abbas, Naveed; Saba, Tanzila; Mahmood, Toqeer; Kolivand, Hoshang

2018-04-10

Splitting the rouleaux RBCs from single RBCs and its further subdivision is a challenging area in computer-assisted diagnosis of blood. This phenomenon is applied in complete blood count, anemia, leukemia, and malaria tests. Several automated techniques are reported in the state of art for this task but face either under or over splitting problems. The current research presents a novel approach to split Rouleaux red blood cells (chains of RBCs) precisely, which are frequently observed in the thin blood smear images. Accordingly, this research address the rouleaux splitting problem in a realistic, efficient and automated way by considering the distance transform and local maxima of the rouleaux RBCs. Rouleaux RBCs are splitted by taking their local maxima as the centres to draw circles by mid-point circle algorithm. The resulting circles are further mapped with single RBC in Rouleaux to preserve its original shape. The results of the proposed approach on standard data set are presented and analyzed statistically by achieving an average recall of 0.059, an average precision of 0.067 and F-measure 0.063 are achieved through ground truth with visual inspection. © 2018 Wiley Periodicals, Inc.

Modifying the red cell surface: towards an ABO-universal blood supply.

PubMed

Olsson, Martin L; Clausen, Henrik

2008-01-01

Eliminating the risk for ABO-incompatible transfusion errors and simplifying logistics by creating a universal blood inventory is a challenging idea. Goldstein and co-workers pioneered the field of enzymatic conversion of blood group A and B red blood cells (RBCs) to O (ECO). Using alpha-galactosidase from coffee beans to produce B-ECO RBCs, proof of principle for this revolutionary concept was achieved in clinical trials. However, because this enzyme has poor kinetic properties and low pH optimum the process was not economically viable. Conversion of group A RBCs was only achieved with the weak A2 subgroup with related enzymes having acidic pH optima. More recently, the identification of entirely new families of bacterial exoglycosidases with remarkably improved kinetic properties for cleaving A and B antigens has reinvigorated the field. Enzymatic conversion of groups A, B and AB RBCs with these novel enzymes resulting in ECO RBCs typing as O can now be achieved with low enzyme protein consumption, short incubation times and at neutral pH. Presently, clinical trials evaluating safety and efficacy of ECO RBCs are ongoing. Here, we review the status of the ECO technology, its impact and potential for introduction into clinical component preparation laboratories.

[Optimization of lyophilization procedures for freeze-drying of human red blood cells].

PubMed

Chen, Lin-feng; Liu, Jing-han; Wang, De-qing; Ouyang, Xi-lin; Zhuang, Yuan; Che, Ji; Yu, Yang; Li, Hui

2010-09-01

To investigate the different parameters of the lyophilization procedures that affect the recovery of the rehydrated red blood cells (RBCs). Human RBCs loaded in tubes were cooled with 4 different modes and subjected to water bath at 25 degrees celsius;. The morphological changes of the RBCs were observed to assess the degree of vitrification, and the specimens were placed in the freeze-dryer with the temperature set up at 40, -50, -60, -70 and -80 degrees celsius;. The rates of temperature rise of the main and secondary drying in the lyophilization procedures were compared, and the water residue in the specimens was determined. The protectant did not show ice crystal in the course of freezing and thawing. No significant difference was found in the recovery rate of the rehydrated RBCs freeze-dried at the minimum temperature of -70 degrees celsius; and -80 degrees celsius; (P > 0.05). The E procedure resulted in the maximum recovery of the RBCs (83.14% ± 9.55%) and Hb (85.33% ± 11.42%), showing significant differences from the other groups(P < 0.01 or 0.05). The recovery of the RBCs showed a positive correlation to the water residue in the samples. Fast cooling in liquid nitrogen and shelf precooling at -70 degrees celsius; with a moderate rate of temperature rise in lyophylization and a start dry temperature close to the shelf equilibrium temperature produce optimal freeze-drying result of human RBCs.

Divergent calcium signaling in RBCs from Tropidurus torquatus (Squamata – Tropiduridae) strengthen classification in lizard evolution

PubMed Central

Beraldo, Flávio H; Garcia, Célia RS

2007-01-01

Background We have previously reported that a Teiid lizard red blood cells (RBCs) such as Ameiva ameiva and Tupinambis merianae controls intracellular calcium levels by displaying multiple mechanisms. In these cells, calcium stores could be discharged not only by: thapsigargin, but also by the Na+/H+ ionophore monensin, K+/H+ ionophore nigericin and the H+ pump inhibitor bafilomycin as well as ionomycin. Moreover, these lizards possess a P2Y-type purinoceptors that mobilize Ca2+ from intracellular stores upon ATP addition. Results Here we report, that RBCs from the tropidurid lizard Tropidurus torquatus store Ca2+ in endoplasmic reticulum (ER) pool but unlike in the referred Teiidae, these cells do not store calcium in monensin-nigericin sensitive pools. Moreover, mitochondria from T. torquatus RBCs accumulate Ca2+. Addition of ATP to a calcium-free medium does not increase the [Ca2+]c levels, however in a calcium medium we observe an increase in cytosolic calcium. This is an indication that purinergic receptors in these cells are P2X-like. Conclusion T. torquatus RBCs present different mechanisms from Teiid lizard red blood cells (RBCs), for controlling its intracellular calcium levels. At T. torquatus the ion is only stored at endoplasmic reticulum and mitochondria. Moreover activation of purinergic receptor, P2X type, was able to induce an influx of calcium from extracelullar medium. These studies contribute to the understanding of the evolution of calcium homeostasis and signaling in nucleated RBCs. PMID:17716375

Divergent calcium signaling in RBCs from Tropidurus torquatus (Squamata--Tropiduridae) strengthen classification in lizard evolution.

PubMed

Beraldo, Flávio H; Garcia, Célia R S

2007-08-23

We have previously reported that a Teiid lizard red blood cells (RBCs) such as Ameiva ameiva and Tupinambis merianae controls intracellular calcium levels by displaying multiple mechanisms. In these cells, calcium stores could be discharged not only by: thapsigargin, but also by the Na+/H+ ionophore monensin, K+/H+ ionophore nigericin and the H+ pump inhibitor bafilomycin as well as ionomycin. Moreover, these lizards possess a P2Y-type purinoceptors that mobilize Ca2+ from intracellular stores upon ATP addition. Here we report, that RBCs from the tropidurid lizard Tropidurus torquatus store Ca2+ in endoplasmic reticulum (ER) pool but unlike in the referred Teiidae, these cells do not store calcium in monensin-nigericin sensitive pools. Moreover, mitochondria from T. torquatus RBCs accumulate Ca2+. Addition of ATP to a calcium-free medium does not increase the [Ca2+]c levels, however in a calcium medium we observe an increase in cytosolic calcium. This is an indication that purinergic receptors in these cells are P2X-like. T. torquatus RBCs present different mechanisms from Teiid lizard red blood cells (RBCs), for controlling its intracellular calcium levels. At T. torquatus the ion is only stored at endoplasmic reticulum and mitochondria. Moreover activation of purinergic receptor, P2X type, was able to induce an influx of calcium from extracellular medium. These studies contribute to the understanding of the evolution of calcium homeostasis and signaling in nucleated RBCs.

Differential Targeting of Gβγ-Subunit Signaling with Small Molecules

NASA Astrophysics Data System (ADS)

Bonacci, Tabetha M.; Mathews, Jennifer L.; Yuan, Chujun; Lehmann, David M.; Malik, Sundeep; Wu, Dianqing; Font, Jose L.; Bidlack, Jean M.; Smrcka, Alan V.

2006-04-01

G protein βγ subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gβγ subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gβγ subunit functions. Several compounds bound to Gβγ subunits with affinities from 0.1 to 60 μM and selectively modulated functional Gβγ-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor-dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein-coupled receptor signaling that may represent an important therapeutic strategy.

Multicenter observational prehospital resuscitation on helicopter study.

PubMed

Holcomb, John B; Swartz, Michael D; DeSantis, Stacia M; Greene, Thomas J; Fox, Erin E; Stein, Deborah M; Bulger, Eileen M; Kerby, Jeffrey D; Goodman, Michael; Schreiber, Martin A; Zielinski, Martin D; O'Keeffe, Terence; Inaba, Kenji; Tomasek, Jeffrey S; Podbielski, Jeanette M; Appana, Savitri N; Yi, Misung; Wade, Charles E

2017-07-01

Earlier use of in-hospital plasma, platelets, and red blood cells (RBCs) has improved survival in trauma patients with severe hemorrhage. Retrospective studies have associated improved early survival with prehospital blood product transfusion (PHT). We hypothesized that PHT of plasma and/or RBCs would result in improved survival after injury in patients transported by helicopter. Adult trauma patients transported by helicopter from the scene to nine Level 1 trauma centers were prospectively observed from January to November 2015. Five helicopter systems had plasma and/or RBCs, whereas the other four helicopter systems used only crystalloid resuscitation. All patients meeting predetermined high-risk criteria were analyzed. Patients receiving PHT were compared with patients not receiving PHT. Our primary analysis compared mortality at 3 hours, 24 hours, and 30 days, using logistic regression to adjust for confounders and site heterogeneity to model patients who were matched on propensity scores. Twenty-five thousand one hundred eighteen trauma patients were admitted, 2,341 (9%) were transported by helicopter, of which 1,058 (45%) met the highest-risk criteria. Five hundred eighty-five of 1,058 patients were flown on helicopters carrying blood products. In the systems with blood available, prehospital median systolic blood pressure (125 vs 128) and Glasgow Coma Scale (7 vs 14) was significantly lower, whereas median Injury Severity Score was significantly higher (21 vs 14). Unadjusted mortality was significantly higher in the systems with blood products available, at 3 hours (8.4% vs 3.6%), 24 hours (12.6% vs 8.9%), and 30 days (19.3% vs 13.3%). Twenty-four percent of eligible patients received a PHT. A median of 1 unit of RBCs and plasma were transfused prehospital. Of patients receiving PHT, 24% received only plasma, 7% received only RBCs, and 69% received both. In the propensity score matching analysis (n = 109), PHT was not significantly associated with mortality at any time point, although only 10% of the high-risk sample were able to be matched. Because of the unexpected imbalance in systolic blood pressure, Glasgow Coma Scale, and Injury Severity Score between systems with and without blood products on helicopters, matching was limited, and the results of this study are inconclusive. With few units transfused to each patient and small outcome differences between groups, it is likely large, multicenter, randomized studies will be required to detect survival differences in this important population. Level II.

Multicenter Observational Prehospital Resuscitation on Helicopter Study (PROHS)

PubMed Central

Holcomb, John B.; Swartz, Michael D.; DeSantis, Stacia M.; Greene, Thomas J.; Fox, Erin E.; Stein, Deborah M.; Bulger, Eileen M.; Kerby, Jeffrey D.; Goodman, Michael; Schreiber, Martin A.; Zielinski, Martin D.; O’Keeffe, Terence; Inaba, Kenji; Tomasek, Jeffrey S.; Podbielski, Jeanette M.; Appana, Savitri; Yi, Misung; Wade, Charles E.

2017-01-01

BACKGROUND Earlier use of in-hospital plasma, platelets and red blood cells (RBCs) has improved survival in trauma patients with severe hemorrhage. Retrospective studies have associated improved early survival with prehospital blood product transfusion (PHT). We hypothesized that PHT of plasma and/or RBCs would result in improved survival after injury in patients transported by helicopter. METHODS Adult trauma patients transported by helicopter from the scene to nine Level 1 trauma centers were prospectively observed from Jan–Nov 2015. Five helicopter systems had plasma and/or RBCs while the other four helicopter systems used only crystalloid resuscitation. All patients meeting predetermined high risk criteria were analyzed. Patients receiving PHT were compared to patients not receiving PHT. Our primary analysis compared mortality at 3 hours, 24 hours, and 30 days, using logistic regression to adjust for confounders and site heterogeneity to model patients who were matched on propensity scores. RESULTS 25,118 trauma patients were admitted, 2341 (9%) were transported by helicopter, of which 1058 (45%) met the highest risk criteria. 585/1058 patients were flown on helicopters carrying blood products. In the systems with blood available, prehospital median systolic blood pressure (125 vs 128) and GCS (7 vs 14) was significantly lower, while median ISS was significantly higher (21 vs 14). Unadjusted mortality was significantly higher in the systems with blood products available, at 3 (8.4% vs 3.6%), 24 (12.6% vs 8.9%) hours and 30 days (19.3% vs 13.3%). 24% of eligible patients received a prehospital transfusion. A median of 1 unit of RBCs and plasma were transfused prehospital. Of patients receiving PHT, 24% received only plasma, 7% received only RBCs and 69% received both. In the propensity score matching analysis (n=109), PHT was not significantly associated with mortality at any time point, although only 10% of the high risk sample were able to be matched. CONCLUSIONS Because of the unexpected imbalance in systolic blood pressure, GCS and ISS between systems with and without blood products on helicopters, matching was limited and the results of this study are inconclusive. With few units transfused to each patient and small outcome differences between groups, it is likely large, multicenter, randomized studies will be required to detect survival differences in this important population. PMID:28383476

Metabolomics of AS-1 RBC storage

PubMed Central

Roback, John D.; Josephson, Cassandra D.; Waller, Edmund K.; Newman, James L.; Karatela, Sulaiman; Uppal, Karan; Jones, Dean; Zimring, James C.; Dumont, Larry J.

2014-01-01

Background Population based investigations suggest that red blood cells (RBCs) are therapeutically effective when collected, processed and stored for up to 42 days under validated conditions prior to transfusion. However, some retrospective clinical studies have shown worse patient outcomes when transfused RBCs have been stored for the longest times. Furthermore, studies of RBC persistence in the circulation after transfusion have suggested that considerable donor-to-donor variability exists, and may affect transfusion efficacy. To understand the limitations of current blood storage technologies and to develop approaches to improve RBC storage and transfusion efficacy, we investigated the global metabolic alterations that occur when RBCs are stored in AS-1 (AS1-RBC). Methods Leukoreduced AS1-RBC units prepared from 9 volunteer research donors (12 total donated units) were serially sampled for metabolomics analysis over 42 days of refrigerated storage. Samples were tested by GC/MS and LC/MS/MS, and specific biochemical compounds were identified by comparison to a library of purified standards. Results Over three experiments, 185–264 defined metabolites were quantified in stored RBC samples. Kinetic changes in these biochemicals confirmed known alterations in glycolysis and other pathways previously identified in RBCs stored in SAGM (SAGM-RBC). Furthermore, we identified additional alterations not previously seen in SAGM-RBCs (e.g., stable pentose phosphate pathway flux, progressive decreases in oxidized glutathione), and we delineated changes occurring in other metabolic pathways not previously studied (e.g., S-adenosyl methionine cycle). These data are presented in the context of a detailed comparison with previous studies of SAGM-RBCs from human donors and murine AS1-RBCs. Conclusion Global metabolic profiling of AS1-RBCs revealed a number of biochemical alterations in stored blood that may affect RBC viability during storage as well as therapeutic effectiveness of stored RBCs in transfusion recipients. Significance These results provide future opportunities to more clearly pinpoint the metabolic defects during RBC storage, to identify biomarkers for donor screening and prerelease RBC testing, and to develop improved RBC storage solutions and methodologies. PMID:24636780

Length of Storage of Red Blood Cells and Patient Survival After Blood Transfusion: A Binational Cohort Study.

PubMed

Halmin, Märit; Rostgaard, Klaus; Lee, Brian K; Wikman, Agneta; Norda, Rut; Nielsen, Kaspar René; Pedersen, Ole B; Holmqvist, Jacob; Hjalgrim, Henrik; Edgren, Gustaf

2017-02-21

Possible negative effects, including increased mortality, among persons who receive stored red blood cells (RBCs) have recently garnered considerable attention. Despite many studies, including 4 randomized trials, no consensus exists. To study the association between the length of RBC storage and mortality in a large population-based cohort of patients who received transfusions, allowing detection of small yet clinically significant effects. Binational cohort study. All transfusion recipients in Sweden and Denmark. 854 862 adult patients who received transfusions from 2003 to 2012. Patients were followed from first blood transfusion. Relative and absolute risks for death in 30 days or 1 year in relation to length of RBC storage were assessed by using 3 independent analytic approaches. All analyses were conducted by using Cox proportional hazards regression. Regardless of the analytic approach, no association was found between the length of RBC storage and mortality. The difference in 30-day cumulative mortality between patients receiving blood stored for 30 to 42 days and those receiving blood stored for 10 to 19 days was -0.2% (95% CI, -0.5% to 0.1%). Even among patients who received more than 6 units of RBCs stored for 30 days or longer, the hazard ratio of death was 1.00 (CI, 0.96 to 1.05) compared with those who received no such units. Observational study; risk of confounding by indication. Consistent with previous randomized trials, this study found no association between the length of storage of transfused RBCs and patient mortality. Results were homogeneous, with differences in absolute mortality consistently less than 1% among the most extreme exposure categories. These findings suggest that the current practice of storing RBCs for up to 42 days does not need to be changed. The Swedish Research Council, Swedish Heart-Lung Foundation, Swedish Society for Medical Research, Strategic Research Program in Epidemiology at Karolinska Institutet, and Danish Council for Independent Research.

α-Synuclein Heterocomplexes with β-Amyloid Are Increased in Red Blood Cells of Parkinson’s Disease Patients and Correlate with Disease Severity

PubMed Central

Daniele, Simona; Frosini, Daniela; Pietrobono, Deborah; Petrozzi, Lucia; Lo Gerfo, Annalisa; Baldacci, Filippo; Fusi, Jonathan; Giacomelli, Chiara; Siciliano, Gabriele; Trincavelli, Maria Letizia; Franzoni, Ferdinando; Ceravolo, Roberto; Martini, Claudia; Bonuccelli, Ubaldo

2018-01-01

Neurodegenerative disorders (NDs) are characterized by abnormal accumulation/misfolding of specific proteins, primarily α-synuclein (α-syn), β-amyloid1–42 (Aβ1–42) and tau, in both brain and peripheral tissues. In addition to oligomers, the role of the interactions of α-syn with Aβ or tau has gradually emerged. Nevertheless, despite intensive research, NDs have no accepted peripheral markers for biochemical diagnosis. In this respect, Red Blood Cells (RBCs) are emerging as a valid peripheral model for the study of aging-related pathologies. Herein, a small cohort (N = 28) of patients affected by Parkinson’s disease (PD) and age-matched controls were enrolled to detect the content of α-syn (total and oligomeric), Aβ1–42 and tau (total and phosphorylated) in RBCs. Moreover, the presence of α-syn association with tau and Aβ1–42 was explored by co-immunoprecipitation/western blotting in the same cells, and quantitatively confirmed by immunoenzymatic assays. For the first time, PD patients were demonstrated to exhibit α-syn heterocomplexes with Aβ1–42 and tau in peripheral tissues; interestingly, α-syn-Aβ1–42 concentrations were increased in PD subjects with respect to healthy controls (HC), and directly correlated with disease severity and motor deficits. Moreover, total-α-syn levels were decreased in PD subjects and inversely related to their motor deficits. Finally, an increase of oligomeric-α-syn and phosphorylated-tau was observed in RBCs of the enrolled patients. The combination of three parameters (total-α-syn, phosphorylated-tau and α-syn-Aβ1–42 concentrations) provided the best fitting predictive index for discriminating PD patients from controls. Nevertheless further investigations should be required, overall, these data suggest α-syn hetero-aggregates in RBCs as a putative tool for the diagnosis of PD. PMID:29520218

The haemagglutination activity of equine herpesvirus type 1 glycoprotein C.

PubMed

Andoh, Kiyohiko; Hattori, Shiho; Mahmoud, Hassan Y A H; Takasugi, Maaya; Shimoda, Hiroshi; Bannai, Hiroshi; Tsujimura, Koji; Matsumura, Tomio; Kondo, Takashi; Kirisawa, Rikio; Mochizuki, Masami; Maeda, Ken

2015-01-02

Equine herpesvirus type 1 (EHV-1) has haemagglutination (HA) activity toward equine red blood cells (RBCs), but the identity of its haemagglutinin is unknown. To identify the haemagglutinin of EHV-1, the major glycoproteins of EHV-1 were expressed in 293T cells, and the cells or cell lysates were mixed with equine RBCs. The results showed that only EHV-1 glycoprotein C (gC)-producing cells adsorbed equine RBCs, and that the lysate of EHV-1 gC-expressing cells agglutinated equine RBCs. EHV-1 lacking gC did not show HA activity. HA activity was inhibited by monoclonal antibodies (MAbs) specific for gC, but not by antibodies directed against other glycoproteins. In addition, HA activity was not inhibited by the addition of heparin. These results indicate that EHV-1 gC can bind equine RBCs irrespective of heparin, in contrast to other herpesvirus gC proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

Effect of ascorbic acid on storage of Greyhound erythrocytes.

PubMed

Fontes, Jorge A; Banerjee, Uddyalok; Iazbik, M Cristina; Marín, Liliana M; Couto, C Guillermo; Palmer, Andre F

2015-09-01

To assess changes in biochemical and biophysical properties of canine RBCs during cold (1° to 6°C) storage in a licensed RBC additive solution (the RBC preservation solution designated AS-1) supplemented with ascorbic acid. Blood samples from 7 neutered male Greyhounds; all dogs had negative results when tested for dog erythrocyte antigen 1.1. Blood was collected into citrate-phosphate-dextrose and stored in AS-1. Stored RBCs were supplemented with 7.1mM ascorbic acid or with saline (0.9% NaCl) solution (control samples). Several biochemical and biophysical properties of RBCs were measured, including percentage hemolysis, oxygen-hemoglobin equilibrium, and the kinetic rate constants for O2 dissociation, carbon monoxide association, and nitric oxide dioxygenation. Greyhound RBCs stored in AS-1 supplemented with ascorbic acid did not have significantly decreased hemolysis, compared with results for the control samples, during the storage period. In this study, ascorbic acid did not reduce hemolysis during storage. Several changes in stored canine RBCs were identified as part of the hypothermic storage lesion.

The Effects of Ethanol on the Morphological and Biochemical Properties of Individual Human Red Blood Cells.

PubMed

Lee, Sang Yun; Park, Hyun Joo; Best-Popescu, Catherine; Jang, Seongsoo; Park, Yong Keun

2015-01-01

Here, we report the results of a study on the effects of ethanol exposure on human red blood cells (RBCs) using quantitative phase imaging techniques at the level of individual cells. Three-dimensional refractive index tomograms and dynamic membrane fluctuations of RBCs were measured using common-path diffraction optical tomography, from which morphological (volume, surface area, and sphericity); biochemical (hemoglobin (Hb) concentration and Hb content); and biomechanical (membrane fluctuation) parameters were retrieved at various concentrations of ethanol. RBCs exposed to the ethanol concentration of 0.1 and 0.3% v/v exhibited cell sphericities higher than those of normal cells. However, mean surface area and sphericity of RBCs in a lethal alcoholic condition (0.5% v/v) are not statistically different with those of healthy RBCs. Meanwhile, significant decreases of Hb content and concentration in RBC cytoplasm at the lethal condition were observed. Furthermore, dynamic fluctuation of RBC membranes increased significantly upon ethanol treatments, indicating ethanol-induced membrane fluidization.

A genome-editing strategy to treat β-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition.

PubMed

Traxler, Elizabeth A; Yao, Yu; Wang, Yong-Dong; Woodard, Kaitly J; Kurita, Ryo; Nakamura, Yukio; Hughes, Jim R; Hardison, Ross C; Blobel, Gerd A; Li, Chunliang; Weiss, Mitchell J

2016-09-01

Disorders resulting from mutations in the hemoglobin subunit beta gene (HBB; which encodes β-globin), mainly sickle cell disease (SCD) and β-thalassemia, become symptomatic postnatally as fetal γ-globin expression from two paralogous genes, hemoglobin subunit gamma 1 (HBG1) and HBG2, decreases and adult β-globin expression increases, thereby shifting red blood cell (RBC) hemoglobin from the fetal (referred to as HbF or α2γ2) to adult (referred to as HbA or α2β2) form. These disorders are alleviated when postnatal expression of fetal γ-globin is maintained. For example, in hereditary persistence of fetal hemoglobin (HPFH), a benign genetic condition, mutations attenuate γ-globin-to-β-globin switching, causing high-level HbF expression throughout life. Co-inheritance of HPFH with β-thalassemia- or SCD-associated gene mutations alleviates their clinical manifestations. Here we performed CRISPR-Cas9-mediated genome editing of human blood progenitors to mutate a 13-nt sequence that is present in the promoters of the HBG1 and HBG2 genes, thereby recapitulating a naturally occurring HPFH-associated mutation. Edited progenitors produced RBCs with increased HbF levels that were sufficient to inhibit the pathological hypoxia-induced RBC morphology found in SCD. Our findings identify a potential DNA target for genome-editing-mediated therapy of β-hemoglobinopathies.

An automated cell analysis sensing system based on a microfabricated rheoscope for the study of red blood cells physiology.

PubMed

Bransky, Avishay; Korin, Natanel; Nemirovski, Yael; Dinnar, Uri

2006-08-15

An automated rheoscope has been developed, utilizing a microfabricated glass flow cell, high speed camera and advanced image-processing software. RBCs suspended in a high viscosity medium were filmed flowing through a microchannel. Under these conditions, RBCs exhibit different orientations and deformations according to their location in the velocity profile. The rheoscope system produces valuable data such as velocity profile of RBCs, spatial distribution within a microchannel and deformation index (DI) curves. The variation of DI across the channel height, due to change in shear stress, was measured carrying implications for diffractometry methods. These curves of DI were taken at a constant flow rate and cover most of the relevant shear stress spectrum. This is an improvement of the existing techniques for deformability measurements and may serve as a diagnostic tool for certain blood disorders. The DI curves were compared to measurements of the flowing RBCs velocity profile. In addition, we found that RBCs flowing in a microchannel are mostly gathered in the center of the flow and maintain a characteristic spatial distribution. The spatial distribution in this region changes slightly with increasing flow rate. Hence, the system described, provides means for examining the behavior of individual RBCs, and may serve as a microfabricated diagnostic device for deformability measurement.

Erythrocytes-based synthetic delivery systems: transition from conventional to novel engineering strategies.

PubMed

Bhateria, Manisha; Rachumallu, Ramakrishna; Singh, Rajbir; Bhatta, Rabi Sankar

2014-08-01

Erythrocytes (red blood cells [RBCs]) and artificial or synthetic delivery systems such as liposomes, nanoparticles (NPs) are the most investigated carrier systems. Herein, progress made from conventional approach of using RBC as delivery systems to novel approach of using synthetic delivery systems based on RBC properties will be reviewed. We aim to highlight both conventional and novel approaches of using RBCs as potential carrier system. Conventional approaches which include two main strategies are: i) directly loading therapeutic moieties in RBCs; and ii) coupling them with RBCs whereas novel approaches exploit structural, mechanical and biological properties of RBCs to design synthetic delivery systems through various engineering strategies. Initial attempts included coupling of antibodies to liposomes to specifically target RBCs. Knowledge obtained from several studies led to the development of RBC membrane derived liposomes (nanoerythrosomes), inspiring future application of RBC or its structural features in other attractive delivery systems (hydrogels, filomicelles, microcapsules, micro- and NPs) for even greater potential. In conclusion, this review dwells upon comparative analysis of various conventional and novel engineering strategies in developing RBC based drug delivery systems, diversifying their applications in arena of drug delivery. Regardless of the challenges in front of us, RBC based delivery systems offer an exciting approach of exploiting biological entities in a multitude of medical applications.

Experimental analysis on viscoelasticity-induced migration of RBCs using digital holographic microscopy

NASA Astrophysics Data System (ADS)

Go, Taesik; Byeon, Hyeokjun; Lee, Sang Joon

2016-11-01

Migration of particles in viscoelastic fluids has recently received large attention, because the generated elastic forces in viscoelastic fluids give rise to a simple focusing pattern over a wide range of flow rates. In this study, the vertical focusing and alignment of rigid spherical particles, normal and hardened RBCs in a viscoelastic fluid were experimentally investigated by employing a digital in-line holographic microscopy (DIHM). By the elastic forces, the three different particles are pushed away from the walls and concentrated in the midplane of the rectangular microchannel. Furthermore, most of both RBCs maintain face-on orientation in the microchannel. The effects of deformability of RBC on the viscoelasticity-induced migration and orientation in the channel were also examined. In contrary to non-deformable particles, normal RBCs are dispersed as flow rate increases. In the region near side wall of the microchannel, normal RBCs have edge-on orientation with a large angle of inclination, compared to hardened RBCs. These findings have a strong potential in the design of microfluidic devices for deformability-based separation of cells in viscoelastic fluid flows and label-free diagnoses of certain hematological diseases. This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea government (MSIP) (No. 2008-0061991).

Application of image flow cytometry for the characterization of red blood cell morphology

NASA Astrophysics Data System (ADS)

Pinto, Ruben N.; Sebastian, Joseph A.; Parsons, Michael; Chang, Tim C.; Acker, Jason P.; Kolios, Michael C.

2017-02-01

Red blood cells (RBCs) stored in hypothermic environments for the purpose of transfusion have been documented to undergo structural and functional changes over time. One sign of the so-called RBC storage lesion is irreversible damage to the cell membrane. Consequently, RBCs undergo a morphological transformation from regular, deformable biconcave discocytes to rigid spheroechinocytes. The spherically shaped RBCs lack the deformability to efficiently enter microvasculature, thereby reducing the capacity of RBCs to oxygenate tissue. Blood banks currently rely on microscope techniques that include fixing, staining and cell counting in order to morphologically characterize RBC samples; these methods are labor intensive and highly subjective. This study presents a novel, high-throughput RBC morphology characterization technique using image flow cytometry (IFC). An image segmentation template was developed to process 100,000 images acquired from the IFC system and output the relative spheroechinocyte percentage. The technique was applied on samples extracted from two blood bags to monitor the morphological changes of the RBCs during in vitro hypothermic storage. The study found that, for a given sample of RBCs, the IFC method was twice as fast in data acquisition, and analyzed 250-350 times more RBCs than the conventional method. Over the lifespan of the blood bags, the mean spheroechinocyte population increased by 37%. Future work will focus on expanding the template to segregate RBC images into more subpopulations for the validation of the IFC method against conventional techniques; the expanded template will aid in establishing quantitative links between spheroechinocyte increase and other RBC storage lesion characteristics.

Identification and characterization of a newly recognized population of high-Na+, low-K+, low-density sickle and normal red cells.

PubMed

Bookchin, R M; Etzion, Z; Sorette, M; Mohandas, N; Skepper, J N; Lew, V L

2000-07-05

We describe a population of sickle cell anemia red cells (SS RBCs) ( approximately 4%) and a smaller fraction of normal RBCs (<0.03%) that fail to dehydrate when permeabilized to K(+) with either valinomycin or elevated internal Ca(2+). The nonshrinking, valinomycin-resistant (val-res) fractions, first detected by flow cytometry of density-fractionated SS RBCs, constituted up to 60% of the lightest, reticulocyte-rich (R1) cell fraction, and progressively smaller portions of the slightly denser R2 cells and discocytes. R1 val-res RBCs had a mean cell hemoglobin concentration of approximately 21 g of Hb per dl, and many had an elongated shape like "irreversibly sickled cells," suggesting a dense SS cell origin. Of three possible explanations for val-res cells, failure of valinomycin to K(+)-permeabilize the cells, low co-ion permeability, or reduced driving K(+) gradient, the latter proved responsible: Both SS and normal val-res RBCs were consistently high-Na(+) and low-K(+), even when processed entirely in Na-free media. Ca(2+) + A23187-induced K(+)-permeabilization of SS R1 fractions revealed a similar fraction of cal-res cells, whose (86)Rb uptake showed both high Na/K pump and leak fluxes. val-res/cal-res RBCs might represent either a distinct erythroid genealogy, or an "end-stage" of normal and SS RBCs. This paper focuses on the discovery, basic characterization, and exclusion of artifactual origin of this RBC fraction. Many future studies will be needed to clarify their mechanism of generation and full pathophysiological significance.

Tracking Resilience to Infections by Mapping Disease Space

PubMed Central

Thomas Tate, Ann; Rath, Poonam; Cumnock, Katherine; Schneider, David S.

2016-01-01

Infected hosts differ in their responses to pathogens; some hosts are resilient and recover their original health, whereas others follow a divergent path and die. To quantitate these differences, we propose mapping the routes infected individuals take through “disease space.” We find that when plotting physiological parameters against each other, many pairs have hysteretic relationships that identify the current location of the host and predict the future route of the infection. These maps can readily be constructed from experimental longitudinal data, and we provide two methods to generate the maps from the cross-sectional data that is commonly gathered in field trials. We hypothesize that resilient hosts tend to take small loops through disease space, whereas nonresilient individuals take large loops. We support this hypothesis with experimental data in mice infected with Plasmodium chabaudi, finding that dying mice trace a large arc in red blood cells (RBCs) by reticulocyte space as compared to surviving mice. We find that human malaria patients who are heterozygous for sickle cell hemoglobin occupy a small area of RBCs by reticulocyte space, suggesting this approach can be used to distinguish resilience in human populations. This technique should be broadly useful in describing the in-host dynamics of infections in both model hosts and patients at both population and individual levels. PMID:27088359

Supernatants and lipids from stored red blood cells activate pulmonary microvascular endothelium through the BLT2 receptor and protein kinase C activation.

PubMed

Silliman, Christopher C; Kelher, Marguerite R; Khan, Samina Y; West, F Bernadette; McLaughlin, Nathan J D; Elzi, David J; England, Kelly; Bjornsen, Jason; Kuldanek, Susan A; Banerjee, Anirban

2017-11-01

Although transfusion is a lifesaving intervention, it may be associated with significant morbidity in injured patients. We hypothesize that stored red blood cells (RBCs) induce proinflammatory activation of human pulmonary microvascular endothelial cells (HMVECs) resulting in neutrophil (PMN) adhesion and predisposition to acute lung injury (ALI). Ten units of RBCs were collected; 50% (by weight) were leukoreduced (LR-RBCs) and the remainder was unmodified and stored in additive solution-5 (AS-5). An additional 10 units of RBCs were collected, leukoreduced, and stored in AS-3. HMVECs were incubated with [10%-40%] FINAL of the supernatants on Day (D)1 to D42 of storage, lipid extracts, and purified lipids. Endothelial surface expression of intercellular adhesion molecule-1 (ICAM-1), interleukin (IL)-8 release, and PMN adhesion to HMVECs were measured. HMVEC signaling via the BLT2 receptor was evaluated. Supernatants and lipids were also employed as the first event in a two-event model of ALI. The supernatants [10%-40%] FINAL from D21 LR-RBCs and D42 RBCs and LR-RBCs and the lipids from D42 stored in AS-5 induced increased ICAM-1 surface expression on endothelium, IL-8 release, and PMN adhesion. In addition, the supernatants [20%-40%] FINAL from D21 and D42 RBCs in AS-5 also increased endothelial surface expression of ICAM-1. D42 supernatants and lipids also caused coprecipitation of β-arrestin-1 with BLT2, protein kinase C (PKC)β I , and PKCδ and served as the first event in a two-event rodent model of ALI. Lipids that accumulate during RBC storage activate endothelium and predispose to ALI, which may explain some of the adverse events associated with the transfusion of critically injured patients. © 2017 AABB.

Whole-plant growth and N utilization in transgenic rice plants with increased or decreased Rubisco content under different CO2 partial pressures.

PubMed

Sudo, Emi; Suzuki, Yuji; Makino, Amane

2014-11-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) strongly limits photosynthesis at lower CO2 concentration [CO2] whereas [corrected] Rubisco limitation is cancelled by elevated [CO2]. Therefore, increase or reduction in Rubisco content by transformation with a sense or an antisense RBCS construct are expected to alter the biomass production under different CO2 levels. RBCS-sense (125% Rubisco of wild-type) and -antisense (35% Rubisco of wild-type) rice (Oryza sativa L.) plants were grown for 63 days at three different CO2 levels: low [CO2] (28 Pa), normal [CO2] (40 Pa) and elevated [CO2] (120 Pa). The biomass of RBCS-sense plants was 32% and 15% greater at low [CO2] and normal [CO2] than that of the wild-type plants, respectively, but did not differ at elevated [CO2]. Conversely, the biomass of RBCS-antisense plants was the smallest at low [CO2]. Thus, overproduction of Rubisco was effective for biomass production at low [CO2]. Greater biomass production at low [CO2] in RBCS-sense plants was caused by an increase in the net assimilation rate, and associated with an increase in the amount of N uptake. Furthermore, Rubisco overproduction in RBCS-sense plants was also promoted at low [CO2]. Although it seems that low [CO2]-growth additionally stimulates the effect of RBCS overexpression, such a phenomenon observed at low [CO2] was mediated through an increase in total leaf N content. Thus, the dependence of the growth improvement in RBCS-sense rice on growth [CO2] was closely related to the degree of Rubisco overproduction which was accompanied not only by leaf N content but also by whole plant N content. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Identification of diverse defense mechanisms in rainbow trout red blood cells in response to halted replication of VHS virus

PubMed Central

Nombela, Ivan; Puente-Marin, Sara; Chico, Veronica; Villena, Alberto J.; Carracedo, Begoña; Ciordia, Sergio; Mena, Maria Carmen; Mercado, Luis; Perez, Luis; Coll, Julio; Ortega-Villaizan, Maria del Mar

2018-01-01

Background: It has been described that fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them and are their main target cell. The immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues has not yet been explored and is the objective of our study. Methods: Rainbow trout RBCs were obtained from peripheral blood, ficoll purified and exposed to Viral Haemorrhagic Septicaemia virus (VHSV). Immune response was evaluated by means of RT-qPCR, flow cytometry, immunofluorescence and isobaric tag for relative and absolute quantification (iTRAQ) protein profiling. Results: VHSV N gene transcripts incremented early postexposure and were drastically decreased after 6 hours postexposure (hpe). The expression of type I interferon ( ifn1) gene was significantly downregulated at early postexposure (3 hpe), together with a gradual downregulation of interferon-inducible mx and pkr genes until 72 hpe. Type I IFN protein was downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs, previously exposed to UV-inactivated VHSV, and TSS (stromal cell line from spleen) revealed IFN crosstalk between both cell types. On the other hand, anti-microbial peptide β-defensin 1 and neutrophil chemotactic factor interleukin 8 were slightly upregulated in VHSV-exposed RBCs. iTRAQ profiling revealed that VHSV exposure can induce a global protein downregulation in rainbow trout RBCs, mainly related to RNA stability and proteasome pathways. Antioxidant/antiviral response is also suggested to be involved in the response of rainbow trout RBCs to VHSV. Conclusions: A variety of mechanisms are proposed to be implicated in the antiviral response of rainbow trout RBCs against VHSV halted infection. Ongoing research is focused on understanding the mechanisms in detail. PMID:29527292

Overnight, room temperature hold of whole blood followed by 42-day storage of red blood cells in additive solution-7.

PubMed

Dumont, Larry J; Cancelas, Jose A; Maes, Lou Ann; Rugg, Neeta; Whitley, Pamela; Herschel, Louise; Siegal, Alan H; Szczepiorkowski, Zbigniew M; Hess, John R; Zia, Majid

2015-03-01

Overnight, room temperature hold (ONH) of whole blood before component processing offers several benefits. This study evaluated the storage and in vivo recovery characteristics of ONH red blood cells (RBCs) stored in additive solution-7 (AS-7). We conducted a three-center, three-arm evaluation of a new blood collection system with AS-7 compared to leukoreduced RBCs processed within 8 hours and stored in AS-1 (control). Whole blood (500 ± 50 mL) from healthy research subjects (n = 240) was held at room temperature 0 to 2 hours, 6 to 8 hours, or ONH (18-24 hr) before component processing and storage at 1 to 6 °C. RBCs were evaluated on Days 42 and 56 with a panel of in vitro assays. Subsets of the AS-7-stored RBCs were evaluated for (51) Cr 24-hour in vivo recovery and long-term survival. Adenosine triphosphate (ATP) levels in ONH RBCs were not different than AS-7 RBCs prepared within 8 hours. ATP was higher in the ONH group on Day 42 than control, and ATP was maintained in all AS-7 groups through Day 56. ONH units had 0.36 ± 0.14% on Day 42 hemolysis (60/60 < 0.8%), and 0.54 ± 0.22% on Day 56 (10/60 > 0.8%, 2/60 > 1%). In vivo recoveries of stored RBCs were not different between the AS-7 arms at 42 days (p = 0.16; 27/27 ONH units > 75%), but the Day 56 ONH was significantly less than ONH on Day 42 (p = 0.008; 7/28 < 75%). Overnight hold of whole blood at room temperature before component processing meets current regulatory requirements when RBCs are stored up to 42 days in AS-7. © 2014 AABB.

Adverse Effects of Hemorrhagic Shock Resuscitation with Stored Blood are Ameliorated by Inhaled Nitric Oxide in Lambs

PubMed Central

Baron, David M.; Beloiartsev, Arkadi; Nakagawa, Akito; Martyn, Trejeeve; Stowell, Christopher P.; Malhotra, Rajeev; Mayeur, Claire; Bloch, Kenneth D.; Zapol, Warren M.

2013-01-01

Objectives Transfusion of stored red blood cells (RBCs) is associated with increased morbidity and mortality in trauma patients. Plasma hemoglobin scavenges nitric oxide (NO), which can cause vasoconstriction, induce inflammation and activate platelets. We hypothesized that transfusion of RBCs stored for prolonged periods would induce adverse effects (pulmonary vasoconstriction, tissue injury, inflammation, and platelet activation) in lambs subjected to severe hemorrhagic shock, and that concurrent inhalation of NO would prevent these adverse effects. Design Animal study. Setting Research laboratory at the Massachusetts General Hospital, Boston, MA. Subjects Seventeen awake Polypay-breed lambs. Interventions Lambs were subjected to 2 h of hemorrhagic shock by acutely withdrawing 50% of their blood volume. Lambs were resuscitated with autologous RBCs stored for 2 h or less (fresh) or 39±2 (mean±SD) days (stored). Stored RBCs were administered with or without breathing NO (80 ppm) during resuscitation and for 21 h thereafter. Measurements and Main Results We measured hemodynamic and oxygenation parameters, markers of tissue injury and inflammation, plasma hemoglobin concentrations, and platelet activation. Peak pulmonary arterial pressure was higher after resuscitation with stored than with fresh RBCs (24±4 vs. 14±2 mmHg, p<0.001) and correlated with peak plasma hemoglobin concentrations (R2=0.56, p=0.003). At 21 h after resuscitation, pulmonary myeloperoxidase activity was higher in lambs resuscitated with stored than with fresh RBCs (11±2 vs. 4±1 U/g, p=0.007). Furthermore, transfusion of stored RBCs increased plasma markers of tissue injury and sensitized platelets to adenosine diphosphate activation. Breathing NO prevented the pulmonary hypertension, and attenuated the pulmonary myeloperoxidase activity, as well as tissue injury and sensitization of platelets to adenosine diphosphate. Conclusions Our data suggest that resuscitation of lambs from hemorrhagic shock with autologous stored RBCs induces pulmonary hypertension and inflammation, which can be ameliorated by breathing NO. PMID:23887236

INRA, a new high-frequency antigen in the INDIAN (IN023) blood group system

PubMed Central

Joshi, Sanmukh R.; Sheladiya, Ankita; Mendapara-Dobariya, Kinjal V.

2017-01-01

BACKGROUND: The INDIAN blood group system comprises 4 antigens sensitive to enzymes and 2-aminoethyl isothiouronium bromide (AET). AIM: The patient's antibody was investigated for its specificity to the high-frequency antigens (HFA) of this system. MATERIAL AND METHODS: Low ionic strength solution (LISS)-tube/LISS-indirect antiglobulin test (IAT) methods were used. The patient's red blood cells (RBCs) were tested with antisera to HFA. Her antibody was tested with RBCs lacking the HFA. Furthermore, it was tested with RBCs as untreated or treated with enzyme or AET. The genetic sequence was studied for mutation in CD44 gene that encodes INDIAN antigens. RESULTS: The patient was grouped A1B, RhD+, antibody screening test positive, direct antiglobulin test negative. A negative autocontrol test had suggested to the alloantibody being present. Antibody had agglutinated RBCs in LISS-tube at RT and by LISS-IAT at 37°C. The RBCs of the 11-cell panel, those lacking HFA and from 50 random donors, were agglutinated by her antibody indicating its specificity to the HFA, though the RBCs of Lu (a-b-)/In (Lu) type showed a weaker reaction. The patient's RBCs were agglutinated by antisera to a number of the enzyme-sensitive HFA, including those of INDIAN blood groups. The antibody showed reduced reactivity with the RBCs treated with papain, chymotrypsin, and AET but resistant to trypsin and dithiothreitol. The patient's genetic sequence revealed a novel homozygous mutation 449G>A in exon 5 of CD44. CONCLUSION: The antibody to enzyme sensitive HFA was tested for serological and molecular genetics studies and found to be directed to the novel HFA, named as INRA of the INDIAN blood group system and was assigned a numerical symbol IN: 005 by the International Society of Blood Transfusion (ISBT). PMID:28970678

Identification of a Kinase in Wheat Germ that Phosphorylates the Large Subunit of Initiation Factor 4F 1

PubMed Central

Humphreys, Jean; Browning, Karen S.; Ravel, Joanne M.

1988-01-01

A kinase has been isolated from wheat (Triticum aestivum) germ that phosphorylates the 220 kilodaltons (kD) subunit of wheat germ initiation factor (eIF) 4F, the 80 kD subunit of eIF-4B (an isozyme form of eIF-4F) and eIF-4G (the functional equivalent to mammalian eIF-4B). The kinase elutes from Sephacryl S-200 slightly in front of ovalbumin. The kinase phosphorylates casein and histone IIA to a small extent, but does not phosphorylate phosvitin. Of the wheat germ initiation factors, elongation factors, and small and large ribosomal subunits, only eIF-4F, eIF-4B, and eIF-4G are phosphorylated to a significant extent. The kinase phosphorylates eIF-4F to the extent of two phosphates per mole of the 220 kD subunit and phosphorylates eIF-4B to the extent of one phosphate per mole of the 80 kD subunit. The 26 kD subunit of eIF-4F and the 28 kD subunit of eIF-4B are not phosphorylated by the kinase. The kinase phosphorylates the 59 kD component of eIF-4G to the extent of 0.25 phosphate per mole of eIF-4G. Phosphorylation of eIF-4F and eIF-4B does not affect their ability to support the binding of mRNA to small ribosomal subunits in vitro. Images Fig. 2 Fig. 3 PMID:16666331

Multiple effects of S13 in modulating the strength of intersubunit interactions in the ribosome during translation.

PubMed

Cukras, Anthony R; Green, Rachel

2005-05-27

The ribosomal protein S13 is found in the head region of the small subunit, where it interacts with the central protuberance of the large ribosomal subunit and with the P site-bound tRNA through its extended C terminus. The bridging interactions between the large and small subunits are dynamic, and are thought to be critical in orchestrating the molecular motions of the translation cycle. S13 provides a direct link between the tRNA-binding site and the movements in the head of the small subunit seen during translocation, thereby providing a possible pathway of signal transduction. We have created and characterized an rpsM(S13)-deficient strain of Escherichia coli and have found significant defects in subunit association, initiation and translocation through in vitro assays of S13-deficient ribosomes. Targeted mutagenesis of specific bridge and tRNA contact elements in S13 provides evidence that these two interaction domains play critical roles in maintaining the fidelity of translation. This ribosomal protein thus appears to play a non-essential, yet important role by modulating subunit interactions in multiple steps of the translation cycle.

Functional Analysis of a Wheat AGPase Plastidial Small Subunit with a Truncated Transit Peptide.

PubMed

Yang, Yang; Gao, Tian; Xu, Mengjun; Dong, Jie; Li, Hanxiao; Wang, Pengfei; Li, Gezi; Guo, Tiancai; Kang, Guozhang; Wang, Yonghua

2017-03-01

ADP-glucose pyrophosphorylase (AGPase), the key enzyme in starch synthesis, consists of two small subunits and two large subunits with cytosolic and plastidial isoforms. In our previous study, a cDNA sequence encoding the plastidial small subunit (TaAGPS1b) of AGPase in grains of bread wheat ( Triticum aestivum L.) was isolated and the protein subunit encoded by this gene was characterized as a truncated transit peptide (about 50% shorter than those of other plant AGPS1bs). In the present study, TaAGPS1b was fused with green fluorescent protein (GFP) in rice protoplast cells, and confocal fluorescence microscopy observations revealed that like other AGPS1b containing the normal transit peptide, TaAGPS1b-GFP was localized in chloroplasts. TaAGPS1b was further overexpressed in a Chinese bread wheat cultivar, and the transgenic wheat lines exhibited a significant increase in endosperm AGPase activities, starch contents, and grain weights. These suggested that TaAGPS1b subunit was targeted into plastids by its truncated transit peptide and it could play an important role in starch synthesis in bread wheat grains.

Earthing (Grounding) the Human Body Reduces Blood Viscosity—a Major Factor in Cardiovascular Disease

PubMed Central

Chevalier, Gaétan; Sinatra, Stephen T.; Delany, Richard M.

2013-01-01

Abstract Objectives Emerging research is revealing that direct physical contact of the human body with the surface of the earth (grounding or earthing) has intriguing effects on human physiology and health, including beneficial effects on various cardiovascular risk factors. This study examined effects of 2 hours of grounding on the electrical charge (zeta potential) on red blood cells (RBCs) and the effects on the extent of RBC clumping. Design/interventions Subjects were grounded with conductive patches on the soles of their feet and palms of their hands. Wires connected the patches to a stainless-steel rod inserted in the earth outdoors. Small fingertip pinprick blood samples were placed on microscope slides and an electric field was applied to them. Electrophoretic mobility of the RBCs was determined by measuring terminal velocities of the cells in video recordings taken through a microscope. RBC aggregation was measured by counting the numbers of clustered cells in each sample. Settings/location Each subject sat in a comfortable reclining chair in a soundproof experiment room with the lights dimmed or off. Subjects Ten (10) healthy adult subjects were recruited by word-of-mouth. Results Earthing or grounding increased zeta potentials in all samples by an average of 2.70 and significantly reduced RBC aggregation. Conclusions Grounding increases the surface charge on RBCs and thereby reduces blood viscosity and clumping. Grounding appears to be one of the simplest and yet most profound interventions for helping reduce cardiovascular risk and cardiovascular events. PMID:22757749

Earthing (grounding) the human body reduces blood viscosity-a major factor in cardiovascular disease.

PubMed

Chevalier, Gaétan; Sinatra, Stephen T; Oschman, James L; Delany, Richard M

2013-02-01

Emerging research is revealing that direct physical contact of the human body with the surface of the earth (grounding or earthing) has intriguing effects on human physiology and health, including beneficial effects on various cardiovascular risk factors. This study examined effects of 2 hours of grounding on the electrical charge (zeta potential) on red blood cells (RBCs) and the effects on the extent of RBC clumping. SUBJECTS were grounded with conductive patches on the soles of their feet and palms of their hands. Wires connected the patches to a stainless-steel rod inserted in the earth outdoors. Small fingertip pinprick blood samples were placed on microscope slides and an electric field was applied to them. Electrophoretic mobility of the RBCs was determined by measuring terminal velocities of the cells in video recordings taken through a microscope. RBC aggregation was measured by counting the numbers of clustered cells in each sample. Each subject sat in a comfortable reclining chair in a soundproof experiment room with the lights dimmed or off. Ten (10) healthy adult subjects were recruited by word-of-mouth. Earthing or grounding increased zeta potentials in all samples by an average of 2.70 and significantly reduced RBC aggregation. Grounding increases the surface charge on RBCs and thereby reduces blood viscosity and clumping. Grounding appears to be one of the simplest and yet most profound interventions for helping reduce cardiovascular risk and cardiovascular events.

Exploring the Possibility of Cryopreservation of Feline and Canine Erythrocytes by Rapid Freezing with Penetrating and Non-Penetrating Cryoprotectants

PubMed Central

Pervushina, Olga; Hofmann, Nicola; Glasmacher, Birgit; Zhegunov, Gennadiy

2017-01-01

Efficient application of veterinary blood transfusion approaches for small companion animals requires readily available supply of the donor material. This can be achieved by developing of effective biobanking technologies allowing long-term storage of donor blood components via cryopreservation. Transfusion of an erythrocyte concentrate allows the successful correction of various hematological pathologies, severe bleeding, and etc. While in the past there were several approaches to cryopreserve red blood cells of dogs, to our knowledge there is virtually no data on cryopreservation of feline erythrocytes. In this paper, we performed a comprehensive parameter optimization for low temperature storage of RBCs of both species. Here, the efficiency of single-component and multicomponent cryoprotective media as well as necessary time of pre-incubation with penetrating and non-penetrating cryoprotectants prior to rapid freezing is analyzed. This study showed that glycerol was not sufficient for cryopreservation of red blood cells of the studied species under the investigated conditions. Application of 10% (v/v) ME2SO allowed for a significant reduction of canine and feline erythrocytes hemolysis after thawing. 17.5% hydroxyethyl starch demonstrated the highest cryoprotective activity for both species. It was found that dog RBCs should be incubated in cryoprotective media for 30 min at 22°C prior to freezing, while for cat RBCs 20 min is sufficient. Combination of CPAs was less effective. Presented data may be considered in further studies in veterinary transfusion and blood banking optimization. PMID:28072844

Exploring the Possibility of Cryopreservation of Feline and Canine Erythrocytes by Rapid Freezing with Penetrating and Non-Penetrating Cryoprotectants.

PubMed

Pogozhykh, Denys; Pakhomova, Yuliya; Pervushina, Olga; Hofmann, Nicola; Glasmacher, Birgit; Zhegunov, Gennadiy

2017-01-01

Efficient application of veterinary blood transfusion approaches for small companion animals requires readily available supply of the donor material. This can be achieved by developing of effective biobanking technologies allowing long-term storage of donor blood components via cryopreservation. Transfusion of an erythrocyte concentrate allows the successful correction of various hematological pathologies, severe bleeding, and etc. While in the past there were several approaches to cryopreserve red blood cells of dogs, to our knowledge there is virtually no data on cryopreservation of feline erythrocytes. In this paper, we performed a comprehensive parameter optimization for low temperature storage of RBCs of both species. Here, the efficiency of single-component and multicomponent cryoprotective media as well as necessary time of pre-incubation with penetrating and non-penetrating cryoprotectants prior to rapid freezing is analyzed. This study showed that glycerol was not sufficient for cryopreservation of red blood cells of the studied species under the investigated conditions. Application of 10% (v/v) ME2SO allowed for a significant reduction of canine and feline erythrocytes hemolysis after thawing. 17.5% hydroxyethyl starch demonstrated the highest cryoprotective activity for both species. It was found that dog RBCs should be incubated in cryoprotective media for 30 min at 22°C prior to freezing, while for cat RBCs 20 min is sufficient. Combination of CPAs was less effective. Presented data may be considered in further studies in veterinary transfusion and blood banking optimization.

Impact of Blood Mixing and ABO Compatibility on Platelet-Leukocyte Aggregations and Platelet P-Selectin Expression: An in Vitro Study.

PubMed

Huang, Go-Shine; Hu, Mei-Hua; Lin, Tso-Chou; Tsai, Yi-Ting; Lin, Chih-Yuan; Ke, Hung-Yen; Zheng, Xu-Zhi; Lin, Yi-Chang; Tsai, Chien-Sung

2018-05-01

Effects of blood transfusions on platelet- and leukocyte-related inflammation are unclear. We simulated transfusion using in vitro blood mixing to evaluate platelet-leukocyte aggregations (PLA) and platelet P-selectin expression, and the mechanism of PLA. Donor packed red blood cells (pRBCs) were obtained from a blood bank. Recipient whole blood samples were obtained from patients undergoing cardiac surgery. Blood sample mixtures were divided into four groups: group M, cross-matched blood type mixing; group O, donor type O with other blood type mixing (A, B, or AB); group S, ABO type-specific uncross-matched blood mixing; and group I, ABO incompatibility mixing. Donor pRBCs were added to recipient blood to reach 1%, 5%, and 10% (vol/vol) concentrations. Blood sample mixtures were analyzed to determine the PLA; P-selectin expression; and leukocyte CD11a, CD11b, and CD18 subunits of integrin expression. Analysis of variance tests were used to analyze differences. PLA significantly increased only in groups O and I (P = 0.003 and P < 0.001). Subpopulations of leukocytes significantly increased in all groups. There were no significant differences among the four groups (P = 0.578) in PLA increase. Although there was no significant effect on P-selectin expression (P = 1.000) and leukocyte CD11a and CD18 expression (P = 0.999, P = 0.422) within and between the groups, there was an increase in CD11b expression (P = 0.018). Blood mixing can increase PLA, especially in platelet-neutrophil and platelet-monocyte aggregations, possibly through nonhemolytic reactions. The CD11b integrin with CD18 may play a role in the formation of PLA.

ALTERATIONS OF PROPERTIES OF RED BLOOD CELLS MEMBRANES PROTEINS OF DIFFERENT AGE AND SEX VOLUNTEERS.

PubMed

Pruidze, N; Khetsuriani, R; Sujashvili, R; Ioramashvili, I; Arabuli, M; Sanikidze, T

2015-01-01

Considering the age and sex-dependent trend in the manifestation of various diseases, as well as an important pathogenic role of circulatory disorders, we decided to study the age-dependent changes in the physical properties of RBCs membrane proteins (their electric charge and molecular weight) in healthy people of different sex (males and females) and age. Blood of 56 healthy volunteers (Tbilisi, Georgia) of different sex and gender was studied (the patients were divided in 8 groups (7 patients in each groups): 1 - 18-25 years old male, 2 - 18-25 years old female, 3 - 25-44 years old male, 4 - 25-44 years old female, 5 - 44-60 years old male, 6 - 44-60 years old female; 7 - 60-80 years old male, 8 - 70-80 years old female). In groups 6 and 8 were women in menopause was determined according 12 months of amenorrhea. Individuals often consume alcohol addicts, pregnant women and patients with chronic diseases were excluded from the study. The study protocol was approved by Ethical Committee of the Tbilisi State Medical University. RBCs membrane proteins have been extracted from human heparinized blood and their mobility was studied by electrophoretic method. The electrophoretic mobility of RBCs membrane proteins decreases with age of healthy volunteers, that indicates decrease of total charge of proteins, depending on the electrically charged amino acids content. In female patients the electrophoretic mobility of the RBCs membrane proteins especially intensively decreases in period of menopause. Increase of molecular weight of proteins (100-200 kDa) from RBCs' membranes of alder age group was manifested. Intensively decrease electrophoretic mobility of erythrocytes membrane proteins from female patients in period of menopause indicates on estrogen related mechanism of the regulation of membrane protein conformation and composition in females. Increased content of high molecular weight proteins in the RBCs membranes from patients of older age groups may be caused to disorders of protein-protein interaction mechanisms, their ubiquitinylation or oligomerisation and formation of high molecular weight complexes of inactivated proteins in aged RBCs. These processes play important role in regulation of the RBCs shape and stability. Identified sex- and age-related alterations in RBCs membranes proteins affect the rheological properties of blood and can be considered as the etiologic and pathogenic markers of various diseases.

In vivo survival of apheresis RBCs, frozen with 40-percent (wt/vol) glycerol, deglycerolized in the ACP 215, and stored at 4 degrees C in AS-3 for up to 21 days.

PubMed

Valeri, C R; Ragno, G; Pivacek, L; O'Neill, E M

2001-07-01

The FDA has approved the storage of frozen RBCs at -80 degrees C for 10 years and the postwash storage at 4 degrees C for no more than 24 hours. The 4 degrees C postwash storage period is limited to 24 hours, because the current deglycerolization systems are functionally open systems. Two units of RBCs were collected from each of 13 healthy male volunteers. The RBCs were collected in CP2D by the FDA-approved protocol for an automated apheresis device (MCS, LN8150, Haemonetics) and were stored at 4 degrees C in AS-3 for 6 days. Using a single disposable glycerolization set in an automated, functionally closed system (ACP 215, Haemonetics) each unit was transferred to a 1000-mL PVC plastic bag and glycerolized to a concentration of 40-percent (wt/vol) glycerol and frozen at -80 degrees C. A single disposable deglycerolization set in the ACP 215 was used to deglycerolize the 2 units from the same donor. The deglycerolized RBCs were stored at 4 degrees C in AS-3 for as long as 21 days. The mean +/- SD freeze-thaw-wash recovery value was 89.4 +/- 3 percent. The residual hemolysis in the RBCs stored at 4 degrees C in AS-3 for 21 days after deglycerolization was 0.9 +/- 0.2 percent, and the units were negative for both aerobic and anaerobic bacteria. The mean Nageotte WBC count was 9 x 10(6) per unit. When the deglycerolized RBCs were given as an autologous transfusion after storage at 4 degrees C in AS-3 for the 7- to 18-day period, the mean +/- SD 24-hour posttransfusion survival was 77 +/- 7 percent, and the index of therapeutic effectiveness was 69 +/- 8 percent. Two units of human RBCs collected from a single donor by apheresis in the MCS using an LN8150 set can be glycerolized sequentially with a single disposable set and deglycerolized sequentially with another single disposable set in the ACP 215. The previously frozen RBCs stored in AS-3 for 7 to 18 days at 4 degrees C had acceptable hemolysis and an acceptable mean 24-hour posttransfusion survival value and index of therapeutic effectiveness.

AS-7 improved in vitro quality of red blood cells prepared from whole blood held overnight at room temperature.

PubMed

Veale, Margaret F; Healey, Geraldine; Sran, Amrita; Payne, Katherine A; Zia, Majid; Sparrow, Rosemary L

2015-01-01

Extended room temperature (RT) hold of whole blood (WB) may affect the quality of red blood cell (RBC) components produced from these donations. The availability of better RBC additive solutions (ASs) may help reduce the effects. A new AS, AS-7 (SOLX, Haemonetics Corporation), was investigated for improved in vitro quality of RBCs prepared from WB held overnight at RT. Sixteen WB units were held for 21.4 hours ± 40 minutes at 22°C on cooling plates before processing. Each pair of ABO-matched WB units were pooled, divided into a WB filter pack containing saline-adenine-glucose-mannitol (control) and a LEUKOSEP WB-filter pack containing SOLX, and processed according to manufacturer's instructions. RBCs were stored at 2 to 6°C and sampled weekly until expiry. Glycophorin A (GPA+) and annexin V-binding microparticles (MPs) were quantitated using flow cytometry. Osmotic fragility, intracellular pH (pHi), adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG), and routine quality variables were measured. Adhesion of RBCs to human endothelial cells (ECs) was evaluated by flow perfusion under low shear stress (0.5 dyne/cm(2) ), similar to low blood flow in microvessels. ATP and 2,3-DPG levels were improved for SOLX-RBCs. SOLX-RBCs maintained higher pHi, increased resistance to hypotonic stress, and reduced numbers of GPA+ MPs. No significant difference was observed between annexin V binding to MPs or adhesion of RBCs to ECs under shear stress. SOLX-stored RBCs showed increased osmotic resistance, pHi, and reduced GPA+ MPs and together with higher ATP and 2,3-DPG levels demonstrated improved in vitro RBC quality measures during 42 days of storage. © 2014 AABB.

Design of microfluidic channels for magnetic separation of malaria-infected red blood cells

PubMed Central

Wu, Wei-Tao; Martin, Andrea Blue; Gandini, Alberto; Aubry, Nadine; Massoudi, Mehrdad; Antaki, James F.

2016-01-01

This study is motivated by the development of a blood cell filtration device for removal of malaria-infected, parasitized red blood cells (pRBCs). The blood was modeled as a multi-component fluid using the computational fluid dynamics discrete element method (CFD-DEM), wherein plasma was treated as a Newtonian fluid and the red blood cells (RBCs) were modeled as soft-sphere solid particles which move under the influence of drag, collisions with other RBCs, and a magnetic force. The CFD-DEM model was first validated by a comparison with experimental data from Han et al. 2006 (Han and Frazier 2006) involving a microfluidic magnetophoretic separator for paramagnetic deoxygenated blood cells. The computational model was then applied to a parametric study of a parallel-plate separator having hematocrit of 40% with a 10% of the RBCs as pRBCs. Specifically, we investigated the hypothesis of introducing an upstream constriction to the channel to divert the magnetic cells within the near-wall layer where the magnetic force is greatest. Simulations compared the efficacy of various geometries upon the stratification efficiency of the pRBCs. For a channel with nominal height of 100 µm, the addition of an upstream constriction of 80% improved the proportion of pRBCs retained adjacent to the magnetic wall (separation efficiency) by almost 2 fold, from 26% to 49%. Further addition of a downstream diffuser reduced remixing, hence improved separation efficiency to 72%. The constriction introduced a greater pressure drop (from 17 to 495 Pa), which should be considered when scaling-up this design for a clinical-sized system. Overall, the advantages of this design include its ability to accommodate physiological hematocrit and high throughput – which is critical for clinical implementation as a blood-filtration system. PMID:27761107

A theoretical model for optical oximetry at the capillary-level by optical coherence tomography (Conference Presentation)

NASA Astrophysics Data System (ADS)

Liu, Rongrong; Spicer, Graham; Chen, Siyu; Zhang, Hao F.; Yi, Ji; Backman, Vadim

2017-02-01

Oxygen saturation (sO2) of RBCs in capillaries can indirectly assess local tissue oxygenation and metabolic function. For example, the altered retinal oxygenation in diabetic retinopathy and local hypoxia during tumor development in cancer are reflected by abnormal sO2 of local capillary networks. However, it is far from clear whether accurate label-free optical oximetry (i.e. measuring hemoglobin sO2) is feasible from dispersed red blood cells (RBCs) at the single-capillary level. The sO2-dependent hemoglobin absorption contrast present in optical scattering signal is complicated by geometry-dependent scattering from RBCs. Here we provide a theoretical model to calculate the backscattering spectra of single RBCs based on the first-order Born approximation, considering the orientation, size variation, and deformation of RBCs. We show that the oscillatory spectral behavior of RBC geometries is smoothed by variations in cell size and orientation, resulting in clear sO2-dependent spectral contrast. In addition, this spectral contrast persists with different deformations of RBCs, allowing the sO2 of individual RBCs in capillaries to be characterized. The theoretical model is verified by Mie theory and experiments using visible light optical coherence tomography (vis-OCT). Thus, this study shows for the first time the feasibility of, and provides a theoretical model for, label-free optical oximetry at the single-capillary level by backscattering-based imaging modalities, challenging the popular view that such measurements are impossible at the single-capillary level. This is promising for in vivo backscattering-based optical oximetry at the single-capillary level, to measure local capillary sO2 for early diagnosis, progression monitoring, and treatment evaluation of diabetic retinopathy and cancer.

Pathogen inactivation by riboflavin and ultraviolet light illumination accelerates the red blood cell storage lesion and promotes eryptosis.

PubMed

Qadri, Syed M; Chen, Deborah; Schubert, Peter; Perruzza, Darian L; Bhakta, Varsha; Devine, Dana V; Sheffield, William P

2017-03-01

Pathogen reduction treatment using riboflavin and ultraviolet light illumination (Mirasol) effectively reduces the risk of transfusion-transmitted infections. This treatment is currently licensed for only platelets and plasma products, while its application to whole blood (WB) to generate pathogen-inactivated red blood cells (RBCs) is under development. RBC storage lesion, constituting numerous morphologic and biochemical changes, influences RBC quality and limits shelf life. Stored RBCs further show enhanced susceptibility to RBC programmed cell death (eryptosis) characterized by increased cytosolic Ca 2+ -provoked membrane phosphatidylserine (PS) externalization. Using a "pool-and-split" approach, we examined multiple variables of RBC storage lesion and eryptosis in RBC units, derived from Mirasol-treated or untreated WB, after 4 to 42 days of storage, under blood bank conditions. In comparison to untreated RBC units, Mirasol treatment significantly altered membrane microvesiculation, supernatant hemoglobin, osmotic fragility, and intracellular adenosine triphosphate levels but did not influence membrane CD47 expression and 2,3-diphosphoglycerate levels. Mirasol-treated RBCs showed significantly higher PS exposure after 42, but not after not more than 21, days of storage, which was accompanied by enhanced cytosolic Ca 2+ activity, ceramide abundance, and oxidative stress, but not p38 kinase activation. Mirasol treatment significantly augmented PS exposure, Ca 2+ entry, and protein kinase C activation after energy depletion, a pathophysiologic cell stressor. Mirasol-treated RBCs were, however, more resistant to cell shrinkage. Prolonged storage of Mirasol-treated RBCs significantly increases the proportion of eryptotic RBCs, while even short-term storage enhances the susceptibility of RBCs to stress-induced eryptosis, which could reduce posttransfusion RBC recovery in patients. © 2016 AABB.

Macrophages clear refrigerator storage-damaged red blood cells and subsequently secrete cytokines in vivo, but not in vitro, in a murine model.

PubMed

Wojczyk, Boguslaw S; Kim, Nina; Bandyopadhyay, Sheila; Francis, Richard O; Zimring, James C; Hod, Eldad A; Spitalnik, Steven L

2014-12-01

In mice, refrigerator-stored red blood cells (RBCs) are cleared by extravascular hemolysis and induce cytokine production. To enhance understanding of this phenomenon, we sought to model it in vitro. Ingestion of refrigerator-stored murine RBCs and subsequent cytokine production were studied using J774A.1 mouse macrophage cells and primary murine splenic macrophages. Wild-type and Ccl2-GFP reporter mice were used for RBC clearance in vivo. Although J774A.1 cells and primary macrophages preferentially ingested refrigerator-stored RBCs in vitro, compared to freshly isolated RBCs, neither produced increased cytokines after erythrophagocytosis. In contrast, phagocytosis of refrigerator-stored RBCs in vivo induced increases in circulating monocyte chemoattractant protein-1 (MCP-1) and keratinocyte chemoattractant (KC) and correspondingly increased mRNA levels in mouse spleen and liver. In the spleen, these were predominantly expressed by CD11b+ cells. Using Ccl2-GFP reporter mice, the predominant splenic population responsible for MCP-1 mRNA production was tissue-resident macrophages (i.e., CD45+, CD11b+, F4/80+, Ly6c+, and CD11c(low) cells). J774A.1 cells and primary macrophages selectively ingested refrigerator-stored RBCs by phagocytosis. Although cytokine expression was not enhanced, this approach could be used to identify the relevant receptor-ligand combination(s). In contrast, cytokine levels increased after phagocytosis of refrigerator-stored RBCs in vivo. These were primarily cleared in the liver and spleen, which demonstrated increased MCP-1 and KC mRNA expression. Finally, in mouse spleen, tissue-resident macrophages were predominantly involved in MCP-1 mRNA production. The differences between cytokine production in vitro and in vivo are not yet well understood. © 2014 AABB.

Identification of lipids that accumulate during the routine storage of prestorage leukoreduced red blood cells and cause acute lung injury

PubMed Central

Silliman, Christopher C.; Moore, Ernest E.; Kelher, Marguerite R.; Khan, Samina Y.; Gellar, Lauren; Elzi, David J.

2011-01-01

BACKGROUND Lipids accumulate during the storage of red blood cells (RBCs), prime neutrophils (PMNs), and have been implicated in transfusion-related acute lung injury (TRALI). These lipids are composed of two classes: nonpolar lipids and lysophosphatidylcholines based on their retention time on separation by high-pressure liquid chromatography. Prestorage leukoreduction significantly decreases white blood cell and platelet contamination of RBCs; therefore, it is hypothesized that prestorage leukoreduction changes the classes of lipids that accumulate during storage, and these lipids prime PMNs and induce acute lung injury (ALI) as the second event in a two-event in vivo model. STUDY DESIGN AND METHODS RBC units were divided: 50% was leukoreduced (LR-RBCs), stored, and sampled on Day 1 and at the end of storage, Day 42. Priming activity was evaluated on isolated PMNs, and the purified lipids from Day 1 or Day 42 were used as the second event in the in vivo model. RESULTS The plasma and lipids from RBCs and LR-RBCs primed PMNs, and the LR-RBC activity decreased with longer storage. Unlike RBCs, nonpolar lipids comprised the PMN-priming activity from stored LR-RBCs. Mass spectroscopy identified these lipids as arachidonic acid and 5-, 12-, and 15-hydroxyeicsotetranoic acid. At concentrations from Day 42, but not Day 1, three of four of these lipids individually, and the mixture, primed PMNs. The mixture also caused ALI as the second event in a two-event model of TRALI. CONCLUSION We conclude that the nonpolar lipids that accumulate during LR-RBC storage may represent the agents responsible for antibody-negative TRALI. PMID:21615744

Binary release of ascorbic acid and lecithin from core-shell nanofibers on blood-contacting surface for reducing long-term hemolysis of erythrocyte.

PubMed

Shi, Qiang; Fan, Qunfu; Ye, Wei; Hou, Jianwen; Wong, Shing-Chung; Xu, Xiaodong; Yin, Jinghua

2015-01-01

There is an urgent need to develop blood-contacting biomaterials with long-term anti-hemolytic capability. To obtain such biomaterials, we coaxially electrospin [ascorbic acid (AA) and lecithin]/poly (ethylene oxide) (PEO) core-shell nanofibers onto the surface of styrene-b-(ethylene-co-butylene)-b-styrene elastomer (SEBS) that has been grafted with poly (ethylene glycol) (PEG) chains. Our strategy is based on that the grafted layers of PEG render the surface hydrophilic to reduce the mechanical injure to red blood cells (RBCs) while the AA and lecithin released from nanofibers on blood-contacting surface can actively interact with RBCs to decrease the oxidative damage to RBCs. We demonstrate that (AA and lecithin)/PEO core-shell structured nanofibers have been fabricated on the PEG grafted surface. The binary release of AA and lecithin in the distilled water is in a controlled manner and lasts for almost 5 days; during RBCs preservation, AA acts as an antioxidant and lecithin as a lipid supplier to the membrane of erythrocytes, resulting in low mechanical fragility and hemolysis of RBCs, as well as high deformability of stored RBCs. Our work thus makes a new approach to fabricate blood-contacting biomaterials with the capability of long-term anti-hemolysis. Copyright © 2014 Elsevier B.V. All rights reserved.

The G6PD flow-cytometric assay is a reliable tool for diagnosis of G6PD deficiency in women and anaemic subjects.

PubMed

Bancone, Germana; Kalnoky, Michael; Chu, Cindy S; Chowwiwat, Nongnud; Kahn, Maria; Malleret, Benoit; Wilaisrisak, Pornpimon; Rénia, Laurent; Domingo, Gonzalo J; Nosten, Francois

2017-08-29

Glucose-6-phosphate dehydrogenase (G6PD) activity is essential for redox equilibrium of red blood cells (RBCs) and, when compromised, the RBCs are more susceptible to haemolysis. 8-aminoquinolines (primaquine and tafenoquine) are used for the radical curative treatment of Plasmodium vivax malaria and can cause haemolysis in G6PD deficient subjects. Haemolytic risk is dependent on treatment dose and patient G6PD status but ultimately it correlates with the number of G6PD deficient RBCs. The G6PD spectrophotometric assay reliably identifies deficient subjects but is less reliable in heterozygous females, especially when other blood conditions are present. In this work we analysed samples with a range of G6PD phenotypes and haematologic conditions from 243 healthy volunteers of Asian or African-American heritage using both the spectrophotomeric assay and the G6PD flow-cytometric assay. Overall 18.5% of subjects (29.3% of Asian females) presented with anaemia, associated with decreased RBCs volume (MCV) and reticulocytosis; the flow-cytometric assay showed good correlation with the spectrophotometric assay (Pearson's r 0.918-0.957) and was less influenced by haemoglobin concentration, number of RBCs and number of reticulocytes. This resulted in more precise quantification of the number of G6PD deficient RBCs and presumably higher predictive power of drug induced haemolytic risk.

A mathematical model for lactate transport to red blood cells.

PubMed

Wahl, Patrick; Yue, Zengyuan; Zinner, Christoph; Bloch, Wilhelm; Mester, Joachim

2011-03-01

A simple mathematical model for the transport of lactate from plasma to red blood cells (RBCs) during and after exercise is proposed based on our experimental studies for the lactate concentrations in RBCs and in plasma. In addition to the influx associated with the plasma-to-RBC lactate concentration gradient, it is argued that an efflux must exist. The efflux rate is assumed to be proportional to the lactate concentration in RBCs. This simple model is justified by the comparison between the model-predicted results and observations: For all 33 cases (11 subjects and 3 different warm-up conditions), the model-predicted time courses of lactate concentrations in RBC are generally in good agreement with observations, and the model-predicted ratios between lactate concentrations in RBCs and in plasma at the peak of lactate concentration in RBCs are very close to the observed values. Two constants, the influx rate coefficient C (1) and the efflux rate coefficient C (2), are involved in the present model. They are determined by the best fit to observations. Although the exact electro-chemical mechanism for the efflux remains to be figured out in the future research, the good agreement of the present model with observations suggests that the efflux must get stronger as the lactate concentration in RBCs increases. The physiological meanings of C (1) and C (2) as well as their potential applications are discussed.

SOD2 deficiency in hematopoietic cells in mice results in reduced red blood cell deformability and increased heme degradation

PubMed Central

Mohanty, Joy G.; Nagababu, Enika; Friedman, Jeffrey S.; Rifkind, Joseph M.

2013-01-01

Among the three types of super oxide dismutases (SODs) known, SOD2 deficiency is lethal in neonatal mice owing to cardiomyopathy caused by severe oxidative damage. SOD2 is found in red blood cell (RBC) precursors, but not in mature RBCs. To investigate the potential damage to mature RBCs resulting from SOD2 deficiency in precursor cells, we studied RBCs from mice in which fetal liver stem cells deficient in SOD2 were capable of efficiently rescuing lethally irradiated host animals. These transplanted animals lack SOD2 only in hematopoietically generated cells and live longer than SOD2 knockouts. In these mice, approximately 2.8% of their total RBCs in circulation are iron-laden reticulocytes, with numerous siderocytic granules and increased protein oxidation similar to that seen in sideroblastic anemia. We have studied the RBC deformability and oxidative stress in these animals and the control group by measuring them with a microfluidic ektacytometer and assaying fluorescent heme degradation products with a fluorimeter, respectively. In addition, the rate of hemoglobin oxidation in RBCs from these mice and the control group were measured spectrophotometrically. The results show that RBCs from these SOD2-deficient mice have reduced deformability, increased heme degradation products, and an increased rate of hemoglobin oxidation compared with control animals, indicative of increased RBC oxidative stress. PMID:23142655

Extracellular glucose can fuel metabolism in red blood cells from high glycemic Atlantic cod (Gadus morhua) but not low glycemic short-horned sculpin (Myoxocephalus scorpius).

PubMed

Driedzic, William R; Clow, Kathy A; Short, Connie E

2014-11-01

Energy metabolism was assessed in red blood cells (RBCs) from Atlantic cod and short-horned sculpin, two species that have markedly different levels of blood glucose. The objective was to determine whether the level of extracellular glucose has an impact on rates of glucose metabolism. The blood glucose level was 2.5 mmol l(-1) in Atlantic cod and 0.2 mmol l(-1) in short-horned sculpin, respectively. Oxygen consumption, lactate production and glucose utilization were measured in whole blood and related to grams of RBCs. Glucose utilization was assessed by measuring both glucose disappearance and the production of (3)H2O from [2-(3)H]-glucose. RBCs from both species have an aerobic-based metabolism. In Atlantic cod, extracellular glucose is sufficient to provide the sum of glucosyl equivalents to support both oxidative metabolism and lactate production. In contrast, extracellular glucose can account for only 10% of the metabolic rate in short-horned sculpin RBCs. In both species, about 70% of glucose enters the RBCs via facilitated transport. The difference in rates of extracellular glucose utilization is related to the extremely low levels of blood glucose in short-horned sculpin. In this species energy metabolism by RBCs must be supported by alternative fuels. © 2014. Published by The Company of Biologists Ltd.

A microfabricated bio-sensor for erythrocytes deformability and volume distributions analysis

NASA Astrophysics Data System (ADS)

Bransky, Avishay; Korin, Natanel; Nemirovski, Yael; Dinnar, Uri

2007-12-01

The deformability of erythrocytes is of great importance for oxygen delivery in the microcirculation. Reduced RBC deformability is associated with several types of hemolytic anaemias, malaria, sepsis and diabetes. Aging of erythrocytes is also associated with loss of deformability as well as reduction in cell volume. An automated rheoscope has been developed, utilizing a microfabricated glass flow cell, high speed camera and advanced image-processing software. RBCs suspended in a high viscosity medium were filmed flowing through a microchannel. The system produces valuable data such as velocity profiles of RBCs, spatial distribution within the microchannel, cell volume and deformation index (DI) curves. The variation of DI across the channel height, due to change in shear stress, was measured for the first time. Such DI curves were obtained for normal and Thalassemia RBCs and their diagnostic potential was demonstrated. The spatial distribution and velocity of RBCs and rigid microspheres were measured. Both RBC and rigid spheres showed enhanced inward lateral migration, however the RBCs form a depletion region at the center of flow. The volume and surface area of the flowing cells have been estimated based on a fluid mechanics model and experimental results and fell within the normal range. Hence, the system developed, provides means for examining the behavior of individual RBCs in microchannels, and may serve as a microfabricated diagnostic device for deformability and volume measurements.

Trains of Red Blood Cells in a bi-dimensional microflows

NASA Astrophysics Data System (ADS)

Viallat, Annie; Iss, Cecile; Held, Delphine; Badens, Catherine; Charrier, Anne; Helfer, Emmanuèle; CINaM Team; Dpt de Génétique Médicale Team

2017-11-01

In the vascular microcirculation RBC distribution is uneven in the direction normal to the blood flow, as first evidenced by the existence of a cell-free layer near the vessel wall. In addition, the most rigid cells such as white blood cells and platelets are known to segregate to the walls while flowing in wide channels. We use microfluidic bi-dimensional channels (60 µm wide, 8 µm high, 5 mm long) to explore the flow structure in RBC suspensions at several hematocrits, flow rates and RBC rigidities. We observe the dynamical formation of RBC clusters and their motion along the flow direction. We study healthy RBCs, RBCs stiffened with glutaraldehyde, mixture of healthy and stiffened RBCs and RBC from sickle cell patients. Initially dispersed healthy RBCs organize, while flowing along the channel, into series of parallel trains. The train length depends on RBC hematocrit and flow rate. Stiffened RBCs do not cluster and mainly display tumbling motion like rigid disks. They destabilize existing trains and are preferentially observed close to the walls. We compared our results to that observed in microcapillaries, where trains of RBCs entirely fill in width the microchannel. This work has been carried out thanks to the support of the A*MIDEX project (n° ANR-11-IDEX-0001-02) funding by the ''Investissements d'Avenir'' French Government program, ma,ged by ANR.

Morphology and Small-Subunit Ribosomal DNA Sequence of Henneguya Adiposa (Myxosporea) From Ictalurus punctatus (Siluriformes)

USDA-ARS?s Scientific Manuscript database

The original description of Henneguya adiposa, a myxozoan parasitizing channel catfish Ictalurus punctatus, is supplemented with new data on spore morphology, including photomicrographs and line drawings, as well as 18S small-subunit (SSU) ribosomal DNA (rDNA) sequence. Elongate, translucent, linear...

Red blood cells aggregability measurement of coagulating blood in extracorporeal circulation system with multiple-frequency electrical impedance spectroscopy.

PubMed

Li, Jianping; Sapkota, Achyut; Kikuchi, Daisuke; Sakota, Daisuke; Maruyama, Osamu; Takei, Masahiro

2018-07-30

Red blood cells (RBCs) aggregability A G of coagulating blood in extracorporeal circulation system has been investigated under the condition of pulsatile flow. Relaxation frequency f c from the multiple-frequency electrical impedance spectroscopy is utilized to obtain RBCs aggregability A G . Compared with other methods, the proposed multiple-frequency electrical impedance method is much easier to obtain non-invasive measurement with high speed and good penetrability performance in biology tissues. Experimental results show that, RBCs aggregability A G in coagulating blood falls down with the thrombus formation while that in non-coagulation blood almost keeps the same value, which has a great agreement with the activated clotting time (ACT) fibrinogen concertation (F bg ) tests. Modified Hanai formula is proposed to quantitatively analyze the influence of RBCs aggregation on multiple-frequency electrical impedance measurement. The reduction of RBCs aggregability A G is associated with blood coagulation reaction, which indicates the feasibility of the high speed, compact and cheap on-line thrombus measurement biosensors in extracorporeal circulation systems. Copyright © 2018 Elsevier B.V. All rights reserved.

Oxidation-induced calcium-dependent dehydration of normal human red blood cells.

PubMed

Shcherbachenko, Irina M; Lisovskaya, Irina L; Tikhonov, Vladimir P

2007-05-01

Phenazine-methosulphate (PMS) is a strong oxidant that induces reactive oxygen species (ROS) formation in cells. Though it has been shown that PMS increases the red blood cell (RBC) membrane permeability to K(+), the hypotheses on the mechanism of PMS-induced effects are contradictory and there are no data on volume changes induced by this oxidant. Therefore, the influence of the PMS + ascorbate oxidative system on the volume of normal human RBCs was studied. In a Ca(2 + )-containing medium, PMS + ascorbate caused dehydration (shrinking) of RBCs judged by: (1) changes in the density and osmotic resistance distributions of RBCs, and (2) a decrease in their low-angle scattering assessed by FACS analysis. The dehydration resulted from activation of the Gardos channels, was PMS and ascorbate concentration-dependent, was associated with broadening of the density and osmotic resistance distributions of the RBCs, and decreased in the presence of the taxifolin and rutin antioxidants. These findings contribute to a better understanding of the physiology and pathology of oxidatively-modified RBCs and may be of practical significance in estimating the antioxidant activity of various substances.

Humanized mouse model of glucose 6-phosphate dehydrogenase deficiency for in vivo assessment of hemolytic toxicity.

PubMed

Rochford, Rosemary; Ohrt, Colin; Baresel, Paul C; Campo, Brice; Sampath, Aruna; Magill, Alan J; Tekwani, Babu L; Walker, Larry A

2013-10-22

Individuals with glucose 6-phosphate dehydrogenase (G6PD) deficiency are at risk for the development of hemolytic anemia when given 8-aminoquinolines (8-AQs), an important class of antimalarial/antiinfective therapeutics. However, there is no suitable animal model that can predict the clinical hemolytic potential of drugs. We developed and validated a human (hu)RBC-SCID mouse model by giving nonobese diabetic/SCID mice daily transfusions of huRBCs from G6PD-deficient donors. Treatment of SCID mice engrafted with G6PD-deficient huRBCs with primaquine, an 8-AQ, resulted in a dose-dependent selective loss of huRBCs. To validate the specificity of this model, we tested known nonhemolytic antimalarial drugs: mefloquine, chloroquine, doxycycline, and pyrimethamine. No significant loss of G6PD-deficient huRBCs was observed. Treatment with drugs known to cause hemolytic toxicity (pamaquine, sitamaquine, tafenoquine, and dapsone) resulted in loss of G6PD-deficient huRBCs comparable to primaquine. This mouse model provides an important tool to test drugs for their potential to cause hemolytic toxicity in G6PD-deficient populations.

Unexplained agglutination of stored red blood cells in Alsever's solution caused by the gram-negative bacterium Serratia liquefaciens.

PubMed

Martincic, I; Mastronardi, C; Chung, A; Ramirez-Arcos, S

2008-01-01

Alsever's solution has been used for decades as a preservative solution for storage of RBCs. From October 2005 to January 2006, unexplained hemagglutination of approximately 10 to 20 percent of RBCs stored for several days in a modified version of Alsever's solution was noticed in quality control testing at the Canadian Blood Services Serology Laboratory. An investigation, including microbial testing, was initiated to determine the cause of the unexplained hemagglutination. The gram-negative bacterium Serratia liquefaciens was isolated from supernatant solutions of agglutinated RBCs. Further characterization of this strain revealed that it has the ability to form biofilms; presents high levels of resistance to chloramphenicol, neomycin, and gentamicin; and causes mannose-sensitive hemagglutination. The source of S. liquefaciens contamination in RBC supernatants was not found. However, this bacterium has not been isolated since January 2006 after enhanced cleaning practices were implemented in the serology laboratory where the RBCs are stored. This biofilm-forming, antibiotic-resistant S. liquefaciens strain could be directly linked to the unexplained hemagglutination observed in stored RBCs.

Structural Basis of PP2A Inhibition by Small t Antigen

PubMed Central

Cho, Uhn Soo; Morrone, Seamus; Sablina, Anna A; Arroyo, Jason D; Hahn, William C; Xu, Wenqing

2007-01-01

The SV40 small t antigen (ST) is a potent oncoprotein that perturbs the function of protein phosphatase 2A (PP2A). ST directly interacts with the PP2A scaffolding A subunit and alters PP2A activity by displacing regulatory B subunits from the A subunit. We have determined the crystal structure of full-length ST in complex with PP2A A subunit at 3.1 Å resolution. ST consists of an N-terminal J domain and a C-terminal unique domain that contains two zinc-binding motifs. Both the J domain and second zinc-binding motif interact with the intra-HEAT-repeat loops of HEAT repeats 3–7 of the A subunit, which overlaps with the binding site of the PP2A B56 subunit. Intriguingly, the first zinc-binding motif is in a position that may allow it to directly interact with and inhibit the phosphatase activity of the PP2A catalytic C subunit. These observations provide a structural basis for understanding the oncogenic functions of ST. PMID:17608567

MUC1 and MUC4: Switching the Emphasis from Large to Small

PubMed Central

Carraway, Kermit L.

2011-01-01

Summation The MUC1 and MUC4 membrane mucins are each composed of a large alpha (α) and a small beta (β) subunit. The α subunits are fully exposed at the cell surface and contain variable numbers of repeated amino acid sequences that are heavily glycosylated. In contrast, the β subunits are much smaller and are anchored within the cell membrane, with their amino-terminal portions exposed at the cell surface and their carboxy-terminal tails facing the cytosol. Studies over the last several years are challenging the long-held belief that α subunits play the predominant role in cancer by conferring cellular properties that allow tumor cells to evade immune recognition and destruction. Indeed, the β subunits of MUC1 and MUC4 have emerged as oncogenes, as they engage signaling pathways responsible for tumor initiation and progression. Thus, a switch in the emphasis from the large α to the small β subunits offers attractive possibilities for successful clinical application. Such a focus shift is further supported by the absence of allelic polymorphism and variable glycosylation in the β subunit as well as by the presence of the β subunit in most MUC1 and MUC4 isoforms expressed by tumors. MUC1α, also known as CA15.3, is a Food and Drug Administration-approved serum biomarker for breast cancer, but its use is no longer recommended by the American Society of Clinical Oncology. However, comparison of β subunit expression in normal and malignant breast tissues may offer a novel approach to the exploitation of membrane mucins as biomarkers, as MUC1β-induced gene signatures with prognostic and predictive values in breast cancer have been reported. Preclinical studies with peptides that interfere with MUC1β oncogenic functions also look promising. PMID:21728842

Capillary red blood cell velocimetry by phase-resolved optical coherence tomography.

PubMed

Tang, Jianbo; Erdener, Sefik Evren; Fu, Buyin; Boas, David A

2017-10-01

We present a phase-resolved optical coherence tomography (OCT) method to extend Doppler OCT for the accurate measurement of the red blood cell (RBC) velocity in cerebral capillaries. OCT data were acquired with an M-mode scanning strategy (repeated A-scans) to account for the single-file passage of RBCs in a capillary, which were then high-pass filtered to remove the stationary component of the signal to ensure an accurate measurement of phase shift of flowing RBCs. The angular frequency of the signal from flowing RBCs was then quantified from the dynamic component of the signal and used to calculate the axial speed of flowing RBCs in capillaries. We validated our measurement by RBC passage velocimetry using the signal magnitude of the same OCT time series data.

A1C Test

MedlinePlus

... Mutation Mycophenolic Acid Mycoplasma Myoglobin Nicotine and Cotinine Non-High Density Lipoprotein Cholesterol Opioid Testing Osmolality Ova ... as older RBCs die and younger RBCs (with non-glycated hemoglobin) take their place. This test may ...

Red blood cell oxidative stress impairs oxygen delivery and induces red blood cell aging.

PubMed

Mohanty, Joy G; Nagababu, Enika; Rifkind, Joseph M

2014-01-01

Red Blood Cells (RBCs) need to deform and squeeze through narrow capillaries. Decreased deformability of RBCs is, therefore, one of the factors that can contribute to the elimination of aged or damaged RBCs from the circulation. This process can also cause impaired oxygen delivery, which contributes to the pathology of a number of diseases. Studies from our laboratory have shown that oxidative stress plays a significant role in damaging the RBC membrane and impairing its deformability. RBCs are continuously exposed to both endogenous and exogenous sources of reactive oxygen species (ROS) like superoxide and hydrogen peroxide (H2O2). The bulk of the ROS are neutralized by the RBC antioxidant system consisting of both non-enzymatic and enzymatic antioxidants including catalase, glutathione peroxidase and peroxiredoxin-2. However, the autoxidation of hemoglobin (Hb) bound to the membrane is relatively inaccessible to the predominantly cytosolic RBC antioxidant system. This inaccessibility becomes more pronounced under hypoxic conditions when Hb is partially oxygenated, resulting in an increased rate of autoxidation and increased affinity for the RBC membrane. We have shown that a fraction of peroxyredoxin-2 present on the RBC membrane may play a major role in neutralizing these ROS. H2O2 that is not neutralized by the RBC antioxidant system can react with the heme producing fluorescent heme degradation products (HDPs). We have used the level of these HDP as a measure of RBC oxidative Stress. Increased levels of HDP are detected during cellular aging and various diseases. The negative correlation (p < 0.0001) between the level of HDP and RBC deformability establishes a contribution of RBC oxidative stress to impaired deformability and cellular stiffness. While decreased deformability contributes to the removal of RBCs from the circulation, oxidative stress also contributes to the uptake of RBCs by macrophages, which plays a major role in the removal of RBCs from circulation. The contribution of oxidative stress to the removal of RBCs by macrophages involves caspase-3 activation, which requires oxidative stress. RBC oxidative stress, therefore, plays a significant role in inducing RBC aging.

Small subunits of RNA polymerase: localization, levels and implications for core enzyme composition.

PubMed

Doherty, Geoff P; Fogg, Mark J; Wilkinson, Anthony J; Lewis, Peter J

2010-12-01

Bacterial RNA polymerases (RNAPs) contain several small auxiliary subunits known to co-purify with the core α, β and β' subunits. The ω subunit is conserved between Gram-positive and Gram-negative bacteria, while the δ subunit is conserved within, but restricted to, Gram-positive bacteria. Although various functions have been assigned to these subunits via in vitro assays, very little is known about their in vivo roles. In this work we constructed a pair of vectors to investigate the subcellular localization of the δ and ω subunits in Bacillus subtilis with respect to the core RNAP. We found these subunits to be closely associated with RNAP involved in transcribing both mRNA and rRNA operons. Quantification of these subunits revealed δ to be present at equimolar levels with RNAP and ω to be present at around half the level of core RNAP. For comparison, the localization and quantification of RNAP β' and ω subunits in Escherichia coli was also investigated. Similar to B. subtilis, β' and ω closely associated with the nucleoid and formed subnucleoid regions of high green fluorescent protein intensity, but, unlike ω in B. subtilis, ω levels in E. coli were close to parity with those of β'. These results indicate that δ is likely to be an integral RNAP subunit in Gram-positives, whereas ω levels differ substantially between Gram-positives and -negatives. The ω subunit may be required for RNAP assembly and subsequently be turned over at different rates or it may play roles in Gram-negative bacteria that are performed by other factors in Gram-positives.

High-resolution three-dimensional imaging of red blood cells parasitized by Plasmodium falciparum and in situ hemozoin crystals using optical diffraction tomography

NASA Astrophysics Data System (ADS)

Kim, Kyoohyun; Yoon, HyeOk; Diez-Silva, Monica; Dao, Ming; Dasari, Ramachandra R.; Park, YongKeun

2014-01-01

We present high-resolution optical tomographic images of human red blood cells (RBC) parasitized by malaria-inducing Plasmodium falciparum (Pf)-RBCs. Three-dimensional (3-D) refractive index (RI) tomograms are reconstructed by recourse to a diffraction algorithm from multiple two-dimensional holograms with various angles of illumination. These 3-D RI tomograms of Pf-RBCs show cellular and subcellular structures of host RBCs and invaded parasites in fine detail. Full asexual intraerythrocytic stages of parasite maturation (ring to trophozoite to schizont stages) are then systematically investigated using optical diffraction tomography algorithms. These analyses provide quantitative information on the structural and chemical characteristics of individual host Pf-RBCs, parasitophorous vacuole, and cytoplasm. The in situ structural evolution and chemical characteristics of subcellular hemozoin crystals are also elucidated.

Quantitative investigation of red blood cell three-dimensional geometric and chemical changes in the storage lesion using digital holographic microscopy.

PubMed

Jaferzadeh, Keyvan; Moon, Inkyu

2015-11-01

Quantitative phase information obtained by digital holographic microscopy (DHM) can provide new insight into the functions and morphology of single red blood cells (RBCs). Since the functionality of a RBC is related to its three-dimensional (3-D) shape, quantitative 3-D geometric changes induced by storage time can help hematologists realize its optimal functionality period. We quantitatively investigate RBC 3-D geometric changes in the storage lesion using DHM. Our experimental results show that the substantial geometric transformation of the biconcave-shaped RBCs to the spherocyte occurs due to RBC storage lesion. This transformation leads to progressive loss of cell surface area, surface-to-volume ratio, and functionality of RBCs. Furthermore, our quantitative analysis shows that there are significant correlations between chemical and morphological properties of RBCs.

High-resolution three-dimensional imaging of red blood cells parasitized by Plasmodium falciparum and in situ hemozoin crystals using optical diffraction tomography

PubMed Central

Kim, Kyoohyun; Yoon, HyeOk; Diez-Silva, Monica; Dao, Ming; Dasari, Ramachandra R.

2013-01-01

Abstract. We present high-resolution optical tomographic images of human red blood cells (RBC) parasitized by malaria-inducing Plasmodium falciparum (Pf)-RBCs. Three-dimensional (3-D) refractive index (RI) tomograms are reconstructed by recourse to a diffraction algorithm from multiple two-dimensional holograms with various angles of illumination. These 3-D RI tomograms of Pf-RBCs show cellular and subcellular structures of host RBCs and invaded parasites in fine detail. Full asexual intraerythrocytic stages of parasite maturation (ring to trophozoite to schizont stages) are then systematically investigated using optical diffraction tomography algorithms. These analyses provide quantitative information on the structural and chemical characteristics of individual host Pf-RBCs, parasitophorous vacuole, and cytoplasm. The in situ structural evolution and chemical characteristics of subcellular hemozoin crystals are also elucidated. PMID:23797986

Nitrite transport into pig erythrocytes and its potential biological role.

PubMed

Jensen, F B

2005-07-01

To study nitrite transport and its oxygenation dependency in pig erythrocytes, as this is fundamental to the possible participation of nitrite in blood flow regulation via its reduction to nitric oxide by deoxygenated haemoglobin (Hb). Pig red blood cells (RBCs) were tonometer-equilibrated to physiological pCO2 in oxygenated and deoxygenated states. Nitrite was added and the kinetics of NO2- influx and methaemoglobin (metHb) formation were assessed at variable temperature and haematocrit. Nitrite quickly permeated and equilibrated across the membrane, and then continued to enter RBCs as a consequence of its intracellular removal (via reactions with Hb to form nitrate and metHb in oxygenated cells, and NO and metHb in deoxygenated cells). The membrane permeation as such showed little oxygenation dependency, but as metHb formation was significantly higher in oxygenated than deoxygenated RBCs, nitrite transport tended to be largest into oxygenated RBCs. This contrasts with a preferential permeation of deoxygenated RBCs in some fish species. Nitrite transport showed low temperature sensitivity but was speeded up at low haematocrit via more rapid intracellular nitrite removal (metHb formation). Nitrite influx was not affected by inhibitors of facilitated diffusion (DIDS, phloretin and PCMB) and may occur via conductive transport. Extracellular pH was stable during nitrite transport. Nitrite extensively permeates both oxygenated and deoxygenated pig RBCs, which may enable a dual function of nitrite entry: viz. conversion to NO at low pO2 to promote blood flow and detoxification to non-toxic nitrate at inappropriate high nitrite levels.

Assessment of red blood cell deformability in type 2 diabetes mellitus and diabetic retinopathy by dual optical tweezers stretching technique.

PubMed

Agrawal, Rupesh; Smart, Thomas; Nobre-Cardoso, João; Richards, Christopher; Bhatnagar, Rhythm; Tufail, Adnan; Shima, David; Jones, Phil H; Pavesio, Carlos

2016-03-15

A pilot cross sectional study was conducted to investigate the role of red blood cells (RBC) deformability in type 2 diabetes mellitus (T2DM) without and with diabetic retinopathy (DR) using a dual optical tweezers stretching technique. A dual optical tweezers was made by splitting and recombining a single Nd:YAG laser beam. RBCs were trapped directly (i.e., without microbead handles) in the dual optical tweezers where they were observed to adopt a "side-on" orientation. RBC initial and final lengths after stretching were measured by digital video microscopy, and a Deformability index (DI) calculated. Blood from 8 healthy controls, 5 T2DM and 7 DR patients with respective mean age of 52.4 yrs, 51.6 yrs and 52 yrs was analysed. Initial average length of RBCs for control group was 8.45 ± 0.25 μm, 8.68 ± 0.49 μm for DM RBCs and 8.82 ± 0.32 μm for DR RBCs (p < 0.001). The DI for control group was 0.0698 ± 0.0224, and that for DM RBCs was 0.0645 ± 0.03 and 0.0635 ± 0.028 (p < 0.001) for DR group. DI was inversely related to basal length of RBCs (p =  .02). DI of RBC from DM and DR patients was significantly lower in comparison with normal healthy controls. A dual optical tweezers method can hence be reliably used to assess RBC deformability.

Effects of nitric oxide and its congeners on sickle red blood cell deformability.

PubMed

Belanger, Andrea M; Keggi, Christian; Kanias, Tamir; Gladwin, Mark T; Kim-Shapiro, Daniel B

2015-10-01

Sickle cell disease (SCD) is characterized by hemoglobin polymerization upon deoxygenation. Polymerization causes the sickle cells to become rigid and misshapen (sickling). Red blood cell (RBC) dehydration greatly increases polymerization. Cycles of sickling and unsickling cause an influx of calcium that leads to loss of potassium via the calcium-activated Gardos channel, which dehydrates the cells leading to increased polymerization. In this study the effects of nitric oxide (NO) and its congeners on RBC deformability were examined, focusing on sickle RBCs (sRBCs). RBCs from patients with SCD and from nonpatients were exposed to various compounds that release NO or its congeners. Intracellular calcium was increased using a calcium ionophore or cycling of oxygen tension for sRBCs. Deformability was measured by laser-assisted osmotic gradient ektacytometry. Consistent with a previous report, sodium nitroprusside (SNP) was found to protect against calcium-induced loss of deformability in normal RBCs, but (contrary to some previous reports) no effect of any NO donors was observed when calcium influx was not induced. Importantly, in studies of deoxygenation-induced dehydration of sRBCs, SNP resulted in substantial improvements in deformability (p = 0.036) and hydration (p = 0.024). Sodium nitrite showed similar trends. SNP was shown to have no effect on calcium influx, but reduced potassium efflux. These data suggest that SNP and perhaps certain nitrogen oxides (like nitrite) inhibit the Gardos channel and may be able to protect sickle cells from dehydration and thereby improve outcome in the disease. © 2015 AABB.

Temperature transition of human hemoglobin at body temperature: effects of calcium.

PubMed Central

Kelemen, C; Chien, S; Artmann, G M

2001-01-01

We studied the effects of calcium ion concentration on the temperature dependence of rheological behavior of human red blood cells (RBCs) and concentrated hemoglobin solutions. Our previous study (G. M. Artmann, C. Kelemen, D. Porst, G. Büldt, and S. Chien, 1998, Biophys. J., 75:3179-3183) showed a critical temperature (Tc) of 36.4 +/- 0.3 degrees C at which the RBCs underwent a transition from non-passage to passage through 1.3 microm micropipettes in response to an aspiration pressure of -2.3 kPa. An increase in intracellular Ca2+ concentration by using the ionophore A23187 reduced the passability of intact RBCs through small micropipettes above T(c); the micropipette diameter needed for >90% passage increased to 1.7 microm. Viscometry of concentrated hemoglobin solutions (45 and 50 g/dl) showed a sudden viscosity transition at 36 +/- 1 degrees C (Tc(eta)) at all calcium concentrations investigated. Below Tc(eta), the viscosity value of the concentrated hemoglobin solution at 1.8 mM Ca(2+) was higher than that at other concentrations (0.2 microM, 9 mM, and 18 mM). Above Tc(eta), the viscosity was almost Ca2+ independent. At 1.8 mM Ca2+ and 36 +/- 1 degrees C, the activation energy calculated from the viscometry data showed a strong dependence on the hemoglobin concentration. We propose that the transition of rheological behavior is attributable to a high-to-low viscosity transition mediated by a partial release of the hemoglobin-bound water. PMID:11371439

Detection and characterization of red blood cell (RBC) aggregation with photoacoustics

NASA Astrophysics Data System (ADS)

Hysi, Eno; Saha, Ratan K.; Rui, Min; Kolios, Michael C.

2012-02-01

Red blood cells (RBCs) aggregate in the presence of increased plasma fibrinogen and low shear forces during blood flow. RBC aggregation has been observed in deep vein thrombosis, sepsis and diabetes. We propose using photoacoustics (PA) as a non-invasive imaging modality to detect RBC aggregation. The theoretical and experimental feasibility of PA for detecting and characterizing aggregation was assessed. A simulation study was performed to generate PA signals from non-aggregated and aggregated RBCs using a frequency domain approach and to study the PA signals' dependence on hematocrit and aggregate size. The effect of the finite bandwidth nature of transducers on the PA power spectra was also investigated. Experimental confirmation of theoretical results was conducted using porcine RBC samples exposed to 1064 nm optical wavelength using the Imagio Small Animal PA imaging system (Seno Medical Instruments, Inc., San Antonio, TX). Aggregation was induced with Dextran-70 (Sigma-Aldrich, St. Louis, MO) and the effect of hematocrit and aggregation level was investigated. The theoretical and experimental PA signal amplitude increased linearly with increasing hematocrit. The theoretical dominant frequency content of PA signals shifted towards lower frequencies (<30 MHz) and 9 dB enhancements in spectral power were observed as the size of aggregates increased compared to non-aggregating RBCs. Calibration of the PA spectra with the transducer response obtained from a 200 nm gold film was performed to remove system dependencies. Analysis of the spectral parameters from the calibrated spectra suggested that PA can assess the degree of aggregation at multiple hematocrit and aggregation levels.

The Relation Between Capillary Transit Times and Hemoglobin Saturation Heterogeneity. Part 1: Theoretical Models

PubMed Central

Lücker, Adrien; Secomb, Timothy W.; Weber, Bruno; Jenny, Patrick

2018-01-01

Capillary dysfunction impairs oxygen supply to parenchymal cells and often occurs in Alzheimer's disease, diabetes and aging. Disturbed capillary flow patterns have been shown to limit the efficacy of oxygen extraction and can be quantified using capillary transit time heterogeneity (CTH). However, the transit time of red blood cells (RBCs) through the microvasculature is not a direct measure of their capacity for oxygen delivery. Here we examine the relation between CTH and capillary outflow saturation heterogeneity (COSH), which is the heterogeneity of blood oxygen content at the venous end of capillaries. Models for the evolution of hemoglobin saturation heterogeneity (HSH) in capillary networks were developed and validated using a computational model with moving RBCs. Two representative situations were selected: a Krogh cylinder geometry with heterogeneous hemoglobin saturation (HS) at the inflow, and a parallel array of four capillaries. The heterogeneity of HS after converging capillary bifurcations was found to exponentially decrease with a time scale of 0.15–0.21 s due to diffusive interaction between RBCs. Similarly, the HS difference between parallel capillaries also drops exponentially with a time scale of 0.12–0.19 s. These decay times are substantially smaller than measured RBC transit times and only weakly depend on the distance between microvessels. This work shows that diffusive interaction strongly reduces COSH on a small spatial scale. Therefore, we conclude that CTH influences COSH yet does not determine it. The second part of this study will focus on simulations in microvascular networks from the rodent cerebral cortex. Actual estimates of COSH and CTH will then be given. PMID:29755365

The Relation Between Capillary Transit Times and Hemoglobin Saturation Heterogeneity. Part 1: Theoretical Models.

PubMed

Lücker, Adrien; Secomb, Timothy W; Weber, Bruno; Jenny, Patrick

2018-01-01

Capillary dysfunction impairs oxygen supply to parenchymal cells and often occurs in Alzheimer's disease, diabetes and aging. Disturbed capillary flow patterns have been shown to limit the efficacy of oxygen extraction and can be quantified using capillary transit time heterogeneity (CTH). However, the transit time of red blood cells (RBCs) through the microvasculature is not a direct measure of their capacity for oxygen delivery. Here we examine the relation between CTH and capillary outflow saturation heterogeneity (COSH), which is the heterogeneity of blood oxygen content at the venous end of capillaries. Models for the evolution of hemoglobin saturation heterogeneity (HSH) in capillary networks were developed and validated using a computational model with moving RBCs. Two representative situations were selected: a Krogh cylinder geometry with heterogeneous hemoglobin saturation (HS) at the inflow, and a parallel array of four capillaries. The heterogeneity of HS after converging capillary bifurcations was found to exponentially decrease with a time scale of 0.15-0.21 s due to diffusive interaction between RBCs. Similarly, the HS difference between parallel capillaries also drops exponentially with a time scale of 0.12-0.19 s. These decay times are substantially smaller than measured RBC transit times and only weakly depend on the distance between microvessels. This work shows that diffusive interaction strongly reduces COSH on a small spatial scale. Therefore, we conclude that CTH influences COSH yet does not determine it. The second part of this study will focus on simulations in microvascular networks from the rodent cerebral cortex. Actual estimates of COSH and CTH will then be given.

Superradiant instabilities in the Kerr-mirror and Kerr-AdS black holes with Robin boundary conditions

NASA Astrophysics Data System (ADS)

Ferreira, Hugo R. C.; Herdeiro, Carlos A. R.

2018-04-01

It has been recently observed that a scalar field with Robin boundary conditions (RBCs) can trigger both a superradiant and a bulk instability for a Bañados-Teitelboim-Zanelli (BTZ) black hole (BH) [1]. To understand the generality and scrutinize the origin of this behavior, we consider here the superradiant instability of a Kerr BH confined either in a mirrorlike cavity or in anti-de Sitter (AdS) space, triggered also by a scalar field with RBCs. These boundary conditions are the most general ones that ensure the cavity/AdS space is an isolated system and include, as a particular case, the commonly considered Dirichlet boundary conditions (DBCs). Whereas the superradiant modes for some RBCs differ only mildly from the ones with DBCs, in both cases, we find that as we vary the RBCs the imaginary part of the frequency may attain arbitrarily large positive values. We interpret this growth as being sourced by a bulk instability of both confined geometries when certain RBCs are imposed to either the mirrorlike cavity or the AdS boundary, rather than by energy extraction from the BH, in analogy with the BTZ behavior.

Transport of diseased red blood cells in the spleen

NASA Astrophysics Data System (ADS)

Peng, Zhangli; Pivkin, Igor; Dao, Ming

2012-11-01

A major function of the spleen is to remove old and diseased red blood cells (RBCs) with abnormal mechanical properties. We investigated this mechanical filtering mechanism by combining experiments and computational modeling, especially for red blood cells in malaria and sickle cell disease (SCD). First, utilizing a transgenic line for 3D confocal live imaging, in vitro capillary assays and 3D finite element modeling, we extracted the mechanical properties of both the RBC membrane and malaria parasites for different asexual malaria stages. Secondly, using a non-invasive laser interferometric technique, we optically measured the dynamic membrane fluctuations of SCD RBCs. By simulating the membrane fluctuation experiment using the dissipative particle dynamics (DPD) model, we retrieved mechanical properties of SCD RBCs with different shapes. Finally, based on the mechanical properties obtained from these experiments, we simulated the full fluid-structure interaction problem of diseased RBCs passing through endothelial slits in the spleen under different fluid pressure gradients using the DPD model. The effects of the mechanical properties of the lipid bilayer, the cytoskeleton and the parasite on the critical pressure of splenic passage of RBCs were investigated separately. This work is supported by NIH and Singapore-MIT Alliance for Science and Technology (SMART).

Humanized mouse model of glucose 6-phosphate dehydrogenase deficiency for in vivo assessment of hemolytic toxicity

PubMed Central

Rochford, Rosemary; Ohrt, Colin; Baresel, Paul C.; Campo, Brice; Sampath, Aruna; Magill, Alan J.; Tekwani, Babu L.; Walker, Larry A.

2013-01-01

Individuals with glucose 6-phosphate dehydrogenase (G6PD) deficiency are at risk for the development of hemolytic anemia when given 8-aminoquinolines (8-AQs), an important class of antimalarial/antiinfective therapeutics. However, there is no suitable animal model that can predict the clinical hemolytic potential of drugs. We developed and validated a human (hu)RBC-SCID mouse model by giving nonobese diabetic/SCID mice daily transfusions of huRBCs from G6PD-deficient donors. Treatment of SCID mice engrafted with G6PD-deficient huRBCs with primaquine, an 8-AQ, resulted in a dose-dependent selective loss of huRBCs. To validate the specificity of this model, we tested known nonhemolytic antimalarial drugs: mefloquine, chloroquine, doxycycline, and pyrimethamine. No significant loss of G6PD-deficient huRBCs was observed. Treatment with drugs known to cause hemolytic toxicity (pamaquine, sitamaquine, tafenoquine, and dapsone) resulted in loss of G6PD-deficient huRBCs comparable to primaquine. This mouse model provides an important tool to test drugs for their potential to cause hemolytic toxicity in G6PD-deficient populations. PMID:24101478

Rheological alteration of erythrocytes exposed to carbon nanotubes.

PubMed

Heo, Yujin; Li, Cheng-Ai; Kim, Duckjong; Shin, Sehyun

2017-01-01

Single-walled carbon nanotubes (SWNTs) have been increasingly used in a variety of biomedical applications, such as in vivo delivery of drugs and tumor imaging. Potential exposure of SWNTs to human red blood cells (RBCs) may cause serious toxicity including alteration of mechanical properties of cells. The present study investigated the cellular response to exposure of SWNTs with measuring rheological characteristics of RBCs, including hemolysis, deformability, aggregation, and morphological changes. RBCs were exposed to two different dispersion-state samples (i.e. individual SWNTs and bundled SWNTs) in chitosan hydroxyphenyl acetamide (CHPA) solutions. The concentrations of SWNTs were carefully chosen to avoid any hemorheological alterations due to hemolysis. Rheological characteristics were measured using microfluidic-laser diffractometry and aggregometry. Our results show that the bundled SWNTs had higher hemolytic activity than did the individual SWNTs. RBC aggregation apparently decreased as the concentration of SWNTs or incubation time increased. Additionally, bundled SWNTs caused significant alterations in the shape and fusion of RBCs. In conclusion, bundled SWNTs were found to be more toxic than individual SWNTs. These results provide important insights into the interactions between RBCs and SWNTs and will facilitate assessment of the risk of nanomaterial toxicity of blood.

Blood-plasma separation in Y-shaped bifurcating microfluidic channels: A dissipative particle dynamics simulation study

PubMed Central

Li, Xuejin; Popel, Aleksander S.; Karniadakis, George Em

2012-01-01

The motion of a suspension of red blood cells (RBCs) flowing in a Y-shaped bifurcating microfluidic channel is investigated using a validated low-dimensional RBC (LD-RBC) model based on dissipative particle dynamics (DPD). Specifically, the RBC is represented as a closed torus-like ring of ten colloidal particles, which leads to efficient simulations of blood flow in microcirculation over a wide range of hematocrits. Adaptive no-slip wall boundary conditions were implemented to model hydrodynamic flow within a specific wall structure of diverging 3D microfluidic channels, paying attention to controlling density fluctuations. Plasma skimming and the all-or-nothing phenomenon of RBCs in a bifurcating microfluidic channel have been investigated in our simulations for healthy and diseased blood, including the size of cell-free layer on the daughter branches. The feed hematocrit level in the parent channel has considerable influence on blood-plasma separation. Compared to the blood-plasma separation efficiencies of healthy RBCs, malaria-infected stiff RBCs (iRBCs) have a tendency to travel into the low flowrate daughter branch because of their different initial distribution in the parent channel. Our simulation results are consistent with previously published experimental results and theoretical predictions. PMID:22476709

Photoassociation of cold (RbCs)2 tetramers in the ground electronic state

NASA Astrophysics Data System (ADS)

Gacesa, Marko; Côté, Robin

2017-04-01

We theoretically investigate prospects for photoassociative formation of cold (RbCs)2 tetramers from a pair of ultracold RbCs molecules. The long-range region of the potential energy surface (PES) of the lowest electronic state of (RbCs)2 can be affected by orienting both RbCs molecules by an external electric field. In fact, we find a long-range barrier that supports long-range shelf states for relative angles between the dimers' internuclear axes smaller than about 20°. We show that these shelf states can be populated by spontaneous decay from the first excited electronic state which can be efficiently populated by photoassociation from the scattering continuum at ultracold temperatures. The vibrationally excited ground-state tetramer molecules formed this way have sufficiently long lifetimes to allow experimental detection. Moreover, for the relative angles between the dimers close to 20°, the proposed approach may result in production of deeply bound tetramers. Partially supported by the NASA Postdoctoral Program at the NASA Ames Research Center, administered by USRA and the MURI US Army Research Office Grant No. W911NF-14-1-0378 (MG), and by the PIF program of the National Science Foundation Grant No. PHY-141556.

Structural Variation of Type I-F CRISPR RNA Guided DNA Surveillance.

PubMed

Pausch, Patrick; Müller-Esparza, Hanna; Gleditzsch, Daniel; Altegoer, Florian; Randau, Lennart; Bange, Gert

2017-08-17

CRISPR-Cas systems are prokaryotic immune systems against invading nucleic acids. Type I CRISPR-Cas systems employ highly diverse, multi-subunit surveillance Cascade complexes that facilitate duplex formation between crRNA and complementary target DNA for R-loop formation, retention, and DNA degradation by the subsequently recruited nuclease Cas3. Typically, the large subunit recognizes bona fide targets through the PAM (protospacer adjacent motif), and the small subunit guides the non-target DNA strand. Here, we present the Apo- and target-DNA-bound structures of the I-Fv (type I-F variant) Cascade lacking the small and large subunits. Large and small subunits are functionally replaced by the 5' terminal crRNA cap Cas5fv and the backbone protein Cas7fv, respectively. Cas5fv facilitates PAM recognition from the DNA major groove site, in contrast to all other described type I systems. Comparison of the type I-Fv Cascade with an anti-CRISPR protein-bound I-F Cascade reveals that the type I-Fv structure differs substantially at known anti-CRISPR protein target sites and might therefore be resistant to viral Cascade interception. Copyright © 2017 Elsevier Inc. All rights reserved.

Crystallization and preliminary X-ray crystallographic analysis of the small subunit of the heterodimeric laccase POXA3b from Pleurotus ostreatus

PubMed Central

Ferraroni, Marta; Scozzafava, Andrea; Ullah, Sana; Tron, Thierry; Piscitelli, Alessandra; Sannia, Giovanni

2014-01-01

Laccases are multicopper oxidases of great biotechnological potential. While laccases are generally monomeric glycoproteins, the white-rot fungus Pleurotus ostreatus produces two closely related heterodimeric isoenzymes composed of a large subunit, homologous to the other fungal laccases, and a small subunit. The sequence of the small subunit does not show significant homology to any other protein or domain of known function and consequently its function is unknown. The highest similarity to proteins of known structure is to a putative enoyl-CoA hydratase/isomerase from Acinetobacter baumannii, which shows an identity of 27.8%. Diffraction-quality crystals of the small subunit of the heterodimeric laccase POXA3b (sPOXA3b) from P. ostreatus were obtained using the sitting-drop vapour-diffusion method at 294 K from a solution consisting of 1.8 M sodium formate, 0.1 M Tris–HCl pH 8.5. The crystals belonged to the tetragonal space group P41212 or P43212, with unit-cell parameters a = 126.6, c = 53.9 Å. The asymmetric unit contains two molecules related by a noncrystallographic twofold axis. A complete data set extending to a maximum resolution of 2.5 Å was collected at 100 K using a wavelength of 1.140 Å. PMID:24419623

Cop9/signalosome subunits and Pcu4 regulate ribonucleotide reductase by both checkpoint-dependent and -independent mechanisms

PubMed Central

Liu, Cong; Powell, Kelly A.; Mundt, Kirsten; Wu, LeJung; Carr, Antony M.; Caspari, Thomas

2003-01-01

The signalosome is implicated in regulating cullin-dependent ubiquitin ligases. We find that two signalosome subunits, Csn1 and Csn2, are required to regulate ribonucleotide reductase (RNR) through the degradation of a small protein, Spd1, that acts to anchor the small RNR subunit in the nucleus. Spd1 destruction correlates with the nuclear export of the small RNR subunit, which, in turn, correlates with a requirement for RNR in replication and repair. Spd1 degradation is promoted by two separate CSN-dependent mechanisms. During unperturbed S phase, Spd1 degradation is independent of checkpoint proteins. In irradiated G2 cells, Spd1 degradation requires the DNA damage checkpoint. The signalosome copurifies with Pcu4 (cullin 4). Pcu4, Csn1, and Csn2 promote the degradation of Spd1, identifying a new function for the signalosome as a regulator of Pcu4-containing E3 ubiquitin ligase. PMID:12695334

B-type natriuretic peptide and plasma hemoglobin levels following transfusion of shorter-storage versus longer-storage Red Blood Cells: results from the TOTAL randomized trial

PubMed Central

Dhabangi, Aggrey; Ainomugisha, Brenda; Cserti-Gazdewich, Christine; Ddungu, Henry; Kyeyune, Dorothy; Musisi, Ezra; Opoka, Robert; Stowell, Christopher P.; Dzik, Walter H

2016-01-01

Background Prior studies have suggested that transfusion of stored RBCs with increased levels of cell free hemoglobin might reduce the bioavailability of recipient nitric oxide (NO) and cause myocardial strain. Methods Ugandan children (ages 6 to 60 months) with severe anemia and lactic acidosis were randomly assigned to receive RBCs stored 1-10 days versus 25-35 days. B-type natriuretic peptide (BNP), vital signs, renal function tests, and plasma hemoglobin were measured. Most children had either malaria or sickle cell disease and were thus at risk for reduced NO bioavailability. Results 70 patients received RBCs stored 1-10 days and 77 received RBCs stored 25-35 days. The median (IQR) cell free hemoglobin was nearly three times higher in longer-storage RBCs (26.4 [15.5-43.4] μmol/L) than in shorter-storage RBCs (10.8 [7.8-18.6] μmol/L), p<0.0001. Median (IQR) BNP 2 hours post-transfusion was 156 (59-650) pg/mL (shorter-storage) versus 158 (59-425) pg/mL (longer-storage), p=0.76. BNP values 22 hours post-transfusion were 110 (46-337) pg/mL (shorter-storage) versus 96 (49-310) pg/mL (longer-storage), p=0.76. Changes in BNP within individuals from pre-transfusion to 2-hour (or 22-hour) post-transfusion were not significantly different between the study groups. BNP change following transfusion did not correlate with the concentration of cell free hemoglobin in the RBC supernatant. Blood pressure, BUN, creatinine, and change in plasma hemoglobin were not significantly different in the two groups. Conclusion In a randomized trial among children at risk for reduced NO bioavailability, we found that BNP, blood pressure, creatinine, and plasma hemoglobin were not higher in patients receiving RBCs stored for 25-35 days versus 1-10 days. PMID:27302626

Intake of Erythrocytes Required for Reproductive Development of Female Schistosoma japonicum.

PubMed

Wang, Jipeng; Wang, Shuqi; Liu, Xiufeng; Xu, Bin; Chai, Riyi; Zhou, Pan; Ju, Chuan; Sun, Jun; Brindley, Paul J; Hu, Wei

2015-01-01

The reproductive development and maturation of female schistosomes are crucial since their released eggs are responsible for the host immunopathology and transmission of schistosomiasis. However, little is known about the nutrients required by female Schistosoma japonicum during its sexual maturation. We evaluated the promoting effect of several nutrients (calf serum, red blood cells (RBCs), ATP and hypoxanthine) on the reproductive development of pre-adult females at 18 days post infection (dpi) from mixed infections and at 50 dpi from unisexual infections of laboratory mice in basic medium RPMI-1640. We found RBCs, rather than other nutrients, promoted the female sexual maturation and egg production with significant morphological changes. In 27% of females (18 dpi) from mixed infections that paired with males in vitro on day 14, vitelline glands could be positively stained by Fast Blue B; and in 35% of females (50 dpi) from unisexual infections on day 21, mature vitelline cells were observed. Infertile eggs were detected among both groups. To analyze which component of mouse RBCs possesses the stimulating effect, RBCs were fractionated and included in media. However, the RBC fractions failed to stimulate development of the female reproductive organs. In addition, bovine hemoglobin hydrolysate, digested by neutral protease, was found to exhibit the promoting activity instead of untreated bovine hemoglobin. The other protein hydrolysate, lactalbumin hydrolysate, exhibited a similar effect with bovine hemoglobin hydrolysate. Using quantitative RT-PCR, we found the expression levels of four reproduction-related genes were significantly stimulated by RBCs. These data indicate that RBCs provide essential nutrients for the sexual maturation of female S. japonicum and that the protein component of RBCs appeared to constitute the key nutrient. These findings would improve laboratory culture of pre-adult schistosomes to adult worms in medium with well-defined components, which is important to investigate the function of genes related to female sexual maturation.

Intake of Erythrocytes Required for Reproductive Development of Female Schistosoma japonicum

PubMed Central

Wang, Jipeng; Wang, Shuqi; Liu, Xiufeng; Xu, Bin; Chai, Riyi; Zhou, Pan; Ju, Chuan; Sun, Jun; Brindley, Paul J.; Hu, Wei

2015-01-01

The reproductive development and maturation of female schistosomes are crucial since their released eggs are responsible for the host immunopathology and transmission of schistosomiasis. However, little is known about the nutrients required by female Schistosoma japonicum during its sexual maturation. We evaluated the promoting effect of several nutrients (calf serum, red blood cells (RBCs), ATP and hypoxanthine) on the reproductive development of pre-adult females at 18 days post infection (dpi) from mixed infections and at 50 dpi from unisexual infections of laboratory mice in basic medium RPMI-1640. We found RBCs, rather than other nutrients, promoted the female sexual maturation and egg production with significant morphological changes. In 27% of females (18 dpi) from mixed infections that paired with males in vitro on day 14, vitelline glands could be positively stained by Fast Blue B; and in 35% of females (50 dpi) from unisexual infections on day 21, mature vitelline cells were observed. Infertile eggs were detected among both groups. To analyze which component of mouse RBCs possesses the stimulating effect, RBCs were fractionated and included in media. However, the RBC fractions failed to stimulate development of the female reproductive organs. In addition, bovine hemoglobin hydrolysate, digested by neutral protease, was found to exhibit the promoting activity instead of untreated bovine hemoglobin. The other protein hydrolysate, lactalbumin hydrolysate, exhibited a similar effect with bovine hemoglobin hydrolysate. Using quantitative RT-PCR, we found the expression levels of four reproduction-related genes were significantly stimulated by RBCs. These data indicate that RBCs provide essential nutrients for the sexual maturation of female S. japonicum and that the protein component of RBCs appeared to constitute the key nutrient. These findings would improve laboratory culture of pre-adult schistosomes to adult worms in medium with well-defined components, which is important to investigate the function of genes related to female sexual maturation. PMID:25978643

Identification of a soluble guanylate cyclase in RBCs: preserved activity in patients with coronary artery disease.

PubMed

Cortese-Krott, Miriam M; Mergia, Evanthia; Kramer, Christian M; Lückstädt, Wiebke; Yang, Jiangning; Wolff, Georg; Panknin, Christina; Bracht, Thilo; Sitek, Barbara; Pernow, John; Stasch, Johannes-Peter; Feelisch, Martin; Koesling, Doris; Kelm, Malte

2018-04-01

Endothelial dysfunction is associated with decreased NO bioavailability and impaired activation of the NO receptor soluble guanylate cyclase (sGC) in the vasculature and in platelets. Red blood cells (RBCs) are known to produce NO under hypoxic and normoxic conditions; however evidence of expression and/or activity of sGC and downstream signaling pathway including phopshodiesterase (PDE)-5 and protein kinase G (PKG) in RBCs is still controversial. In the present study, we aimed to investigate whether RBCs carry a functional sGC signaling pathway and to address whether this pathway is compromised in coronary artery disease (CAD). Using two independent chromatographic procedures, we here demonstrate that human and murine RBCs carry a catalytically active α 1 β 1 -sGC (isoform 1), which converts 32 P-GTP into 32 P-cGMP, as well as PDE5 and PKG. Specific sGC stimulation by NO+BAY 41-2272 increases intracellular cGMP-levels up to 1000-fold with concomitant activation of the canonical PKG/VASP-signaling pathway. This response to NO is blunted in α1-sGC knockout (KO) RBCs, but fully preserved in α2-sGC KO. In patients with stable CAD and endothelial dysfunction red cell eNOS expression is decreased as compared to aged-matched controls; by contrast, red cell sGC expression/activity and responsiveness to NO are fully preserved, although sGC oxidation is increased in both groups. Collectively, our data demonstrate that an intact sGC/PDE5/PKG-dependent signaling pathway exists in RBCs, which remains fully responsive to NO and sGC stimulators/activators in patients with endothelial dysfunction. Targeting this pathway may be helpful in diseases with NO deficiency in the microcirculation like sickle cell anemia, pulmonary hypertension, and heart failure. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Quality of red blood cells isolated from umbilical cord blood stored at room temperature.

PubMed

Zhurova, Mariia; Akabutu, John; Acker, Jason

2012-01-01

Red blood cells (RBCs) from cord blood contain fetal hemoglobin that is predominant in newborns and, therefore, may be more appropriate for neonatal transfusions than currently transfused adult RBCs. Post-collection, cord blood can be stored at room temperature for several days before it is processed for stem cells isolation, with little known about how these conditions affect currently discarded RBCs. The present study examined the effect of the duration cord blood spent at room temperature and other cord blood characteristics on cord RBC quality. RBCs were tested immediately after their isolation from cord blood using a broad panel of quality assays. No significant decrease in cord RBC quality was observed during the first 65 hours of storage at room temperature. The ratio of cord blood to anticoagulant was associated with RBC quality and needs to be optimized in future. This knowledge will assist in future development of cord RBC transfusion product.

Cell-penetrating peptides meditated encapsulation of protein therapeutics into intact red blood cells and its application

PubMed Central

Wang, Yinsong; Liu, Quan; Chung, Hee Sun; Kwon, Young Min; Shin, Meong Cheol; Lee, Kyuri; Yang, Victor C

2014-01-01

Red blood cells (RBCs) based drug carrier appears to be the most appealing for protein drugs due to their unmatched biocompatability, biodegradability, and long lifespan in the circulation. Numerous methods for encapsulating protein drugs into RBCs were developed, however, most of them induce partial disruption of the cell membrane, resulting in irreversible alterations in both physical and chemical properties of RBCs. Herein, we introduce a novel method for encapsulating proteins into intact RBCs, which was meditated by a cell penetrating peptide (CPP) developed in our lab—low molecular weight protamine (LMWP). L-asparaginase, one of the primary drugs used in treatment of acute lymphoblastic leukemia (ALL), was chosen as a model protein to illustrate the encapsulation into erythrocytes mediated by CPPs. In addition current treatment of ALL using different L-asparaginase delivery and encapsulation methods as well as their associated problems were also reviewed. PMID:24374002

7SL RNA in Vertebrate Red Blood Cells.

PubMed

Talhouarne, Gaëlle J S; Gall, Joseph G

2018-04-23

We report that 7SL, the RNA component of the signal recognition particle (SRP), is an abundant ncRNA in mature red blood cells (RBCs) of human, mouse, and the frog Xenopus. 7SL RNA in RBCs is not associated with the canonical proteins of the SRP. Instead, it co-immunoprecipitates from a lysate of RBCs with a number of membrane-binding proteins. Human and mouse RBCs also contain a previously undescribed 68 nt RNA, sRN7SL, derived from the "S domain" of 7SL RNA. We discuss the possibility that 7SL RNA is selectively protected from nucleases by association with the RBC membrane. Because 7SL is not associated with the canonical proteins of the SRP, it could represent a non-functional remnant of the protein synthetic machinery. Alternatively, it could play a new, as yet undefined role in RBC metabolism. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Infection after injury: association with blood transfusion.

PubMed

Rosemurgy, A S; Hart, M B; Murphy, C G; Albrink, M H; Piazza, A; Leparc, G F; Harris, R E

1992-02-01

This study was undertaken to evaluate the association between red blood cell transfusions and infections in an easily stratified, homogenous group of injured adults. All received their initial transfusions upon arrival to the emergency department. Over 5 years, 390 uncross-matched trauma patients received type "O" red blood cells (RBCs) during initial resuscitation. One hundred fifty-four (39%) died within 7 days because of injuries sustained: 236 (61%) survived at least 7 days. Of these 236, clear differences could be seen between those receiving 6 or fewer or 7 or more units of RBCs. When adjusted for age, sex, and severity of injury (Champion Trauma Score, Injury Severity Score, TRISS), the risk of infection was higher in those receiving 7 or more units of RBCs. Similarly, risk of infection was related to units of RBCs transfused in a dose-related fashion. Blood transfusions should be avoided, if possible. Arbitrary "trigger points" for transfusions should be abandoned.

Effect of whole-body irradiation of mice on the number of background plaque-forming cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, R.E.; Lefkovits, I.; Soeederberg, A.

1983-08-01

Mice were exposed in whole-body fashion to several doses of radiation and killed at various times thereafter for a determination of the number of background plaque-forming cells (PFCs) as assayed on either sheep erythrocytes or bromelain-treated autologous mouse erythrocytes. Increased numbers of both types of PFC were found in the irradiated groups. These increases were dependent on radiation dose and time after exposure. They did not appear to be caused by a disruption of normal lymphocyte traffic or a switch in immunoglobulin isotype. An increased number of PFCs on bromelain-treated mouse RBCs but not on sheep RBCs were found inmore » irradiated congenitally athymic nude mice. On the basis of this and related observations, background PFCs on bromelain-treated mouse RBCs and on sheep RBCs appear to fall under different forms of homeostatic control.« less

Size-Based Separation of Particles and Cells Utilizing Viscoelastic Effects in Straight Microchannels.

PubMed

Liu, Chao; Xue, Chundong; Chen, Xiaodong; Shan, Lei; Tian, Yu; Hu, Guoqing

2015-06-16

Viscoelasticity-induced particle migration has recently received increasing attention due to its ability to obtain high-quality focusing over a wide range of flow rates. However, its application is limited to low throughput regime since the particles can defocus as flow rate increases. Using an engineered carrier medium with constant and low viscosity and strong elasticity, the sample flow rates are improved to be 1 order of magnitude higher than those in existing studies. Utilizing differential focusing of particles of different sizes, here, we present sheathless particle/cell separation in simple straight microchannels that possess excellent parallelizability for further throughput enhancement. The present method can be implemented over a wide range of particle/cell sizes and flow rates. We successfully separate small particles from larger particles, MCF-7 cells from red blood cells (RBCs), and Escherichia coli (E. coli) bacteria from RBCs in different straight microchannels. The proposed method could broaden the applications of viscoelastic microfluidic devices to particle/cell separation due to the enhanced sample throughput and simple channel design.

Effect of transient warming of red blood cells for up to 24 h: in vitro characteristics in CPD/saline-adenine-glucose-mannitol environment.

PubMed

Gulliksson, H; Nordahl-Källman, A-S

2014-01-01

There are few studies on transient warming of red blood cells (RBCs). Occasional storage outside restricted temperature range often results in destroying of the RBC unit, even after a short period of time due to national guidelines. This study evaluates the in vitro effects associated with such accidental warming on RBCs stored in saline-adenine-glucose-mannitol (SAGM) and prepared within 8 h after blood collection. This study includes both repeated short-term exposure of RBCs to room temperature for 6 h as wells as warming for either 6, 12, 18 or 24 h after 1 week or after 3 weeks of storage in two separate studies. RBCs were stored for 42 days. We weekly measured pH, K(+) , glucose, lactate, haemolysis, red cell ATP and 2,3-diphosphoglycerate. The lowest individual ATP value observed in any of the groups of warmed units was 2·6 μmol/g haemoglobin. Increased haemolysis in warmed units was noted in two of the studies. None of the individual units exceeded the European maximum limit of 0·8% haemolysis. Our results suggest that quality of RBCs after transient warming will be maintained at acceptable levels specified in standards and in previous studies. However, increased haemolysis was observed when transient warming occurred during the second part of the storage period of 6 weeks suggesting that RBCs are more vulnerable to warming by the end of storage. © 2013 International Society of Blood Transfusion.

Red blood cell populations and membrane levels of peroxiredoxin 2 as candidate biomarkers to reveal blood doping.

PubMed

Marrocco, Cristina; Pallotta, Valeria; D'alessandro, Angelo; Alves, Gilda; Zolla, Lello

2012-05-01

Blood doping represents one main trend in doping strategies. Blood doping refers to the practice of boosting the number of red blood cells (RBCs) in the bloodstream in order to enhance athletic performance, by means of blood transfusions, administration of erythropoiesis-stimulating substances, blood substitutes, natural or artificial altitude facilities, and innovative gene therapies. While detection of recombinant EPO and homologous transfusion is already feasible through electrophoretic, mass spectrometry or flow cytometry-based approaches, no method is currently available to tackle doping strategies relying on autologous transfusions. We exploited an in vitro model of autologous transfusion through a 1:10 dilution of concentrated RBCs after 30 days of storage upon appropriate dilution in freshly withdrawn RBCs from the same donor. Western blot towards membrane Prdx2 and Percoll density gradients were exploited to assess their suitability as biomarkers of transfusion. Membrane Prdx2 was visible in day 30 samples albeit not in day 0, while it was still visible in the 1:10 dilution of day 30 in day 0 RBCs. Cell gradients also highlighted changes in the profile of the RBC subpopulations upon dilution of stored RBCs in the fresh ones. From this preliminary in vitro investigation it emerges that Prdx2 and RBC populations might be further tested as candidate biomarkers of blood doping through autologous transfusion, though it is yet to be assessed whether the kinetics in vivo of Prdx2 exposure in the membrane of transfused RBCs will endow a sufficient time-window to allow reliable anti-doping testing.

Red blood cell populations and membrane levels of peroxiredoxin 2 as candidate biomarkers to reveal blood doping

PubMed Central

Marrocco, Cristina; Pallotta, Valeria; D’Alessandro, Angelo; Alves, Gilda; Zolla, Lello

2012-01-01

Background Blood doping represents one main trend in doping strategies. Blood doping refers to the practice of boosting the number of red blood cells (RBCs) in the bloodstream in order to enhance athletic performance, by means of blood transfusions, administration of erythropoiesis-stimulating substances, blood substitutes, natural or artificial altitude facilities, and innovative gene therapies. While detection of recombinant EPO and homologous transfusion is already feasible through electrophoretic, mass spectrometry or flow cytometry-based approaches, no method is currently available to tackle doping strategies relying on autologous transfusions. Materials and methods. We exploited an in vitro model of autologous transfusion through a 1:10 dilution of concentrated RBCs after 30 days of storage upon appropriate dilution in freshly withdrawn RBCs from the same donor. Western blot towards membrane Prdx2 and Percoll density gradients were exploited to assess their suitability as biomarkers of transfusion. Results Membrane Prdx2 was visible in day 30 samples albeit not in day 0, while it was still visible in the 1:10 dilution of day 30 in day 0 RBCs. Cell gradients also highlighted changes in the profile of the RBC subpopulations upon dilution of stored RBCs in the fresh ones. Discussion. From this preliminary in vitro investigation it emerges that Prdx2 and RBC populations might be further tested as candidate biomarkers of blood doping through autologous transfusion, though it is yet to be assessed whether the kinetics in vivo of Prdx2 exposure in the membrane of transfused RBCs will endow a sufficient time-window to allow reliable anti-doping testing. PMID:22890272

pH modulation ameliorates the red blood cell storage lesion in a murine model of transfusion.

PubMed

Chang, Alex L; Kim, Young; Seitz, Aaron P; Schuster, Rebecca M; Pritts, Timothy A

2017-05-15

Prolonged storage of packed red blood cells (pRBCs) induces a series of harmful biochemical and metabolic changes known as the RBC storage lesion. RBCs are currently stored in an acidic storage solution, but the effect of pH on the RBC storage lesion is unknown. We investigated the effect of modulation of storage pH on the RBC storage lesion and on erythrocyte survival after transfusion. Murine pRBCs were stored in Additive Solution-3 (AS3) under standard conditions (pH, 5.8), acidic AS3 (pH, 4.5), or alkalinized AS3 (pH, 8.5). pRBC units were analyzed at the end of the storage period. Several components of the storage lesion were measured, including cell-free hemoglobin, microparticle production, phosphatidylserine externalization, lactate accumulation, and byproducts of lipid peroxidation. Carboxyfluorescein-labeled erythrocytes were transfused into healthy mice to determine cell survival. Compared with pRBCs stored in standard AS3, those stored in alkaline solution exhibited decreased hemolysis, phosphatidylserine externalization, microparticle production, and lipid peroxidation. Lactate levels were greater after storage in alkaline conditions, suggesting that these pRBCs remained more metabolically viable. Storage in acidic AS3 accelerated erythrocyte deterioration. Compared with standard AS3 storage, circulating half-life of cells was increased by alkaline storage but decreased in acidic conditions. Storage pH significantly affects the quality of stored RBCs and cell survival after transfusion. Current erythrocyte storage solutions may benefit from refinements in pH levels. Copyright © 2016 Elsevier Inc. All rights reserved.

Structural and Functional Analysis of BipA, a Regulator of Virulence in Enteropathogenic Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fan, Haitian; Hahm, Joseph; Diggs, Stephen

The translational GTPase BipA regulates the expression of virulence and pathogenicity factors in several eubacteria. BipA-dependent expression of virulence factors occurs under starvation conditions, such as encountered during infection of a host. Under these conditions, BipA associates with the small ribosomal subunit. BipA also has a second function to promote the efficiency of late steps in biogenesis of large ribosomal subunits at low temperatures, presumably while bound to the ribosome. During starvation, the cellular concentration of stress alarmone guanosine-3', 5'-bis pyrophosphate (ppGpp) is increased. This increase allows ppGpp to bind to BipA and switch its binding specificity from ribosomes tomore » small ribosomal subunits. A conformational change of BipA upon ppGpp binding could explain the ppGpp regulation of the binding specificity of BipA. Here, we present the structures of the full-length BipA from Escherichia coli in apo, GDP-, and ppGpp-bound forms. The crystal structure and small-angle x-ray scattering data of the protein with bound nucleotides, together with a thermodynamic analysis of the binding of GDP and of ppGpp to BipA, indicate that the ppGpp-bound form of BipA adopts the structure of the GDP form. This suggests furthermore, that the switch in binding preference only occurs when both ppGpp and the small ribosomal subunit are present. Finally, this molecular mechanism would allow BipA to interact with both the ribosome and the small ribosomal subunit during stress response.« less

Structural and Functional Analysis of BipA, a Regulator of Virulence in Enteropathogenic Escherichia coli

DOE PAGES

Fan, Haitian; Hahm, Joseph; Diggs, Stephen; ...

2015-07-10

The translational GTPase BipA regulates the expression of virulence and pathogenicity factors in several eubacteria. BipA-dependent expression of virulence factors occurs under starvation conditions, such as encountered during infection of a host. Under these conditions, BipA associates with the small ribosomal subunit. BipA also has a second function to promote the efficiency of late steps in biogenesis of large ribosomal subunits at low temperatures, presumably while bound to the ribosome. During starvation, the cellular concentration of stress alarmone guanosine-3', 5'-bis pyrophosphate (ppGpp) is increased. This increase allows ppGpp to bind to BipA and switch its binding specificity from ribosomes tomore » small ribosomal subunits. A conformational change of BipA upon ppGpp binding could explain the ppGpp regulation of the binding specificity of BipA. Here, we present the structures of the full-length BipA from Escherichia coli in apo, GDP-, and ppGpp-bound forms. The crystal structure and small-angle x-ray scattering data of the protein with bound nucleotides, together with a thermodynamic analysis of the binding of GDP and of ppGpp to BipA, indicate that the ppGpp-bound form of BipA adopts the structure of the GDP form. This suggests furthermore, that the switch in binding preference only occurs when both ppGpp and the small ribosomal subunit are present. Finally, this molecular mechanism would allow BipA to interact with both the ribosome and the small ribosomal subunit during stress response.« less

Recognition of chimeric small-subunit ribosomal DNAs composed of genes from uncultivated microorganisms

NASA Technical Reports Server (NTRS)

Kopczynski, E. D.; Bateson, M. M.; Ward, D. M.

1994-01-01

When PCR was used to recover small-subunit (SSU) rRNA genes from a hot spring cyanobacterial mat community, chimeric SSU rRNA sequences which exhibited little or no secondary structural abnormality were recovered. They were revealed as chimeras of SSU rRNA genes of uncultivated species through separate phylogenetic analysis of short sequence domains.

Ribosomal small subunit domains radiate from a central core

NASA Astrophysics Data System (ADS)

Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O'Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean

2016-02-01

The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2‧OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit.

Computational prediction of protein interactions related to the invasion of erythrocytes by malarial parasites.

PubMed

Liu, Xuewu; Huang, Yuxiao; Liang, Jiao; Zhang, Shuai; Li, Yinghui; Wang, Jun; Shen, Yan; Xu, Zhikai; Zhao, Ya

2014-11-30

The invasion of red blood cells (RBCs) by malarial parasites is an essential step in the life cycle of Plasmodium falciparum. Human-parasite surface protein interactions play a critical role in this process. Although several interactions between human and parasite proteins have been discovered, the mechanism related to invasion remains poorly understood because numerous human-parasite protein interactions have not yet been identified. High-throughput screening experiments are not feasible for malarial parasites due to difficulty in expressing the parasite proteins. Here, we performed computational prediction of the PPIs involved in malaria parasite invasion to elucidate the mechanism by which invasion occurs. In this study, an expectation maximization algorithm was used to estimate the probabilities of domain-domain interactions (DDIs). Estimates of DDI probabilities were then used to infer PPI probabilities. We found that our prediction performance was better than that based on the information of D. melanogaster alone when information related to the six species was used. Prediction performance was assessed using protein interaction data from S. cerevisiae, indicating that the predicted results were reliable. We then used the estimates of DDI probabilities to infer interactions between 490 parasite and 3,787 human membrane proteins. A small-scale dataset was used to illustrate the usability of our method in predicting interactions between human and parasite proteins. The positive predictive value (PPV) was lower than that observed in S. cerevisiae. We integrated gene expression data to improve prediction accuracy and to reduce false positives. We identified 80 membrane proteins highly expressed in the schizont stage by fast Fourier transform method. Approximately 221 erythrocyte membrane proteins were identified using published mass spectral datasets. A network consisting of 205 interactions was predicted. Results of network analysis suggest that SNARE proteins of parasites and APP of humans may function in the invasion of RBCs by parasites. We predicted a small-scale PPI network that may be involved in parasite invasion of RBCs by integrating DDI information and expression profiles. Experimental studies should be conducted to validate the predicted interactions. The predicted PPIs help elucidate the mechanism of parasite invasion and provide directions for future experimental investigations.

Improved purification of brine-shrimp (Artemia saline) (Na+ + K+)-activated adenosine triphosphatase and amino-acid and carbohydrate analyses of the isolated subunits.

PubMed Central

Peterson, G L; Hokin, L E

1980-01-01

Purification of the (Na+ + K+)-activated ATPase has been improved 2-fold the respect to both purity and yield over the previous method [Peterson, Ewing, Hootman & Conte (1978) J. Biol. Chem. 253, 4762-4770] by using Lubrol WX and non-denaturing concentrations of sodium dodecyl sulphate (SDS). The enzyme was purified 200-fold over the homogenate. The preparation had a specific activity of about 600 mumol of Pi/h per mg of protein, and was about 60% pure according to quantification of Coomassie Blue-stained SDS/polyacrylamide gels. The yield of purified enzyme was about 10 mg of protein per 100g of dry brine-shrimp (Artemia salina) cysts. The method is highly suitable for purification either on a small scale (10-25g of dry cysts) or on a large scale (900g of dry cysts) and methods are described for both. The large (Na+ + K+)-activated ATPase subunit (alpha-subunit) was isolated in pure form by SDS-gel filtration on Bio-Gel A 1.5m. The small subunit (beta-subunit) was eluted with other contaminating proteins on the Bio-Gel column, but was isolated in pure form by extraction from SDS/polyacrylamide gels. The amino acid and carbohydrate compositions of both subunits are reported. The alpha-subunit contained 5.2% carbohydrate by weight, and the beta-subunit 9.2%. Sialic acid was absent from both subunits. Images Fig. 3. Fig. 4. PMID:6272692

Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ε

PubMed Central

Zahurancik, Walter J.; Baranovskiy, Andrey G.; Tahirov, Tahir H.; Suo, Zucai

2015-01-01

Numerous genetic studies have provided compelling evidence to establish DNA polymerase ε (Polε) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polε is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3′ → 5′ exonuclease domain common to many replicative polymerases. In addition, Polε possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Polε heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Polε in vitro. However, similar studies of the human Polε heterote-tramer (hPolε) have been limited by the difficulty of obtaining hPolε in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPolε from insect host cells has allowed for isolation of greater amounts of active hPolε, thus enabling a more detailed kinetic comparison between hPolε and an active N-terminal fragment of the hPolε catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPolε. We observe that the small subunits increase DNA binding by hPolε relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3′ → 5′ exonuclease activity of hPolε is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPolε and sway hPolε toward DNA synthesis rather than proofreading. PMID:25684708

Distinct cellular distributions of Kv4 pore-forming and auxiliary subunits in rat dorsal root ganglion neurons.

PubMed

Matsuyoshi, Hiroko; Takimoto, Koichi; Yunoki, Takakazu; Erickson, Vickie L; Tyagi, Pradeep; Hirao, Yoshihiko; Wanaka, Akio; Yoshimura, Naoki

2012-09-17

Dorsal root ganglia contain heterogeneous populations of primary afferent neurons that transmit various sensory stimuli. This functional diversity may be correlated with differential expression of voltage-gated K(+) (Kv) channels. Here, we examine cellular distributions of Kv4 pore-forming and ancillary subunits that are responsible for fast-inactivating A-type K(+) current. Expression pattern of Kv α-subunit, β-subunit and auxiliary subunit was investigated using immunohistochemistry, in situ hybridization and RT-PCR technique. The two pore-forming subunits Kv4.1 and Kv4.3 show distinct cellular distributions: Kv4.3 is predominantly in small-sized C-fiber neurons, whereas Kv4.1 is seen in DRG neurons in various sizes. Furthermore, the two classes of Kv4 channel auxiliary subunits are also distributed in different-sized cells. KChIP3 is the only significantly expressed Ca(2+)-binding cytosolic ancillary subunit in DRGs and present in medium to large-sized neurons. The membrane-spanning auxiliary subunit DPP6 is seen in a large number of DRG neurons in various sizes, whereas DPP10 is restricted in small-sized neurons. Distinct combinations of Kv4 pore-forming and auxiliary subunits may constitute A-type channels in DRG neurons with different physiological roles. Kv4.1 subunit, in combination with KChIP3 and/or DPP6, form A-type K(+) channels in medium to large-sized A-fiber DRG neurons. In contrast, Kv4.3 and DPP10 may contribute to A-type K(+) current in non-peptidergic, C-fiber somatic afferent neurons. Copyright © 2012 Elsevier Inc. All rights reserved.

In Silico Functional Networks Identified in Fish Nucleated Red Blood Cells by Means of Transcriptomic and Proteomic Profiling.

PubMed

Puente-Marin, Sara; Nombela, Iván; Ciordia, Sergio; Mena, María Carmen; Chico, Verónica; Coll, Julio; Ortega-Villaizan, María Del Mar

2018-04-09

Nucleated red blood cells (RBCs) of fish have, in the last decade, been implicated in several immune-related functions, such as antiviral response, phagocytosis or cytokine-mediated signaling. RNA-sequencing (RNA-seq) and label-free shotgun proteomic analyses were carried out for in silico functional pathway profiling of rainbow trout RBCs. For RNA-seq, a de novo assembly was conducted, in order to create a transcriptome database for RBCs. For proteome profiling, we developed a proteomic method that combined: (a) fractionation into cytosolic and membrane fractions, (b) hemoglobin removal of the cytosolic fraction, (c) protein digestion, and (d) a novel step with pH reversed-phase peptide fractionation and final Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MS/MS) analysis of each fraction. Combined transcriptome- and proteome- sequencing data identified, in silico, novel and striking immune functional networks for rainbow trout nucleated RBCs, which are mainly linked to innate and adaptive immunity. Functional pathways related to regulation of hematopoietic cell differentiation, antigen presentation via major histocompatibility complex class II (MHCII), leukocyte differentiation and regulation of leukocyte activation were identified. These preliminary findings further implicate nucleated RBCs in immune function, such as antigen presentation and leukocyte activation.

In Silico Functional Networks Identified in Fish Nucleated Red Blood Cells by Means of Transcriptomic and Proteomic Profiling

PubMed Central

Puente-Marin, Sara; Ciordia, Sergio; Mena, María Carmen; Chico, Verónica; Coll, Julio

2018-01-01

Nucleated red blood cells (RBCs) of fish have, in the last decade, been implicated in several immune-related functions, such as antiviral response, phagocytosis or cytokine-mediated signaling. RNA-sequencing (RNA-seq) and label-free shotgun proteomic analyses were carried out for in silico functional pathway profiling of rainbow trout RBCs. For RNA-seq, a de novo assembly was conducted, in order to create a transcriptome database for RBCs. For proteome profiling, we developed a proteomic method that combined: (a) fractionation into cytosolic and membrane fractions, (b) hemoglobin removal of the cytosolic fraction, (c) protein digestion, and (d) a novel step with pH reversed-phase peptide fractionation and final Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MS/MS) analysis of each fraction. Combined transcriptome- and proteome- sequencing data identified, in silico, novel and striking immune functional networks for rainbow trout nucleated RBCs, which are mainly linked to innate and adaptive immunity. Functional pathways related to regulation of hematopoietic cell differentiation, antigen presentation via major histocompatibility complex class II (MHCII), leukocyte differentiation and regulation of leukocyte activation were identified. These preliminary findings further implicate nucleated RBCs in immune function, such as antigen presentation and leukocyte activation. PMID:29642539

Regulation of nitrite transport in red blood cells by hemoglobin oxygen fractional saturation.

PubMed

Vitturi, Dario A; Teng, Xinjun; Toledo, José C; Matalon, Sadis; Lancaster, Jack R; Patel, Rakesh P

2009-05-01

Allosteric regulation of nitrite reduction by deoxyhemoglobin has been proposed to mediate nitric oxide (NO) formation during hypoxia. Nitrite is predominantly an anion at physiological pH, raising questions about the mechanism by which it enters the red blood cell (RBC) and whether this is regulated and coupled to deoxyhemoglobin-mediated reduction. We tested the hypothesis that nitrite transport by RBCs is regulated by fractional saturation. Using human RBCs, nitrite consumption was faster at lower fractional saturations, consistent with faster reactions with deoxyheme. A membrane-based regulation was suggested by slower nitrite consumption with intact versus lysed RBCs. Interestingly, upon nitrite addition, intracellular nitrite concentrations attained a steady state that, despite increased rates of consumption, did not change with decreasing oxygen tensions, suggesting a deoxygenation-sensitive step that either increases nitrite import or decreases the rate of nitrite export. A role for anion exchanger (AE)-1 in the control of nitrite export was suggested by increased intracellular nitrite concentrations in RBCs treated with DIDS. Moreover, deoxygenation decreased steady-state levels of intracellular nitrite in AE-1-inhibited RBCs. Based on these data, we propose a model in which deoxyhemoglobin binding to AE-1 inhibits nitrite export under low oxygen tensions allowing for the coupling between deoxygenation and nitrite reduction to NO along the arterial-to-venous gradient.

Red blood cells play a role in reverse cholesterol transport.

PubMed

Hung, Kimberly T; Berisha, Stela Z; Ritchey, Brian M; Santore, Jennifer; Smith, Jonathan D

2012-06-01

Reverse cholesterol transport (RCT) involves the removal of cholesterol from peripheral tissue for excretion in the feces. Here, we determined whether red blood cells (RBCs) can contribute to RCT. We performed a series of studies in apolipoprotein AI-deficient mice where the high-density lipoprotein-mediated pathway of RCT is greatly diminished. RBCs carried a higher fraction of whole blood cholesterol than plasma in apolipoprotein AI-deficient mice, and as least as much of the labeled cholesterol derived from injected foam cells appeared in RBCs compared with plasma. To determine whether RBCs mediate RCT to the fecal compartment, we measured RCT in anemic and control apolipoprotein AI-deficient mice and found that anemia decreased RCT to the feces by over 35% after correcting for fecal mass. Transfusion of [(3)H]cholesterol-labeled RBCs led to robust delivery of the labeled cholesterol to the feces in apolipoprotein AI-deficient hosts. In wild-type mice, the majority of the blood cholesterol mass, as well as [(3)H]cholesterol derived from the injected foam cells, was found in plasma, and anemia did not significantly alter RCT to the feces after correction for fecal mass. The RBC cholesterol pool is dynamic and facilitates RCT of peripheral cholesterol to the feces, particularly in the low high-density lipoprotein state.

Red Blood Cell Function and Dysfunction: Redox Regulation, Nitric Oxide Metabolism, Anemia

PubMed Central

Kuhn, Viktoria; Diederich, Lukas; Keller, T.C. Stevenson; Kramer, Christian M.; Lückstädt, Wiebke; Panknin, Christina; Suvorava, Tatsiana; Isakson, Brant E.; Kelm, Malte

2017-01-01

Abstract Significance: Recent clinical evidence identified anemia to be correlated with severe complications of cardiovascular disease (CVD) such as bleeding, thromboembolic events, stroke, hypertension, arrhythmias, and inflammation, particularly in elderly patients. The underlying mechanisms of these complications are largely unidentified. Recent Advances: Previously, red blood cells (RBCs) were considered exclusively as transporters of oxygen and nutrients to the tissues. More recent experimental evidence indicates that RBCs are important interorgan communication systems with additional functions, including participation in control of systemic nitric oxide metabolism, redox regulation, blood rheology, and viscosity. In this article, we aim to revise and discuss the potential impact of these noncanonical functions of RBCs and their dysfunction in the cardiovascular system and in anemia. Critical Issues: The mechanistic links between changes of RBC functional properties and cardiovascular complications related to anemia have not been untangled so far. Future Directions: To allow a better understanding of the complications associated with anemia in CVD, basic and translational science studies should be focused on identifying the role of noncanonical functions of RBCs in the cardiovascular system and on defining intrinsic and/or systemic dysfunction of RBCs in anemia and its relationship to CVD both in animal models and clinical settings. Antioxid. Redox Signal. 26, 718–742. PMID:27889956

Removal of malaria-infected red blood cells using magnetic cell separators: A computational study

PubMed Central

Kim, Jeongho; Massoudi, Mehrdad; Antaki, James F.; Gandini, Alberto

2012-01-01

High gradient magnetic field separators have been widely used in a variety of biological applications. Recently, the use of magnetic separators to remove malaria-infected red blood cells (pRBCs) from blood circulation in patients with severe malaria has been proposed in a dialysis-like treatment. The capture efficiency of this process depends on many interrelated design variables and constraints such as magnetic pole array pitch, chamber height, and flow rate. In this paper, we model the malaria-infected RBCs (pRBCs) as paramagnetic particles suspended in a Newtonian fluid. Trajectories of the infected cells are numerically calculated inside a micro-channel exposed to a periodic magnetic field gradient. First-order stiff ordinary differential equations (ODEs) governing the trajectory of particles under periodic magnetic fields due to an array of wires are solved numerically using the 1st –5th order adaptive step Runge-Kutta solver. The numerical experiments show that in order to achieve a capture efficiency of 99% for the pRBCs it is required to have a longer length than 80 mm; this implies that in principle, using optimization techniques the length could be adjusted, i.e., shortened to achieve 99% capture efficiency of the pRBCs. PMID:22345827

The pathogenesis of Plasmodium falciparum malaria in humans: insights from splenic physiology

PubMed Central

Safeukui, Innocent; Deplaine, Guillaume; Brousse, Valentine; Prendki, Virginie; Thellier, Marc; Turner, Gareth D.; Mercereau-Puijalon, Odile

2011-01-01

Clinical manifestations of Plasmodium falciparum infection are induced by the asexual stages of the parasite that develop inside red blood cells (RBCs). Because splenic microcirculatory beds filter out altered RBCs, the spleen can innately clear subpopulations of infected or uninfected RBC modified during falciparum malaria. The spleen appears more protective against severe manifestations of malaria in naïve than in immune subjects. The spleen-specific pitting function accounts for a large fraction of parasite clearance in artemisinin-treated patients. RBC loss contributes to malarial anemia, a clinical form associated with subacute progression, frequent splenomegaly, and relatively low parasitemia. Stringent splenic clearance of ring-infected RBCs and uninfected, but parasite-altered, RBCs, may altogether exacerbate anemia and reduce the risks of severe complications associated with high parasite loads, such as cerebral malaria. The age of the patient directly influences the risk of severe manifestations. We hypothesize that coevolution resulting in increased splenic clearance of P. falciparum–altered RBCs in children favors the survival of the host and, ultimately, sustained parasite transmission. This analysis of the RBC–spleen dynamic interactions during P falciparum infection reflects both data and hypotheses, and provides a framework on which a more complete immunologic understanding of malaria pathogenesis may be elaborated. PMID:20852127

Portable microsystem integrates multifunctional dielectrophoresis manipulations and a surface stress biosensor to detect red blood cells for hemolytic anemia.

PubMed

Sang, Shengbo; Feng, Qiliang; Jian, Aoqun; Li, Huiming; Ji, Jianlong; Duan, Qianqian; Zhang, Wendong; Wang, Tao

2016-09-20

Hemolytic anemia intensity has been suggested as a vital factor for the growth of certain clinical complications of sickle cell disease. However, there is no effective and rapid diagnostic method. As a powerful platform for bio-particles testing, biosensors integrated with microfluidics offer great potential for a new generation of portable point of care systems. In this paper, we describe a novel portable microsystem consisting of a multifunctional dielectrophoresis manipulations (MDM) device and a surface stress biosensor to separate and detect red blood cells (RBCs) for diagnosis of hemolytic anemia. The peripheral circuit to power the interdigitated electrode array of the MDM device and the surface stress biosensor test platform were integrated into a portable signal system. The MDM includes a preparing region, a focusing region, and a sorting region. Simulation and experimental results show the RBCs trajectories when they are subjected to the positive DEP force, allowing the successful sorting of living/dead RBCs. Separated RBCs are then transported to the biosensor and the capacitance values resulting from the variation of surface stress were measured. The diagnosis of hemolytic anemia can be realized by detecting RBCs and the portable microsystem provides the assessment to the hemolytic anemia patient.

A study of the transport and immobilisation mechanisms of human red blood cells in a paper-based blood typing device using confocal microscopy.

PubMed

Li, Lizi; Tian, Junfei; Ballerini, David; Li, Miaosi; Shen, Wei

2013-09-07

Recent research on the use of bioactive paper for human blood typing has led to the discovery of a new method for identifying the haemagglutination of red blood cells (RBCs). When a blood sample is introduced onto paper treated with the grouping antibodies, RBCs undergo haemagglutination with the corresponding grouping antibodies, forming agglutinated cell aggregates in the paper. A subsequent washing of the paper with saline buffer could not remove these aggregates from the paper; this phenomenon provides a new method for rapid, visual identification of the antibody-specific haemagglutination reactions and thus the determination of the blood type. This study aims to understand the mechanism of RBC immobilization inside the paper which follows haemagglutination reactions. Confocal microscopy is used to observe the morphology of the free and agglutinated RBCs that are labelled with FITC. Chromatographic elution patterns of both agglutinated and non-agglutinated RBCs are studied to gain insight into the transport behaviour of free RBCs and agglutinated aggregates. This work provides new information about RBC haemagglutination inside the fibre network of paper on a microscopic level, which is important for the future design of paper-based blood typing devices with high sensitivity and assaying speed.

Effects of an angelica extract on human erythrocyte aggregation, deformation and osmotic fragility.

PubMed

Wang, X; Wei, L; Ouyang, J P; Muller, S; Gentils, M; Cauchois, G; Stoltz, J F

2001-01-01

In Chinese traditional medicine, angelica is widely used for its known clinical effects of ameliorating blood microcirculation. But the mechanism of these beneficial effects still remains unclear. In this work the rheological behaviour of human erythrocytes treated by angelica was studied in vitro. Normal RBCs incubated with an angelica extract at different concentrations (5, 10 or 20 mg/ml) for 60 min at 37 degrees C and then their aggregation, deformation and osmotic fragility were measured with different recently developed optical techniques, namely Erythroaggregometer (Regulest, Florange, France), LORCA (Mechatronics, Amsterdam) and Fragilimeter (Regulest, Florange, France). Experimental results show that angelica (20 mg/ml) significantly decreased normal RBCs' aggregation speed (p<0.01) and could inhibit the hyperaggregability caused by dextran 500. However, the strength of normal RBCs aggregates were not influenced by angelica. When a calcium ionophore A23187 (1.9 microM) was used to harden cell membrane, angelica (20 mg/ml) could significantly (p<0.01) protect erythrocytes against the loss of their deformability even it had no effects on normal RBCs deformation. Finally angelica (5 and 10 mg/ml) decreased significantly (p<0.01) normal RBCs osmotic fragility. In conclusion angelica plays a rheologically active role on human erythrocytes, and this study suggests a possible mechanism for angelica's positive effects against certain cardiovascular diseases.

Phospholipid composition of packed red blood cells and that of extracellular vesicles show a high resemblance and stability during storage.

PubMed

Laurén, Eva; Tigistu-Sahle, Feven; Valkonen, Sami; Westberg, Melissa; Valkeajärvi, Anne; Eronen, Juha; Siljander, Pia; Pettilä, Ville; Käkelä, Reijo; Laitinen, Saara; Kerkelä, Erja

2018-01-01

Red blood cells (RBCs) are stored up to 35-42days at 2-6°C in blood banks. During storage, the RBC membrane is challenged by energy depletion, decreasing pH, altered cation homeostasis, and oxidative stress, leading to several biochemical and morphological changes in RBCs and to shedding of extracellular vesicles (EVs) into the storage medium. These changes are collectively known as RBC storage lesions. EVs accumulate in stored RBC concentrates and are, thus, transfused into patients. The potency of EVs as bioactive effectors is largely acknowledged, and EVs in RBC concentrates are suspected to mediate some adverse effects of transfusion. Several studies have shown accumulation of lipid raft-associated proteins in RBC EVs during storage, whereas a comprehensive phospholipidomic study on RBCs and corresponding EVs during the clinical storage period is lacking. Our mass spectrometric and chromatographic study shows that RBCs maintain their major phospholipid (PL) content well during storage despite abundant vesiculation. The phospholipidomes were largely similar between RBCs and EVs. No accumulation of raft lipids in EVs was seen, suggesting that the primary mechanism of RBC vesiculation during storage might not be raft -based. Nonetheless, a slight tendency of EV PLs for shorter acyl chains was observed. Copyright © 2017 Elsevier B.V. All rights reserved.

L-Asparaginase encapsulated intact erythrocytes for treatment of acute lymphoblastic leukemia (ALL).

PubMed

Kwon, Young Min; Chung, Hee Sun; Moon, Cheol; Yockman, James; Park, Yoon Jeong; Gitlin, Scott D; David, Allan E; Yang, Victor C

2009-11-03

As a primary drug for the treatment of acute lymphoblastic leukemia (ALL), encapsulation of L-asparaginase (ASNase) into red blood cells (RBC) has been popular to circumvent immunogenicity from the exogenous protein. Unlike existing methods that perturbs RBC membranes, we introduce a novel method of RBC-incorporation of proteins using the membrane-translocating low molecular weight protamine (LMWP). Confocal study of fluorescence-labeled LMWP-ovalbumin, as a model protein conjugate, has shown significant fluorescence inside RBCs. Surface morphology by scanning electron microscopy of the RBCs loaded with LMWP-ASNase was indistinguishable with normal RBCs. These drug loaded RBCs also closely resembled the profile of the native erythrocytes in terms of osmotic fragility, oxygen dissociation and hematological parameters. The in vivo half-life of enzyme activity after administering 8 units of RBC/LMWP-ASNase in DBA/2 mice was prolonged to 4.5+/-0.5 days whereas that of RBCs loaded with ASNase via a hypotonic method was 2.4+/-0.7 days. Furthermore, the mean survival time of DBA/2 mice bearing mouse lymphoma cell L5178Y was improved by approximately 44% compared to the saline control group after treatment with the RBC loaded enzymes. From these data, an innovative, novel method for encapsulating proteins into intact and fully functional erythrocytes was established for potential treatment of ALL.

Comparison of Voltage Gated K+ Currents in Arterial Myocytes with Heterologously Expressed K v Subunits.

PubMed

Cox, Robert H; Fromme, Samantha

2016-12-01

We have shown that three components contribute to functional voltage gated K + (K v ) currents in rat small mesenteric artery myocytes: (1) Kv1.2 plus Kv1.5 with Kvβ1.2 subunits, (2) Kv2.1 probably associated with Kv9.3 subunits, and (3) Kv7.4 subunits. To confirm and address subunit stoichiometry of the first two, we have compared the biophysical properties of K v currents in small mesenteric artery myocytes with those of K v subunits heterologously expressed in HEK293 cells using whole cell voltage clamp methods. Selective inhibitors of Kv1 (correolide, COR) and Kv2 (stromatoxin, ScTx) channels were used to separate these K v current components. Conductance-voltage and steady state inactivation data along with time constants of activation, inactivation, and deactivation of native K v components were generally well represented by those of Kv1.2-1.5-β1.2 and Kv2.1-9.3 channels. The slope of the steady state inactivation-voltage curve (availability slope) proved to be the most sensitive measure of accessory subunit presence. The availability slope curves exhibited a single peak for both native K v components. Availability slope curves for Kv1.2-1.5-β1.2 and Kv2.1-9.3 channels expressed in human embryonic kidney cells also exhibited a single peak that shifted to more depolarized voltages with increasing accessory to α subunit transfection ratio. Availability slope curves for SxTc-insensitive currents were similar to those of Kv1.2-1.5 expressed with Kvβ1.2 at a 1:5 molar ratio while curves for COR-insensitive currents closely resembled those of Kv2.1 expressed with Kv9.3 at a 1:1 molar ratio. These results support the suggested K v subunit combinations in small mesenteric artery, and further suggest that Kv1 α and Kvβ1.2 but not Kv2.1 and Kv9.3 subunits are present in a saturated (4:4) stoichiometry.

The morphological and chemical characteristics of striatal neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the rat.

PubMed

Waldvogel, H J; Kubota, Y; Trevallyan, S C; Kawaguchi, Y; Fritschy, J M; Mohler, H; Faull, R L

1997-10-01

The distribution, morphology and chemical characteristics of neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the striatum of the basal ganglia in the rat brain were investigated at the light, confocal and electron microscope levels using single, double and triple immunohistochemical labelling techniques. The results showed that alpha1-subunit immunoreactive neurons were sparsely distributed throughout the rat striatum. Double and triple labelling results showed that all the alpha1-subunit-immunoreactive neurons were positive for glutamate decarboxylase and immunoreactive for the beta2,3 and gamma2 subunits of the GABA(A) receptor. Three types of alpha1-subunit-immunoreactive neurons were identified in the striatum on the basis of cellular morphology and chemical characteristics. The most numerous alpha1-subunit-immunoreactive neurons were medium-sized, aspiny neurons with a widely branching dendritic tree. They were parvalbumin-negative and were located mainly in the dorsolateral regions of the striatum. Electron microscopy showed that these neurons had an indented nuclear membrane, typical of striatal interneurons, and were surrounded by small numbers of axon terminals which established alpha1-subunit-immunoreactive synaptic contacts with the soma and dendrites. These cells were classified as type 1 alpha1-subunit-immunoreactive neurons and comprised 75% of the total population of alpha1-subunit-immunoreactive neurons in the striatum. The remaining alpha1-subunit-immunoreactive neurons comprised of a heterogeneous population of large-sized neurons localized in the ventral and medial regions of the striatum. The most numerous large-sized cells were parvalbumin-negative, had two to three relatively short branching dendrites and were designated type 2 alpha1-subunit-immunoreactive neurons. Electron microscopy showed that the type 2 neurons were characterized by a highly convoluted nuclear membrane and were sparsely covered with small axon terminals. The type 2 neurons comprised 20% of the total population of alpha1-subunit-immunoreactive neurons. The remaining large-sized alpha1-immunoreactive cells were designated type 3 cells; they were positive for parvalbumin and were distinguished by long branching dendrites extending dorsally for 600-800 microm into the striatum. These neurons comprised 5% of the total population of alpha1-subunit-immunoreactive neurons and were surrounded by enkephalin-immunoreactive terminals. Electron microscopy showed that the alpha1-subunit type 3 neurons had an indented nuclear membrane and were densely covered with small axon terminals which established alpha1-subunit-immunoreactive symmetrical synaptic contacts with the soma and dendrites. These results provide a detailed characterization of the distribution, morphology and chemical characteristics of the alpha1-subunit-immunoreactive neurons in the rat striatum and suggest that the type 1 and type 2 neurons comprise of separate populations of striatal interneurons while the type 3 neurons may represent the large striatonigral projection neurons described by Bolam et al. [Bolam J. P., Somogyi P., Totterdell S. and Smith A. D. (1981) Neuroscience 6, 2141-2157.].

Successful Blood Transfusion Management of a Living Donor Liver Transplant Recipient in the Presence of Anti-Jra: A Case Report.

PubMed

Kurata, N; Onishi, Y; Kamei, H; Hori, T; Komagome, M; Kato, C; Matsushita, T; Ogura, Y

2017-09-01

A 48-year-old Japanese woman was diagnosed with Budd-Chiari syndrome and transferred for possible living donor liver transplantation (LDLT). Examinations before LDLT revealed that the recipient had anti-Jr a and preformed donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA). Rituximab was administrated at 16 days prior to the patient's scheduled LDLT for the prophylaxis of antibody-mediated rejection by DSA. The clinical significance of anti-Jr a has not been clearly established because of the rarity of this antibody, so we discussed blood transfusion strategy with the Department of Blood Transfusion Service and prepared for Jr a -negative packed red blood cells (RBCs). Intraoperative blood salvage was used during LDLT procedures to reduce the use of packed RBCs. Although post-transplantation graft function was excellent, a total of 44 U of Jr a -negative RBCs were transfused during the entire perioperative period. Because sufficient amounts of Jr a -negative packed RBCs were supplied, Jr a mismatched blood transfusion was avoided. The patient was discharged from our hospital on postoperative day 102 without clinical evidence of any blood transfusion-related adverse events. Although there are some controversies of blood transfusion related to anti-Jr a antibodies, the current strategies of blood transfusion for liver transplantation with anti-Jr a are as follows: (1) sufficient supply and transfusion of Jr a -negative matched packed RBCs and (2) application of intraoperative blood salvage to reduce the total amount of rare blood type RBCs. These strategies may be changed when the mechanism of anti-Jr a alloimmunization is fully understood in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

Life-threatening hemolytic anemia due to an autoanti-Pr cold agglutinin: evidence that glycophorin A antibodies may induce lipid bilayer exposure and cation permeability independent of agglutination.

PubMed

Brain, Michael C; Ruether, Bernard; Valentine, Karen; Brown, Christopher; ter Keurs, Henk

2010-02-01

The hemoglobin of a 29-year-old man fell below 35 g/L over 5 days, despite 14 units of red blood cells (RBCs), due to an anti-Pr cold agglutinin (CA). His hemolytic anemia necessitated respiratory support in intensive care for 4 weeks. The hemolysis was investigated by the effects on blood group-compatible RBCs of this anti-Pr and an anti-I CA and of a rabbit anti-human glycophorin A (GPA) immunoglobulin G (IgG) antibody on Ca(2+) permeability and of phosphatidylethanolamine (PE) exposure. 1) The anti-Pr CA (in a plasmapheresis product from the patient) was absorbed and eluted from RBC ghosts and its immunophenotype was determined by agarose electrophoresis and immunofixation. 2) Ca(2+) permeability was measured by the response of Fluo-3-labeled RBCs to addition of external Ca(2+). 3) Exposed PE was measured with streptavidin-labeled biotinylated peptide Ro 09-0198 (cinnamycin). 1) The patient's anti-Pr CA was a polyclonal IgG. 2) The anti-Pr and the rabbit anti-human glycophorin IgG, but not an anti-I CA, rapidly increased Ca(2+)-dependent fluorescence upon addition of external Ca(2+) in a fraction (15%-25%) of RBCs that also became positive for cinnamycin. 3) Trypsin treatment of RBCs reduced the Ca(2+) influx due to the anti-Pr IgG, but neither trypsin nor neuraminidase changed the responses to the rabbit anti-human GPA IgG. The anti-Pr CA and rabbit anti-human GPA increased exposure of PE and increased membrane Ca(2+) permeability that may have caused hemolysis. The difference in the responses to these antibodies to enzyme treatment of RBCs suggests that they react with different epitopes on GPA.

Factor XIIIa-dependent retention of red blood cells in clots is mediated by fibrin α-chain crosslinking.

PubMed

Byrnes, James R; Duval, Cédric; Wang, Yiming; Hansen, Caroline E; Ahn, Byungwook; Mooberry, Micah J; Clark, Martha A; Johnsen, Jill M; Lord, Susan T; Lam, Wilbur A; Meijers, Joost C M; Ni, Heyu; Ariëns, Robert A S; Wolberg, Alisa S

2015-10-15

Factor XIII(a) [FXIII(a)] stabilizes clots and increases resistance to fibrinolysis and mechanical disruption. FXIIIa also mediates red blood cell (RBC) retention in contracting clots and determines venous thrombus size, suggesting FXIII(a) is a potential target for reducing thrombosis. However, the mechanism by which FXIIIa retains RBCs in clots is unknown. We determined the effect of FXIII(a) on human and murine clot weight and composition. Real-time microscopy revealed extensive RBC loss from clots formed in the absence of FXIIIa activity, and RBCs exhibited transient deformation as they exited the clots. Fibrin band-shift assays and flow cytometry did not reveal crosslinking of fibrin or FXIIIa substrates to RBCs, suggesting FXIIIa does not crosslink RBCs directly to the clot. RBCs were retained in clots from mice deficient in α2-antiplasmin, thrombin-activatable fibrinolysis inhibitor, or fibronectin, indicating RBC retention does not depend on these FXIIIa substrates. RBC retention in clots was positively correlated with fibrin network density; however, FXIIIa inhibition reduced RBC retention at all network densities. FXIIIa inhibition reduced RBC retention in clots formed with fibrinogen that lacks γ-chain crosslinking sites, but not in clots that lack α-chain crosslinking sites. Moreover, FXIIIa inhibitor concentrations that primarily block α-, but not γ-, chain crosslinking decreased RBC retention in clots. These data indicate FXIIIa-dependent retention of RBCs in clots is mediated by fibrin α-chain crosslinking. These findings expose a newly recognized, essential role for fibrin crosslinking during whole blood clot formation and consolidation and establish FXIIIa activity as a key determinant of thrombus composition and size. © 2015 by The American Society of Hematology.

Red blood cells serve as intravascular carriers of myeloperoxidase.

PubMed

Adam, Matti; Gajdova, Silvie; Kolarova, Hana; Kubala, Lukas; Lau, Denise; Geisler, Anne; Ravekes, Thorben; Rudolph, Volker; Tsao, Philip S; Blankenberg, Stefan; Baldus, Stephan; Klinke, Anna

2014-09-01

Myeloperoxidase (MPO) is a heme enzyme abundantly expressed in polymorphonuclear neutrophils. MPO is enzymatically capable of catalyzing the generation of reactive oxygen species (ROS) and the consumption of nitric oxide (NO). Thus MPO has both potent microbicidal and, upon binding to the vessel wall, pro-inflammatory properties. Interestingly, MPO - a highly cationic protein - has been shown to bind to both endothelial cells and leukocyte membranes. Given the anionic surface charge of red blood cells, we investigated binding of MPO to erythrocytes. Red blood cells (RBCs) derived from patients with elevated MPO plasma levels showed significantly higher amounts of MPO by flow cytometry and ELISA than healthy controls. Heparin-induced MPO-release from patient-derived RBCs was significantly increased compared to controls. Ex vivo experiments revealed dose and time dependency for MPO-RBC binding, and immunofluorescence staining as well as confocal microscopy localized MPO-RBC interaction to the erythrocyte plasma membrane. NO-consumption by RBC-membrane fragments (erythrocyte "ghosts") increased with incrementally greater concentrations of MPO during incubation, indicating preserved catalytic MPO activity. In vivo infusion of MPO-loaded RBCs into C57BL/6J mice increased local MPO tissue concentrations in liver, spleen, lung, and heart tissue as well as within the cardiac vasculature. Further, NO-dependent relaxation of aortic rings was altered by RBC bound-MPO and systemic vascular resistance significantly increased after infusion of MPO-loaded RBCs into mice. In summary, we find that MPO binds to RBC membranes in vitro and in vivo, is transported by RBCs to remote sites in mice, and affects endothelial function as well as systemic vascular resistance. RBCs may avidly bind circulating MPO, and act as carriers of this leukocyte-derived enzyme. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effect of Thickness on Light Transmission and Vickers Hardness of Five Bulk-fill Resin-based Composites Using Polywave and Single-peak Light-emitting Diode Curing Lights.

PubMed

Maghaireh, G A; Price, R B; Abdo, N; Taha, N A; Alzraikat, H

2018-06-28

This study compared light transmission through different thicknesses of bulk-fill resin-based composites (RBCs) using a polywave and a single-peak light-emitting diode light-curing unit (LCU). The effect on the surface hardness was also evaluated. Five bulk-fill RBCs were tested. Specimens (n=5) of 1-, 2-, 4-, or 6-mm thickness were photopolymerized for 10 seconds from the top using a polywave (Bluephase Style) or single-peak (Elipar S10) LCU, while a spectrophotometer monitored in real time the transmitted irradiance and radiant exposure reaching the bottom of the specimen. After 24 hours of storage in distilled water at 37°C, the Vickers microhardness (VH) was measured at top and bottom. Results were analyzed using multiple-way analysis of variance, Tukey post hoc tests, and multivariate analysis (α=0.05). The choice of LCU had no significant effect on the total amount of light transmitted through the five bulk-fill RBCs at each thickness. There was a significant decrease in the amount of light transmitted as the thickness increased for all RBCs tested with both LCUs ( p<0.001). Effect of LCU on VH was minimal (η p 2 =0.010). The 1-, 2-, and 4-mm-thick specimens of SDR, X-tra Fill, and Filtek Bulk Restorative achieved a VH bottom/top ratio of approximately 80% when either LCU was used. The total amount of light transmitted through the five bulk-fill RBCs was similar at the different thicknesses using either LCU. The polywave LCU used in this study did not enhance the polymerization of the tested bulk-fill RBCs when compared with the single-peak LCU.

Factor XIIIa-dependent retention of red blood cells in clots is mediated by fibrin α-chain crosslinking

PubMed Central

Byrnes, James R.; Duval, Cédric; Wang, Yiming; Hansen, Caroline E.; Ahn, Byungwook; Mooberry, Micah J.; Clark, Martha A.; Johnsen, Jill M.; Lord, Susan T.; Lam, Wilbur A.; Meijers, Joost C. M.; Ni, Heyu; Ariëns, Robert A. S.

2015-01-01

Factor XIII(a) [FXIII(a)] stabilizes clots and increases resistance to fibrinolysis and mechanical disruption. FXIIIa also mediates red blood cell (RBC) retention in contracting clots and determines venous thrombus size, suggesting FXIII(a) is a potential target for reducing thrombosis. However, the mechanism by which FXIIIa retains RBCs in clots is unknown. We determined the effect of FXIII(a) on human and murine clot weight and composition. Real-time microscopy revealed extensive RBC loss from clots formed in the absence of FXIIIa activity, and RBCs exhibited transient deformation as they exited the clots. Fibrin band-shift assays and flow cytometry did not reveal crosslinking of fibrin or FXIIIa substrates to RBCs, suggesting FXIIIa does not crosslink RBCs directly to the clot. RBCs were retained in clots from mice deficient in α2-antiplasmin, thrombin-activatable fibrinolysis inhibitor, or fibronectin, indicating RBC retention does not depend on these FXIIIa substrates. RBC retention in clots was positively correlated with fibrin network density; however, FXIIIa inhibition reduced RBC retention at all network densities. FXIIIa inhibition reduced RBC retention in clots formed with fibrinogen that lacks γ-chain crosslinking sites, but not in clots that lack α-chain crosslinking sites. Moreover, FXIIIa inhibitor concentrations that primarily block α-, but not γ-, chain crosslinking decreased RBC retention in clots. These data indicate FXIIIa-dependent retention of RBCs in clots is mediated by fibrin α-chain crosslinking. These findings expose a newly recognized, essential role for fibrin crosslinking during whole blood clot formation and consolidation and establish FXIIIa activity as a key determinant of thrombus composition and size. PMID:26324704

How to quantify microparticles in RBCs? A validated flow cytometry method allows the detection of an increase in microparticles during storage.

PubMed

Gamonet, Clémentine; Mourey, Guillaume; Aupet, Sophie; Biichle, Sabéha; Petitjean, Régis; Vidal, Chrystelle; Pugin, Aurore; Naegelen, Christian; Tiberghien, Pierre; Morel, Pascal; Angelot-Delettre, Fanny; Seilles, Estelle; Saas, Philippe; Bardiaux, Laurent; Garnache-Ottou, Francine

2017-03-01

The procoagulant and proinflammatory microparticles (MPs) released during storage of packed red blood cells (pRBCs) can potentially modify transfusion benefits. A robust method to quantify MPs in pRBCs is needed to evaluate their impact in clinical trials. The objective was to validate the preanalytic conditions required to prepare pRBC supernatant as well as a method to quantify and evaluate MP variations over 42 days of pRBC storage.A flow cytometry method with size-calibrated beads was developed and fully validated. Quantification of MPs in pRBCs (n = 109) was assessed during short-term (7 days) and long-term (42 days) storage at 4°C, during short-term storage (8 hours) at room temperature, and after 2 years frozen. Repeatability, reproducibility, and linearity of the quantification method were validated, and variations during conservation are presented. There was high variability in RBC (erythrocyte) MP (ERMP) and platelet MP (PMP) levels between RBC units, depending on the filter used for leukocyte reduction. During the 42 days of storage at 4°C, significant increases in ERMPs and PMPs occurred (from 58 to 138 ERMPs/µL from Day 2 to Day 42; p = 0.0002; and from 326 to 771 PMPs/µL from Day 2 to Day 42; p = 0.00026). We use a robust method to confirm that ERMPs and PMPs are present to various degrees in pRBCs and that storage for 42 days significantly increases their generation. This method is robust enough to allow MP quantification in pRBCs and is adapted to evaluate the clinical impact of transfused MPs in prospective clinical trials. © 2017 AABB.

Priming dose of phenylhydrazine protects against hemolytic and lethal effects of 2-butoxyethanol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Palkar, Prajakta S.; Philip, Binu K.; Reddy, Ramesh N.

2007-11-15

Protection against a high dose of a toxicant by prior exposure to another toxicant is called heteroprotection. Our objective was to establish a heteroprotection model in RBCs. Female Sprague Dawley rats treated with an LD90 dose of 2-butoxyethanol (BE, 1500 mg/kg in water, 5 ml/kg po) 14 days after priming with 0.9% NaCl suffered 90% mortality by 15 days, whereas all rats receiving the LD90 dose of BE 14 days after priming with phenylhydrazine (PHZ, 125 mg/kg in 0.9% NaCl, 3 ml/kg po) survived. Hematocrit decreased from normal 45% to 24% by day 3 after PHZ priming and improved thereafter.more » Increasing the time interval between the priming and LD90 dose to 21 days abolished the heteroprotection. RBCs obtained on days 7 and 14 after PHZ priming unlike those on day 21 were resilient to the hemotoxic metabolite of BE, butoxyacetic acid (BAA). Unaltered hepatic alcohol and aldehyde dehydrogenase activities upon PHZ priming suggested that bioactivation of BE to BAA was unaffected. Lower renal (6 and 12 h) and hepatic (12 h) BAA levels and 3 fold higher excretion of BAA in PHZ-primed rat urine suggested a protective role of toxicokinetics. Higher erythropoietin, reticulocytes, and resiliency of PHZ-primed rat RBCs indicated that newly formed RBCs are resilient to hemolytic BAA. The antioxidant levels in the PHZ-primed rat RBCs did not indicate a protective role in heteroprotection. In conclusion, the resistance of PHZ-primed rats against BE-induced hemotoxicity and lethality is mediated by a combination of altered toxicokinetics, robust erythropoiesis, and resiliency of new RBCs.« less

Dynamical clustering of red blood cells in capillary vessels.

PubMed

Boryczko, Krzysztof; Dzwinel, Witold; Yuen, David A

2003-02-01

We have modeled the dynamics of a 3-D system consisting of red blood cells (RBCs), plasma and capillary walls using a discrete-particle approach. The blood cells and capillary walls are composed of a mesh of particles interacting with harmonic forces between nearest neighbors. We employ classical mechanics to mimic the elastic properties of RBCs with a biconcave disk composed of a mesh of spring-like particles. The fluid particle method allows for modeling the plasma as a particle ensemble, where each particle represents a collective unit of fluid, which is defined by its mass, moment of inertia, translational and angular momenta. Realistic behavior of blood cells is modeled by considering RBCs and plasma flowing through capillaries of various shapes. Three types of vessels are employed: a pipe with a choking point, a curved vessel and bifurcating capillaries. There is a strong tendency to produce RBC clusters in capillaries. The choking points and other irregularities in geometry influence both the flow and RBC shapes, considerably increasing the clotting effect. We also discuss other clotting factors coming from the physical properties of blood, such as the viscosity of the plasma and the elasticity of the RBCs. Modeling has been carried out with adequate resolution by using 1 to 10 million particles. Discrete particle simulations open a new pathway for modeling the dynamics of complex, viscoelastic fluids at the microscale, where both liquid and solid phases are treated with discrete particles. Figure A snapshot from fluid particle simulation of RBCs flowing along a curved capillary. The red color corresponds to the highest velocity. We can observe aggregation of RBCs at places with the most stagnant plasma flow.

Posttransfusion survival (24-hour) and hemolysis of previously frozen, deglycerolized RBCs after storage at 4 degrees C for up to 14 days in sodium chloride alone or sodium chloride supplemented with additive solutions.

PubMed

Valeri, C R; Pivacek, L E; Cassidy, G P; Ragno, G

2000-11-01

Previously frozen human RBCs currently are glycerolized and deglycerolized by the use of open systems that limit storage of the deglycerolized RBCs at 4 degrees C to only 24 hours. Healthy male volunteers who met AABB requirements for blood donors (n = 38) were studied. A volume of 450 mL of blood was collected into CPDA-1. The RBC concentrates were stored at 4 degrees C for 3 to 6 days before being frozen with 40-percent (wt/vol) glycerol and stored at -80 degrees C. The RBCs were deglycerolized, resuspended in 0.9-percent sodium chloride and 0.2-percent glucose (SG) solution or SG solution supplemented with AS-1, AS-3, or AS-5, and stored in the resuspension medium at 4 degrees C for 14 days. The mean +/- SD freeze-thaw-wash process recovery was 90.0 +/- 4.0 percent for all 38 units. The mean 24-hour posttransfusion survival value was 79 percent for deglycerolized RBC stored at 4 degrees C for 7 days in SG alone, SG plus AS-3, or SG plus AS-5. Deglycerolized RBC that were stored at 4 C for 14 days in SG supplemented with AS-1, AS-3, or AS-5 had a mean 24-hour posttransfusion survival of 74 percent. After 7 days of storage of deglycerolized RBCs in SG alone, the mean hemolysis was 3. 7 percent. After 14 days of storage of deglycerolized RBCs in SG supplemented with AS-1, AS-3, or AS-5, the mean hemolysis was 2.5 percent. The levels of hemolysis did not correlate with the 24-hour posttransfusion survival values.

The survival, function, and hemolysis of human RBCs stored at 4 degrees C in additive solution (AS-1, AS-3, or AS-5) for 42 days and then biochemically modified, frozen, thawed, washed, and stored at 4 degrees C in sodium chloride and glucose solution for 24 hours.

PubMed

Valeri, C R; Pivacek, L E; Cassidy, G P; Ragno, G

2000-11-01

A study was done to assess the quality of RBCs stored at 4 degrees C in AS-1, AS-3, or AS-5 for 42 days before biochemical modification and freezing. RBCs were stored at 4 degrees C for 42 days in AS-1, AS-3, or AS-5 and then biochemically modified with pyruvate, inosine, phosphate, and adenine solution (Rejuvesol), frozen with 40-percent (wt/vol) glycerol, and stored at -80 degrees C for at least 2 months. The RBCs were deglycerolized by the use of a cell washer (Haemonetics 115), and stored for 24 hours at 4 degrees C in a 0.9-percent sodium chloride and 0.2-percent glucose solution before the autologous transfusion. The mean freeze-thaw-wash recovery process produced RBC recovery values of 85 percent, with the mean 24-hour posttransfusion survival at 75 percent, and the mean index of therapeutic effectiveness at 64 percent for the RBCs stored at 4 degrees C in AS-1, AS-3, or AS-5 for 42 days before biochemical modification and freezing. All the units exhibited normal or slightly higher than normal 2,3 DPG levels after deglycerolization and postwash storage at 4 degrees C for 24 hours. RBCs stored in AS-1, AS-3, or AS-5 at 4 degrees C for 42 days and then biochemically modified with pyruvate, inosine, phosphate, and adenine and glycerolized, frozen, washed, and stored at 4 degrees C for 24 hours before autologous transfusion had acceptable in vitro and in vivo measurements.

Coinfusion of dextrose-containing fluids and red blood cells does not adversely affect in vitro red blood cell quality.

PubMed

Keir, Amy K; Hansen, Adele L; Callum, Jeannie; Jankov, Robert P; Acker, Jason P

2014-08-01

Transfusion guidelines advise against coinfusing red blood cells (RBCs) with solutions other than 0.9% saline. We evaluated the impact of coinfusion with dextrose-containing fluids (DW) on markers of RBC quality. A pool-and-split design was used to allow conditions to be tested on each pool within 2 hours of irradiation. Three pools at each storage age (5, 14, and 21 days) were created for each phase. In Phase 1, samples were infused through a neonatal transfusion apparatus alone or with treatment solutions: D5W, D10W, D5W/0.2% saline, and 0.9% saline. In Phase 2, samples were incubated alone or in a 1:1 ratio with treatment solutions and tested after 5, 30, and 180 minutes. Hemolysis, supernatant potassium, RBC indices, morphology, and deformability were measured on all samples. In Phase 1, RBCs transfused alone through the apparatus had higher (p<0.01) hematocrit, total hemoglobin, and supernatant potassium compared to all other groups. No statistical differences were identified between groups for other measured variables. In Phase 2, mean corpuscular volume of all samples containing DW increased with incubation length and were higher (p<0.01) than RBCs incubated alone or with 0.9% saline after 30 and 180 minutes. RBCs incubated with D5W and D5W/0.2% saline had greater (p<0.05) hemolysis than RBCs alone after 180 minutes. In vitro characteristics of RBCs coinfused with 0.9% saline or D10W were not adversely impacted. When developing clinical studies in neonates, we recommend use of D10W and a transfusion apparatus that minimizes the contact volume of the coinfusate with the RBC. © 2014 AABB.

Impact on storage quality of red blood cells and platelets by ultrahigh-frequency radiofrequency identification tags.

PubMed

Wang, Quan-Li; Wang, Xiao-Wei; Zhuo, Hai-Long; Shao, Chun-Yan; Wang, Jie; Wang, Hai-Ping

2013-04-01

Compared to ISBT128 code labels, radiofrequency identification (RFID) tags have incomparable advantages and gradually applied in blood management system. However, there is no global standard for the uses of RFID frequency. Even though ISBT recommended high-frequency RFID with 13.56MHz, 820- to 960-MHz ultrahigh frequency (UHF) RFID technology in many ways has even more advantages. For this reason, we studied the effect of UHF RFID tags with 820- to 960-MHz exposure on storage quality of red blood cells (RBCs) and platelets (PLTs). Thirty units of collected and prepared suspended RBCs (sRBCs) and PLTs were divided into two bags, one each for the test and control groups. The sRBCs were stored in 4±2°C refrigerator and the PLTs in a 22±2°C rocking box. The test groups were exposed to RF reader continuously during storage. Sampling at different time points and biologic changes were tested. As the extension of storage and the pH and chlorine levels in the supernatant of sRBCs were reduced, free hemoglobin, potassium, and sodium increased, but were not significant between test and control groups (p>0.05). During the storage period, the pH levels, PLT count, and PLT aggregation rate were decreased in both test and control groups, but were not significant (p>0.05). When exposed to 820- to 960-MHz RF, the biologic and biochemical indexes are not found to be exacerbated during 35 days of storage for sRBCs and 5 days for PLTs, respectively. © 2012 American Association of Blood Banks.

Towards bedside washing of stored red blood cells: a prototype of a simple apparatus based on microscale sedimentation in normal gravity.

PubMed

Khanal, G; Huynh, R A; Torabian, K; Xia, H; Vörös, E; Shevkoplyas, S S

2018-01-01

Infusion of by-products of red blood cell (RBC) storage-induced degradation as well as of the residual plasma proteins and the anticoagulant-preservative solution contained in units of stored blood serve no therapeutic purpose and may be harmful to some patients. Here, we describe a prototype of a gravity-driven system for bedside washing of stored RBCs. Stored RBCs were diluted to 10% haematocrit (Hct) with normal saline, matching the conventional washing procedure. The dilute RBC suspensions were passed through a column of coiled tubing to allow RBC sedimentation in normal gravity, thus separating them from the washing solution. Washed RBCs were collected using bifurcations located along the tubing. Washing efficiency was quantified by measuring Hct, morphology, deformability, free haemoglobin and total-free protein. The gravity-driven washing system operating at 0·5 ml/min produced washed RBCs with final Hct of 36·7 ± 3·4% (32·3-41·2%, n = 10) and waste Hct of 3·4 ± 0·7% (2·4-4·3%, n = 10), while removing 80% of free haemoglobin and 90% of total-free protein. Washing improved the ability of stored RBCs to perfuse an artificial microvascular network by 20%. The efficiency of washing performed using the gravity-driven system was not significantly different than that of conventional centrifugation. This proof-of-concept study demonstrates the feasibility of washing stored RBCs using a simple, disposable system with efficiency comparable to that of conventional centrifugation, and thus represents a significant first step towards enabling low-cost washing of stored blood at bedside. © 2017 International Society of Blood Transfusion.

Ameliorating effects of fluorocarbon emulsion on sickle red blood cell-induced obstruction in an ex vivo vasculature.

PubMed

Kaul, D K; Liu, X; Nagel, R L

2001-11-15

In sickle cell (SS) vaso-occlusion, the culminating event is blockage of blood vessels by sickled red blood cells (SS RBCs). As shown in animal models, SS RBC-induced vaso-occlusion is often partial, allowing for a residual flow, hence oxygen delivery to partially occluded vessels could reduce vaso-occlusion. The efficacy of an oxygenated perflubron-based fluorocarbon emulsion (PFE) was tested for its anti-vaso-occlusive effects in the ex vivo mesocecum vasculature of the rat. Microvascular obstruction was induced by the infusion of deoxygenated SS RBCs into ex vivo preparations with or without pretreatment with platelet-activating factor (PAF). PAF induced enhanced SS RBC-endothelium interactions, leading to greater vaso-occlusion. Microvascular blockage resulted in increased peripheral resistance units (PRU). Deoxygenated SS RBCs caused a persistent 1.5-fold PRU increase in untreated preparations and approximately a 2-fold PRU increase in PAF-treated preparations. The greater PRU in PAF-treated preparations was caused by widespread adhesion and postcapillary blockage. Oxygenated PFE, but not deoxygenated PFE, resulted in PRU decreases to baseline values in both groups of experiments (with or without PAF). The PRU decrease caused by oxygenated PFE infusion was caused by unsickling of SS RBCs in partially occluded vessels, with no antiadhesive effect on already adherent SS RBCs as assessed by intravital microscopy. PFE had no effect on vascular tone. The efficacy of PFE appears to result from its greater capacity to dissolve oxygen (10-fold higher than plasma). The dislodgement of trapped SS RBCs and an increase in wall shear rates will help reverse the partial obstruction. Thus, oxygenated PFE is capable of reducing SS RBC-induced vaso-occlusion, and further development of this approach is advisable.

Additive effect of red blood cell rigidity and adherence to endothelial cells in inducing vascular resistance.

PubMed

Kaul, D K; Koshkaryev, A; Artmann, G; Barshtein, G; Yedgar, S

2008-10-01

To explore the contribution of red blood cell (RBC) deformability and interaction with endothelial cells (ECs) to circulatory disorders, these RBC properties were modified by treatment with hydrogen peroxide (H(2)O(2)), and their effects on vascular resistance were monitored following their infusion into rat mesocecum vasculature. Treatment with 0.5 mM H(2)O(2) increased RBC/EC adherence without significant alteration of RBC deformability. At 5.0 mM H(2)O(2), RBC deformability was considerably reduced, inducing a threefold increase in the number of undeformable cells, whereas RBC/EC adherence was not further affected by the increased H(2)O(2) concentration. This enabled the selective manipulation of RBC adherence and deformability and the testing of their differential effect on vascular resistance. Perfusion of RBCs with enhanced adherence and unchanged deformability (treatment with 0.5 mM H(2)O(2)) increased vascular resistance by about 35% compared with untreated control RBCs. Perfusion of 5.0 mM H(2)O(2)-treated RBCs, with reduced deformability (without additional increase of adherence), further increased vascular resistance by about 60% compared with untreated control RBCs. These results demonstrate the specific effects of elevated adherence and reduced deformability of oxidized RBCs on vascular resistance. These effects can be additive, depending on the oxidation conditions. The oxidation-induced changes applied in this study are moderate compared with those observed in RBCs in pathological states. Yet, they caused a considerable increase in vascular resistance, thus demonstrating the potency of RBC/EC adherence and RBC deformability in determining resistance to blood flow in vivo.

Predicting storage-dependent damage to red blood cells using nitrite oxidation kinetics, peroxiredoxin-2 oxidation, and hemoglobin and free heme measurements.

PubMed

Oh, Joo-Yeun; Stapley, Ryan; Harper, Victoria; Marques, Marisa B; Patel, Rakesh P

2015-12-01

Storage-dependent damage to red blood cells (RBCs) varies significantly. Identifying RBC units that will undergo higher levels of hemolysis during storage may allow for more efficient inventory management decision-making. Oxidative-stress mediates storage-dependent damage to RBCs and will depend on the oxidant:antioxidant balance. We reasoned that this balance or redox tone will serve as a determinant of how a given RBC unit stores and that its assessment in "young" RBCs will predict storage-dependent hemolysis. RBCs were sampled from bags and segments stored for 7 to 42 days. Redox tone was assessed by nitrite oxidation kinetics and peroxiredoxin-2 (Prx-2) oxidation. In parallel, hemolysis was assessed by measuring cell-free hemoglobin (Hb) and free heme (hemin). Correlation analyses were performed to determine if Day 7 measurements predicted either the level of hemolysis at Day 35 or the increase in hemolysis during storage. Higher Day 7 Prx-2 oxidation was associated with higher Day 35 Prx-2 oxidation, suggesting that early assessment of this variable may identify RBCs that will incur the most oxidative damage during storage. RBCs that oxidized nitrite faster on Day 7 were associated with the greatest levels of storage-dependent hemolysis and increases in Prx-2 oxidation. An inverse relationship between storage-dependent changes in oxyhemoglobin and free heme was observed underscoring an unappreciated reciprocity between these molecular species. Moreover, free heme was higher in the bag compared to paired segments, with opposite trends observed for free Hb. Measurement of Prx-2 oxidation and nitrite oxidation kinetics early during RBC storage may predict storage-dependent damage to RBC including hemolysis-dependent formation of free Hb and heme. © 2015 AABB.

DEL phenotype.

PubMed

Kwon, Dong H; Sandler, S G; Flegel, Willy A

2017-09-01

DEL red blood cells (RBCs) type as D- by routine serologic methods and are transfused routinely, without being identified as expressing a very weak D antigen, to D- recipients. DEL RBCs are detected only by adsorption and elution of anti-D or by molecular methods. Most DEL phenotypes have been reported in population studies conducted in East Asia, although DEL phenotypes have been detected also among Caucasian individuals. Approximately 98 percent of DEL phenotypes in East Asians are associated with the RHD*DEL1 or RHD*01EL.01 allele. The prevalence of DEL phenotypes has been reported among D- Han Chinese (30%), Japanese (28%), and Korean (17%) populations. The prevalence of DEL phenotypes is significantly lower among D- Caucasian populations (0.1%). Among the 3-5 percent of African individuals who are D-, there are no reports of the DEL phenotype. Case reports from East Asia indicate that transfusion of DEL RBCs to D- recipients has been associated with D alloimmunization. East Asian immigrants constitute 2.1 percent of the 318.9 million persons residing in the United States, and an estimated 2.8 percent are blood donors. Using these statistics, we estimate that 68-683 units of DEL RBCs from donors of East Asian ancestry are transfused as D- annually in the United States. Given the reports from East Asia of D alloimmunization attributed to transfusion of DEL RBCs, one would expect an occasional report of D alloimmunization in the United States following transfusion of DEL RBCs to a D- recipient. If such cases do occur, the most likely reason that they are not detected is the absence of active post-transfusion monitoring for formation of anti-D.

Prolonged maintenance of 2,3-diphosphoglycerate acid and adenosine triphosphate in red blood cells during storage.

PubMed

de Korte, Dirk; Kleine, Mya; Korsten, Herbert G H; Verhoeven, Arthur J

2008-06-01

Current additive solutions (ASs) for red cells (RBCs) do not maintain a constant level of critical metabolites such as adenosine triphosphate (ATP) and 2,3-diphosphoglycerate acid (2,3-DPG) during cold storage. From the literature it is known that the intracellular pH is an important determinant of RBC metabolism. Therefore, a new, alkaline, AS was developed with the aim to allow cold storage of RBCs with stable product characteristics. Whole blood-derived RBCs (leukoreduced) were resuspended in experimental medium phosphate-adenine-guanosine-glucose-gluconate-mannitol (PAGGG-M; pH 8.2) with and without washing in the same medium. During cold storage several in vitro variables, such as intracellular pH, 2,3-DPG, ATP, and hemolysis, were analyzed. During cold storage, RBCs resuspended in PAGGG-M showed a constant ATP level (approx. 6 mumol/g Hb) and a very limited hemolysis (<0.2%). The 2,3-DPG content showed an increase until Day 21 (150% of initial level), followed by a slow decrease, with at Day 35 still 100 percent of the initial level. RBCs washed in PAGGG-M even showed a continuous increase of 2,3-DPG during 35 days, with a maximum level of 200 percent of the initial value. The effect of PAGGG-M appears to be related to long-lasting effects of the initial intracellular pH shortly after production. Resuspension of RBCs in our alkaline medium PAGGG-M resulted in a RBC unit of high quality during storage for up to at least 35 days, with 2,3-DPG levels of higher than 10 mumol per g Hb, hemolysis of less than 0.2 percent, and ATP levels of higher than 5 mumol per g Hb.

Rejuvenation capacity of red blood cells in additive solutions over long-term storage.

PubMed

Meyer, Erin K; Dumont, Deborah F; Baker, Sharry; Dumont, Larry J

2011-07-01

Red blood cells (RBCs) are Food and Drug Administration (FDA)-approved for 42-day storage with the use of additive solutions (ASs). However, adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) levels in the RBCs decline over this time. These constituents may be restored by treatment with rejuvenation (REJ) solutions. This study was done to assess the response capability of RBCs from 30 to 120 days of storage in three FDA-licensed RBC storage solutions after incubation with a rejuvenating solution of pyruvate, inosine, phosphate, and adenine. Three units each of RBCs in approved AS (AS-1 [Adsol, Fenwal, Inc.], AS-3 [Nutricel, Medsep Corp.], and AS-5 [Optisol, Terumo Corp.]) were stored under standard conditions at 1 to 6°C for up to 120 days. Aliquots (4 mL) on Days 30, 42, 60, 80, 100, and 120 (± 2 days) were REJ by incubating with Rejuvesol (Encyte Corp.). Control untreated and REJ aliquots were extracted using perchloric acid and stored at -80°C until assayed for 2,3-DPG and ATP. RBCs responded to REJ by increasing DPG and ATP contents. The response declined linearly at 0.070 ± 0.008 µmol DPG/g hemoglobin (Hb)/day and 0.035 ± 0.004 µmol ATP/g Hb/day with no differences between ASs. We conclude that Rejuvesol is able to restore ATP and 2,3-DPG levels in RBCs stored up to 120 days in AS. The response diminishes as storage time increases. This rejuvenation (REJ) capability does not seem useful for routine assessment of RBC anabolic capacity in research programs, but may be useful to the investigator when studying unique and novel treatment methods. © 2011 American Association of Blood Banks.

Tangential Flow Filtration of Hemoglobin

PubMed Central

Sun, Guoyong; Harris, David R.

2009-01-01

Bovine and human hemoglobin (bHb and hHb, respectively) was purified from bovine and human red blood cells (bRBCs and hRBCs, respectively) via tangential flow filtration (TFF) in four successive stages. TFF is a fast and simple method to purify Hb from RBCs using filtration through hollow fiber (HF) membranes. Most of the Hb was retained in stage III (100 kDa HF membrane) and displayed methemoglobin levels less than 1%, yielding final concentrations of 318 and 300 mg/mL for bHb and hHb, respectively. Purified Hb exhibited much lower endotoxin levels than their respective RBCs. The purity of Hb was initially assessed via SDS-PAGE, and showed tiny impurity bands for the stage III retentate. The oxygen affinity (P50), and cooperativity coefficient (n) were regressed from the measured oxygen-RBC/Hb equilibrium curves of RBCs and purified Hb. These results suggest that TFF yielded oxygen affinities of bHb and hHb that are comparable to values in the literature. LC-MS was used to measure the molecular weight of the alpha (α) and beta (β) globin chains of purified Hb. No impurity peaks were present in the HPLC chromatograms of purified Hb. The mass of the molecular ions corresponding to the α and β globin chains agreed well with the calculated theoretical mass of the α-and β-globin chains. Taken together, our results demonstrate that HPLC grade Hb can be generated via TFF. In general, this method can be more broadly applied to purify Hb from any source of RBCs. This work is significant, since it outlines a simple method for generating Hb for synthesis and/or formulation of Hb-based oxygen carriers (HBOCs). PMID:19224583

Influence of red blood cell-derived microparticles upon vasoregulation

PubMed Central

Said, Ahmed S.; Doctor, Allan

2017-01-01

Here we review recent data and the evolving understanding of the role of red blood cell-derived microparticles (RMPs) in normal physiology and in disease progression. Microparticles (MPs) are small membrane vesicles derived from various parent cell types. MPs are produced in response to a variety of stimuli through several cytoskeletal and membrane phospholipid changes. MPs have been investigated as potential biomarkers for multiple disease processes and are thought to have biological effects, most notably in: promotion of coagulation, production and handling of reactive oxygen species, immune modulation, angiogenesis, and in apoptosis. Specifically, RMPs are produced normally during RBC maturation and their production is accelerated during processing and storage for transfusion. Several factors during RBC storage are known to trigger RMP production, including: increased intracellular calcium, increased potassium leakage, and energy failure with ATP depletion. Of note, RMP composition differs from that of intact RBCs, and the nature and composition of RMP components are affected by both storage duration and the character of storage solutions. Recognised RMP bioactivities include: promotion of coagulation, immune modulation, and promotion of endothelial adhesion, as well as influence upon vasoregulation via nitric oxide (NO) scavenging. Of particular relevance, RMPs are more avid NO scavengers than intact RBCs and this feature has been proposed as a mechanism for the impaired oxygen delivery homeostasis that has been observed following transfusion. Preliminary human studies demonstrate that circulating RMP abundance increases with RBC transfusion and is associated with altered plasma vasoactivity and abnormal vasoregulation. In summary, RMPs are submicron particles released from stored RBCs, with demonstrated vasoactive properties that appear to disturb oxygen delivery homeostasis. The clinical impact of RMPs in transfusion recipients is an area of continued investigation. PMID:28686154

Influence of red blood cell-derived microparticles upon vasoregulation.

PubMed

Said, Ahmed S; Doctor, Allan

2017-10-01

Here we review recent data and the evolving understanding of the role of red blood cell-derived microparticles (RMPs) in normal physiology and in disease progression. Microparticles (MPs) are small membrane vesicles derived from various parent cell types. MPs are produced in response to a variety of stimuli through several cytoskeletal and membrane phospholipid changes. MPs have been investigated as potential biomarkers for multiple disease processes and are thought to have biological effects, most notably in: promotion of coagulation, production and handling of reactive oxygen species, immune modulation, angiogenesis, and in apoptosis. Specifically, RMPs are produced normally during RBC maturation and their production is accelerated during processing and storage for transfusion. Several factors during RBC storage are known to trigger RMP production, including: increased intracellular calcium, increased potassium leakage, and energy failure with ATP depletion. Of note, RMP composition differs from that of intact RBCs, and the nature and composition of RMP components are affected by both storage duration and the character of storage solutions. Recognised RMP bioactivities include: promotion of coagulation, immune modulation, and promotion of endothelial adhesion, as well as influence upon vasoregulation via nitric oxide (NO) scavenging. Of particular relevance, RMPs are more avid NO scavengers than intact RBCs and this feature has been proposed as a mechanism for the impaired oxygen delivery homeostasis that has been observed following transfusion. Preliminary human studies demonstrate that circulating RMP abundance increases with RBC transfusion and is associated with altered plasma vasoactivity and abnormal vasoregulation. In summary, RMPs are submicron particles released from stored RBCs, with demonstrated vasoactive properties that appear to disturb oxygen delivery homeostasis. The clinical impact of RMPs in transfusion recipients is an area of continued investigation.

COP9 signalosome subunit 7 from Arabidopsis interacts with and regulates the small subunit of ribonucleotide reductase (RNR2).

PubMed

Halimi, Yair; Dessau, Moshe; Pollak, Shaul; Ast, Tslil; Erez, Tamir; Livnat-Levanon, Nurit; Karniol, Baruch; Hirsch, Joel A; Chamovitz, Daniel A

2011-09-01

The COP9 Signalosome protein complex (CSN) is a pleiotropic regulator of plant development and contains eight-subunits. Six of these subunits contain the PCI motif which mediates specific protein interactions necessary for the integrity of the complex. COP9 complex subunit 7 (CSN7) contains an N-terminal PCI motif followed by a C-terminal extension which is also necessary for CSN function. A yeast-interaction trap assay identified the small subunit of ribonucelotide reductase (RNR2) from Arabidopsis as interacting with the C-terminal section of CSN7. This interaction was confirmed in planta by both bimolecular fluorescence complementation and immuoprecipitation assays with endogenous proteins. The subcellular localization of RNR2 was primarily nuclear in meristematic regions, and cytoplasmic in adult cells. RNR2 was constitutively nuclear in csn7 mutant seedlings, and was also primarily nuclear in wild type seedlings following exposure to UV-C. These two results correlate with constitutive expression of several DNA-damage response genes in csn7 mutants, and to increased tolerance of csn7 seedlings to UV-C treatment. We propose that the CSN is a negative regulator of RNR activity in Arabidopsis.

Glucose-6-Phosphate Dehydrogenase-Deficiency in Transfusion Medicine: The Unknown Risks

PubMed Central

Francis, Richard O.; Jhang, Jeffrey S.; Pham, Huy P.; Hod, Eldad A.; Zimring, James C.; Spitalnik, Steven L.

2013-01-01

The hallmark of glucose-6-phosphate dehydrogenase (G6PD) deficiency is red blood cell (RBC) destruction in response to oxidative stress. Patients requiring RBC transfusions may simultaneously receive oxidative medications or have concurrent infections, both of which can induce hemolysis in G6PD-deficient RBCs. Although it is not routine practice to screen healthy blood donors for G6PD deficiency, case reports identified transfusion of G6PD-deficient RBCs as causing hemolysis and other adverse events. In addition, some patient populations may be more at risk for complications associated with transfusions of G6PD-deficient RBCs because they receive RBCs from donors who are more likely to have G6PD deficiency. This review discusses G6PD deficiency, its importance in transfusion medicine, changes in the RBC antioxidant system (of which G6PD is essential) during refrigerated storage, and mechanisms of hemolysis. In addition, as yet unanswered questions that could be addressed by translational and clinical studies are identified and discussed. PMID:23815264

Cell-penetrating peptides meditated encapsulation of protein therapeutics into intact red blood cells and its application.

PubMed

He, Huining; Ye, Junxiao; Wang, Yinsong; Liu, Quan; Chung, Hee Sun; Kwon, Young Min; Shin, Meong Cheol; Lee, Kyuri; Yang, Victor C

2014-02-28

Red blood cells (RBCs) based drug carrier appears to be the most appealing for protein drugs due to their unmatched biocompatability, biodegradability, and long lifespan in the circulation. Numerous methods for encapsulating protein drugs into RBCs were developed, however, most of them induce partial disruption of the cell membrane, resulting in irreversible alterations in both physical and chemical properties of RBCs. Herein, we introduce a novel method for encapsulating proteins into intact RBCs, which was meditated by a cell penetrating peptide (CPP) developed in our lab-low molecular weight protamine (LMWP). l-asparaginase, one of the primary drugs used in treatment of acute lymphoblastic leukemia (ALL), was chosen as a model protein to illustrate the encapsulation into erythrocytes mediated by CPPs. In addition current treatment of ALL using different l-asparaginase delivery and encapsulation methods as well as their associated problems were also reviewed. Copyright © 2013 Elsevier B.V. All rights reserved.

COMPETITION FOR RED BLOOD CELLS CAN ENHANCE PLASMODIUM VIVAX PARASITEMIA IN MIXED-SPECIES MALARIA INFECTIONS

PubMed Central

MCQUEEN, PHILIP G.; MCKENZIE, F. ELLIS

2008-01-01

We assess the consequences of competition for red blood cells (RBCs) in co-infections with the two major agents of human malaria, Plasmodium vivax and Plasmodium falciparum, using differential equations to model the population dynamics of RBCs and parasites. P. vivax parasitizes only the youngest RBCs, but this can reduce the broader RBC population susceptible to P. falciparum. We found that competition for RBCs typically causes one species to suppress the other, depending on their relative reproduction rates and timing of inoculation. However, if the species’ reproduction rates are nearly equal, transient increases in RBC production stimulated by the presence of P. falciparum may boost P. vivax parasitemia above its single-species infection level. Conversely, P. falciparum parasitemia is rarely enhanced above its single-species level. Furthermore, transients in RBC production can induce coupled oscillations in the parasitemia of both species. These results are remarkably robust to changes in model parameters. PMID:16837717

Patient-specific modeling and analysis of dynamic behavior of individual sickle red blood cells under hypoxic conditions

NASA Astrophysics Data System (ADS)

Li, Xuejin; Du, E.; Li, Zhen; Tang, Yu-Hang; Lu, Lu; Dao, Ming; Karniadakis, George

2015-11-01

Sickle cell anemia is an inherited blood disorder exhibiting heterogeneous morphology and abnormal dynamics under hypoxic conditions. We developed a time-dependent cell model that is able to simulate the dynamic processes of repeated sickling and unsickling of red blood cells (RBCs) under physiological conditions. By using the kinetic cell model with parameters derived from patient-specific data, we present a mesoscopic computational study of the dynamic behavior of individual sickle RBCs flowing in a microfluidic channel with multiple microgates. We investigate how individual sickle RBCs behave differently from healthy ones in channel flow, and analyze the alteration of cellular behavior and response to single-cell capillary obstruction induced by cell rheologic rigidification and morphological change due to cell sickling under hypoxic conditions. We also simulate the flow dynamics of sickle RBCs treated with hydroxyurea (HU) and quantify the relative enhancement of hemodynamic performance of HU. This work was supported by the National Institutes of Health (NIH) Grant U01HL114476.

PTP-ε HAS A CRITICAL ROLE IN SIGNALING TRANSDUCTION PATHWAYS AND PHOSPHOPROTEIN NETWORK TOPOLOGY IN RED CELLS

PubMed Central

De Franceschi, Lucia; Biondani, Andrea; Carta, Franco; Turrini, Franco; Laudanna, Carlo; Deana, Renzo; Brunati, Anna Maria; Turretta, Loris; Iolascon, Achille; Perrotta, Silverio; Elson, Ari; Bulato, Cristina; Brugnara, Carlo

2010-01-01

Protein tyrosine phosphatases (PTPs) are crucial components of cellular signal transduction pathways. We report here that red blood cells (RBCs) from mice lacking PTPε (Ptpre−/−) exhibit abnormal morphology and increased Ca2+-activated-K+ channel activity, which was partially blocked by the Src-Family-Kinases (SFKs) inhibitor PP1. In Ptpre−/− mouse RBCs, the activity of Fyn and Yes, two SFKs, were increased, suggesting a functional relationship between SFKs, PTPε and Ca2+-activated-K+-channel. The absence of PTPε markedly affected the RBC membrane tyrosine (Tyr-) phosphoproteome, indicating a perturbation of RBCs signal transduction pathways. Using signaling network computational analysis of the Tyr-phosphoproteomic data, we identified 7 topological clusters. We studied cluster 1, containing Syk-Tyr-kinase: Syk-kinase activity was higher in wild-type than in Ptpre−/− RBCs, validating the network computational analysis and indicating a novel signaling pathway, which involves Fyn and Syk in regulation of red cell morphology. PMID:18924107

Piezo1 links mechanical forces to red blood cell volume.

PubMed

Cahalan, Stuart M; Lukacs, Viktor; Ranade, Sanjeev S; Chien, Shu; Bandell, Michael; Patapoutian, Ardem

2015-05-22

Red blood cells (RBCs) experience significant mechanical forces while recirculating, but the consequences of these forces are not fully understood. Recent work has shown that gain-of-function mutations in mechanically activated Piezo1 cation channels are associated with the dehydrating RBC disease xerocytosis, implicating a role of mechanotransduction in RBC volume regulation. However, the mechanisms by which these mutations result in RBC dehydration are unknown. In this study, we show that RBCs exhibit robust calcium entry in response to mechanical stretch and that this entry is dependent on Piezo1 expression. Furthermore, RBCs from blood-cell-specific Piezo1 conditional knockout mice are overhydrated and exhibit increased fragility both in vitro and in vivo. Finally, we show that Yoda1, a chemical activator of Piezo1, causes calcium influx and subsequent dehydration of RBCs via downstream activation of the KCa3.1 Gardos channel, directly implicating Piezo1 signaling in RBC volume control. Therefore, mechanically activated Piezo1 plays an essential role in RBC volume homeostasis.

The Rheopathobiology of Plasmodium vivax and Other Important Primate Malaria Parasites.

PubMed

Russell, Bruce M; Cooke, Brian M

2017-04-01

Our current understanding of how malaria parasites remodel their host red blood cells (RBCs) and ultimately cause disease is largely based on studies of Plasmodium falciparum. In this review, we expand our knowledge to include what is currently known about pathophysiological changes to RBCs that are infected by non-falciparum malaria parasites. We highlight the potential folly of making generalizations about the rheology of malaria infection, and emphasize the need for more systematic studies into the erythrocytic biology of non-falciparum malaria parasites. We propose that a better understanding of the mechanisms that underlie the changes to RBCs induced by malaria parasites other than P. falciparum may be highly informative for the development of therapeutics that specifically disrupt the altered rheological profile of RBCs infected with either sexual- or asexual-stage parasites, resulting in drugs that block transmission, reduce disease severity, and help delay the onset of resistance to current and future anti-malaria drugs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structure-function of proteins interacting with the α1 pore-forming subunit of high-voltage-activated calcium channels

PubMed Central

Neely, Alan; Hidalgo, Patricia

2014-01-01

Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1) associated with four additional polypeptide chains β, α2, δ, and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels. PMID:24917826

Static and dynamic light scattering of healthy and malaria-parasite invaded red blood cells

NASA Astrophysics Data System (ADS)

Park, Yongkeun; Diez-Silva, Monica; Fu, Dan; Popescu, Gabriel; Choi, Wonshik; Barman, Ishan; Suresh, Subra; Feld, Michael S.

2010-03-01

We present the light scattering of individual Plasmodium falciparum-parasitized human red blood cells (Pf-RBCs), and demonstrate progressive alterations to the scattering signal arising from the development of malaria-inducing parasites. By selectively imaging the electric fields using quantitative phase microscopy and a Fourier transform light scattering technique, we calculate the light scattering maps of individual Pf-RBCs. We show that the onset and progression of pathological states of the Pf-RBCs can be clearly identified by the static scattering maps. Progressive changes to the biophysical properties of the Pf-RBC membrane are captured from dynamic light scattering.

Cryopreservation of red blood cells.

PubMed

Lagerberg, Johan W

2015-01-01

Cryopreservation of red blood cell concentrates (RBCs) is an important method for maintaining an inventory of rare RBC units and managing special transfusion circumstances. The permeating additive glycerol is used as a cryoprotectant to protect RBCs against freezing damage. The use of thawed RBCs was hampered a 24-h outdating period due to potential bacterial contamination when a functionally open system was used for addition and removal of the glycerol. With the introduction of a functionally closed system for the glycerolization and deglycerolization of RBC units, extended post-thaw storage became possible. Here, we describe the cryopreservation of red blood cells according to the high-glycerol method, using a functionally closed processing system.

The efficacy of methylene blue encapsulated in silica nanoparticles compared to naked methylene blue for photodynamic applications.

PubMed

Makhadmeh, Ghaseb Naser; Abdul Aziz, Azlan; Abdul Razak, Khairunisak

2016-05-01

This study analyzed the physical effects of methylene blue (MB) encapsulated within silica nanoparticles (SiNPs) in photodynamic therapy. The optimum concentration of MB needed to destroy red blood cells (RBCs) was determined, and the efficacy of encapsulated MB-SiNPs compared to that of naked MB was verified. The results confirmed the applicability of MB encapsulated in SiNPs on RBCs, and established a relationship between the concentration of the SiNP-encapsulated MB and the time required to rupture 50% of the RBCs (t50). The MB encapsulated in SiNPs exhibited higher efficacy compared to that of naked MB.

Further evaluation of the NWF filter for the purification of Plasmodium vivax-infected erythrocytes.

PubMed

Li, Jiangyan; Tao, Zhiyong; Li, Qian; Brashear, Awtum; Wang, Ying; Xia, Hui; Fang, Qiang; Cui, Liwang

2017-05-17

Isolation of Plasmodium-infected red blood cells (iRBCs) from clinical blood samples is often required for experiments, such as ex vivo drug assays, in vitro invasion assays and genome sequencing. Current methods for removing white blood cells (WBCs) from malaria-infected blood are time-consuming or costly. A prototype non-woven fabric (NWF) filter was developed for the purification of iRBCs, which showed great efficiency for removing WBCs in a pilot study. Previous work was performed with prototype filters optimized for processing 5-10 mL of blood. With the commercialization of the filters, this study aims to evaluate the efficiency and suitability of the commercial NWF filter for the purification of Plasmodium vivax-infected RBCs in smaller volumes of blood and to compare its performance with that of Plasmodipur ® filters. Forty-three clinical P. vivax blood samples taken from symptomatic patients attending malaria clinics at the China-Myanmar border were processed using the NWF filters in a nearby field laboratory. The numbers of WBCs and iRBCs and morphology of P. vivax parasites in the blood samples before and after NWF filtration were compared. The viability of P. vivax parasites after filtration from 27 blood samples was examined by in vitro short-term culture. In addition, the effectiveness of the NWF filter for removing WBCs was compared with that of the Plasmodipur ® filter in six P. vivax blood samples. Filtration of 1-2 mL of P. vivax-infected blood with the NWF filter removed 99.68% WBCs. The densities of total iRBCs, ring and trophozoite stages before and after filtration were not significantly different (P > 0.05). However, the recovery rates of schizont- and gametocyte-infected RBCs, which were minor parasite stages in the clinical samples, were relatively low. After filtration, the P. vivax parasites did not show apparent morphological changes. Culture of 27 P. vivax-infected blood samples after filtration showed that parasites successfully matured into the schizont stage. The WBC removal rates and iRBC recovery rates were not significantly different between the NWF and Plasmodipur ® filters (P > 0.05). When tested with 1-2 mL of P. vivax-infected blood, the NWF filter could effectively remove WBCs and the recovery rates for ring- and trophozoite-iRBCs were high. P. vivax parasites after filtration could be successfully cultured in vitro to reach maturity. The performance of the NWF and Plasmodipur ® filters for removing WBCs and recovering iRBCs was comparable.

Defected red blood cell membranes and direct correlation with the uraemic milieu: the connection with the decreased red blood cell lifespan observed in haemodialysis patients

NASA Astrophysics Data System (ADS)

Stamopoulos, D.; Grapsa, E.; Manios, E.; Gogola, V.; Bakirtzi, N.

2012-12-01

Together with impaired production of erythropoietin and iron deficiency, the decreased lifespan of red blood cells (RBCs) is a main factor contributing to the chronic anaemia observed in haemodialysis (HD) patients. Atomic force microscopy is employed in this work to thoroughly survey the membrane of intact RBCs (iRBCs) of HD patients in comparison to those of healthy donors, aiming to obtain direct information on the structural status of RBCs that can be related to their decreased lifespan. We observed that the iRBC membrane of the HD patients is overpopulated with extended circular defects, termed ‘orifices’, that have typical dimension ranging between 0.2 and 1.0 μm. The ‘orifice’ index—that is, the mean population of ‘orifices’ per top membrane surface—exhibits a pronounced relative increase of order 54 ± 12% for the HD patients as compared to healthy donors. Interestingly, for the HD patients, the ‘orifice’ index, which relates to the structural status of the RBC membrane, correlates strongly with urea concentration, which is a basic index of the uraemic milieu. Thus, these results indicate that the uraemic milieu downgrades the structural status of the RBC membrane, possibly triggering biochemical processes that result in their premature elimination from the circulation. This process could decrease the lifespan of RBCs, as observed in HD patients.

Mannose and fructose metabolism in red blood cells during cold storage in SAGM.

PubMed

Rolfsson, Óttar; Johannsson, Freyr; Magnusdottir, Manuela; Paglia, Giuseppe; Sigurjonsson, Ólafur E; Bordbar, Aarash; Palsson, Sirus; Brynjólfsson, Sigurður; Guðmundsson, Sveinn; Palsson, Bernhard

2017-11-01

Alternate sugar metabolism during red blood cell (RBC) storage is not well understood. Here we report fructose and mannose metabolism in RBCs during cold storage in SAGM and the impact that these monosaccharides have on metabolic biomarkers of RBC storage lesion. RBCs were stored in SAGM containing uniformly labeled 13 C-fructose or 13 C-mannose at 9 or 18 mmol/L concentration for 25 days. RBCs and media were sampled at 14 time points during storage and analyzed using ultraperformance liquid chromatography-mass spectrometry. Blood banking quality assurance measurements were performed. Red blood cells incorporated fructose and mannose during cold storage in the presence of glucose. Mannose was metabolized in preference to glucose via glycolysis. Fructose lowered adenosine triphosphate (ATP) levels and contributed little to ATP maintenance when added to SAGM. Both monosaccharides form the advanced glycation end product glycerate. Mannose activates enzymes in the RBC that take part in glycan synthesis. Fructose or mannose addition to RBC SAGM concentrates may not offset the shift in metabolism of RBCs that occurs after 10 days of storage. Fructose and mannose metabolism at 4°C in SAGM reflects their metabolism at physiologic temperature. Glycerate excretion is a measure of protein deglycosylation activity in stored RBCs. No cytoprotective effect was observed upon the addition of either fructose or mannose to SAGM. © 2017 AABB.

Erythroid pyrimidine 5'-nucleotidase: cloning, developmental expression, and regulation by cAMP and in vivo hypoxia.

PubMed

Mass, Markus; Simo, Erika; Dragon, Stefanie

2003-12-01

A characteristic process of terminal erythroid differentiation is the degradation of ribosomal RNA into mononucleotides. The pyrimidine mononucleotides can be dephosphorylated by pyrimidine 5'-nucleotidase (P5N-I). In humans, a lack of this enzyme causes hemolytic anemia with ribosomal structures and trinucleotides retained in the red blood cells (RBCs). Although the protein/nucleotide sequence of P5N-I is known in mammals, the onset and regulation of P5N-I during erythroid maturation is unknown. However, in circulating chicken embryonic RBCs, the enzyme is induced together with carbonic anhydrase (CAII) and 2,3-bisphosphoglycerate (2,3-BPG) by norepinephrine (NE) and adenosine, which are released by the embryo under hypoxic conditions. Here, we present the chicken P5N-I sequence and the gene expression of P5N-I during RBC maturation; the profile of gene expression follows the enzyme activity with a rise between days 13 and 16 of embryonic development. The p5n-I expression is induced (1) in definitive but not primitive RBCs by stimulation of beta-adrenergic/adenosine receptors, and (2) in definitive RBCs by hypoxic incubation of the chicken embryo. Since embryonic RBCs increase their hemoglobin-oxygen affinity by degradation of nucleotides such as uridine triphosphate (UTP) and cytidine triphosphate (CTP), the induction of p5n-I expression can be seen as an adaptive response to hypoxia.

Theoretical model for optical oximetry at the capillary level: exploring hemoglobin oxygen saturation through backscattering of single red blood cells

NASA Astrophysics Data System (ADS)

Liu, Rongrong; Spicer, Graham; Chen, Siyu; Zhang, Hao F.; Yi, Ji; Backman, Vadim

2017-02-01

Oxygen saturation (sO2) of red blood cells (RBCs) in capillaries can indirectly assess local tissue oxygenation and metabolic function. For example, the altered retinal oxygenation in diabetic retinopathy and local hypoxia during tumor development in cancer are reflected by abnormal sO2 of local capillary networks. However, it is far from clear whether accurate label-free optical oximetry (i.e., measuring hemoglobin sO2) is feasible from dispersed RBCs at the single capillary level. The sO2-dependent hemoglobin absorption contrast present in optical scattering signal is complicated by geometry-dependent scattering from RBCs. We present a numerical study of backscattering spectra from single RBCs based on the first-order Born approximation, considering practical factors: RBC orientations, size variation, and deformations. We show that the oscillatory spectral behavior of RBC geometries is smoothed by variations in cell size and orientation, resulting in clear sO2-dependent spectral contrast. In addition, this spectral contrast persists with different mean cellular hemoglobin content and different deformations of RBCs. This study shows for the first time the feasibility of, and provides a theoretical model for, label-free optical oximetry at the single capillary level using backscattering-based imaging modalities, challenging the popular view that such measurements are impossible at the single capillary level.

Effects of deoxygenation on active and passive Ca2+ transport and on the cytoplasmic Ca2+ levels of sickle cell anemia red cells.

PubMed

Etzion, Z; Tiffert, T; Bookchin, R M; Lew, V L

1993-11-01

Elevated [Ca2+]i in deoxygenated sickle cell anemia (SS) red cells (RBCs) could trigger a major dehydration pathway via the Ca(2+)-sensitive K+ channel. But apart from an increase in calcium permeability, the effects of deoxygenation on the Ca2+ metabolism of sickle cells have not been previously documented. With the application of 45Ca(2+)-tracer flux methods and the combined use of the ionophore A23187, Co2+ ions, and intracellular incorporation of the Ca2+ chelator benz-2, in density-fractionated SS RBCs, we show here for the first time that upon deoxygenation, the mean [Ca2+]i level of SS discocytes was significantly increased, two- to threefold, from a normal range of 9.4 to 11.4 nM in the oxygenated cells, to a range of 21.8 to 31.7 nM in the deoxygenated cells, closer to K+ channel activatory levels. Unlike normal RBCs, deoxygenated SS RBCs showed a two- to fourfold increase in pump-leak Ca2+ turnover. Deoxygenation of the SS RBCs reduced their Ca2+ pump Vmax, more so in reticulocyte- and discocyte-rich than in dense cell fractions, and decreased their cytoplasmic Ca2+ buffering. Analysis of these results suggests that both increased Ca2+ influx and reduced Ca2+ pump extrusion contribute to the [Ca2+]i elevation.

Assessment of prion reduction filters in decreasing infectivity of ultracentrifuged 263K scrapie-infected brain homogenates in "spiked" human blood and red blood cells.

PubMed

Cardone, Franco; Sowemimo-Coker, Samuel; Abdel-Haq, Hanin; Sbriccoli, Marco; Graziano, Silvia; Valanzano, Angelina; Berardi, Vito Angelo; Galeno, Roberta; Puopolo, Maria; Pocchiari, Maurizio

2014-04-01

The safety of red blood cells (RBCs) is of concern because of the occurrence of four transfusion-transmitted variant Creutzfeldt-Jakob disease (vCJD) cases in the United Kingdom. The absence of validated screening tests requires the use of procedures to remove prions from blood to minimize the risk of transmission. These procedures must be validated using infectious prions in a form that is as close as possible to one in blood. Units of human whole blood (WB) and RBCs were spiked with high-speed supernatants of 263K scrapie-infected hamster brain homogenates. Spiked samples were leukoreduced and then passed through prion-removing filters (Pall Corporation). In another experiment, RBCs from 263K scrapie-infected hamsters were treated as above, and residual infectivity was measured by bioassay. The overall removal of infectivity by the filters from prion-spiked WB and RBCs was approximately two orders of magnitude. No infectivity was detected in filtered hamster RBCs endogenously infected with scrapie. The use of prion-removing filters may help to reduce the risk of transfusion-transmitted vCJD. To avoid overestimation of prion removal efficiency in validation studies, it may be more appropriate to use supernates from ultracentrifugation of scrapie-infected hamster brain homogenate rather than the current standard brain homogenates. © 2013 American Association of Blood Banks.

Effect of red blood cell storage time on markers of hemolysis and inflammation in transfused very low birth weight infants.

PubMed

Kalhan, Tamara G; Bateman, David A; Bowker, Rakhee M; Hod, Eldad A; Kashyap, Sudha

2017-12-01

BackgroundProlonged storage of transfused red blood cells (RBCs) is associated with hemolysis in healthy adults and inflammation in animal models. We aimed to determine whether storage duration affects markers of hemolysis (e.g., serum bilirubin, iron, and non-transferrin-bound iron (NTBI)) and inflammation (e.g., interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1) in transfused very low birth weight (VLBW) infants.MethodsBlood samples from 23 independent transfusion events were collected by heel stick before and 2-6 h after transfusion.ResultsSerum iron, total bilirubin, NTBI, and MCP-1 levels were significantly increased after transfusion of RBCs (P<0.05 for each comparison). The storage age of transfused RBCs positively correlated with increases in NTBI following transfusion (P<0.001; R 2 =0.44). No associations between storage duration and changes in the other analytes were observed.ConclusionTransfusion of RBCs into VLBW infants is associated with increased markers of hemolysis and the inflammatory chemokine MCP-1. RBC-storage duration only correlated with increases in NTBI levels following transfusion. NTBI was only observed in healthy adults following 35 days of storage; however, this study suggests that VLBW infants are potentially more susceptible to produce this pathological form of iron, with increased levels observed after transfusion of only 20-day-old RBCs.

Indolic uremic solutes enhance procoagulant activity of red blood cells through phosphatidylserine exposure and microparticle release.

PubMed

Gao, Chunyan; Ji, Shuting; Dong, Weijun; Qi, Yushan; Song, Wen; Cui, Debin; Shi, Jialan

2015-10-28

Increased accumulation of indolic uremic solutes in the blood of uremic patients contributes to the risk of thrombotic events. Red blood cells (RBCs), the most abundant blood cells in circulation, may be a privileged target of these solutes. However, the effect of uremic solutes indoxyl sulfate (IS) and indole-3-acetic acid (IAA) on procoagulant activity (PCA) of erythrocyte is unclear. Here, RBCs from healthy adults were treated with IS and IAA (mean and maximal concentrations reported in uremic patients). Phosphatidylserine (PS) exposure of RBCs and their microparticles (MPs) release were labeled with Alexa Fluor 488-lactadherin and detected by flow cytometer. Cytosolic Ca(2+) ([Ca(2+)]) with Fluo 3/AM was analyzed by flow cytometer. PCA was assessed by clotting time and purified coagulation complex assays. We found that PS exposure, MPs generation, and consequent PCA of RBCs at mean concentrations of IS and IAA enhanced and peaked in maximal uremic concentrations. Moreover, 128 nM lactadherin, a PS inhibitor, inhibited over 90% PCA of RBCs and RMPs. Eryptosis or damage, by indolic uremic solutes was due to, at least partially, the increase of cytosolic [Ca(2+)]. Our results suggest that RBC eryptosis in uremic solutes IS and IAA plays an important role in thrombus formation through releasing RMPs and exposing PS. Lactadherin acts as an efficient anticoagulant in this process.

Red blood cell thickness is evolutionarily constrained by slow, hemoglobin-restricted diffusion in cytoplasm.

PubMed

Richardson, Sarah L; Swietach, Pawel

2016-10-25

During capillary transit, red blood cells (RBCs) must exchange large quantities of CO 2 and O 2 in typically less than one second, but the degree to which this is rate-limited by diffusion through cytoplasm is not known. Gas diffusivity is intuitively assumed to be fast and this would imply that the intracellular path-length, defined by RBC shape, is not a factor that could meaningfully compromise physiology. Here, we evaluated CO 2 diffusivity (D CO2 ) in RBCs and related our results to cell shape. D CO2 inside RBCs was determined by fluorescence imaging of [H + ] dynamics in cells under superfusion. This method is based on the principle that H + diffusion is facilitated by CO 2 /HCO 3 - buffer and thus provides a read-out of D CO2 . By imaging the spread of H + ions from a photochemically-activated source (6-nitroveratraldehyde), D CO2 in human RBCs was calculated to be only 5% of the rate in water. Measurements on RBCs containing different hemoglobin concentrations demonstrated a halving of D CO2 with every 75 g/L increase in mean corpuscular hemoglobin concentration (MCHC). Thus, to compensate for highly-restricted cytoplasmic diffusion, RBC thickness must be reduced as appropriate for its MCHC. This can explain the inverse relationship between MCHC and RBC thickness determined from >250 animal species.

Red blood cell thickness is evolutionarily constrained by slow, hemoglobin-restricted diffusion in cytoplasm

PubMed Central

Richardson, Sarah L.; Swietach, Pawel

2016-01-01

During capillary transit, red blood cells (RBCs) must exchange large quantities of CO2 and O2 in typically less than one second, but the degree to which this is rate-limited by diffusion through cytoplasm is not known. Gas diffusivity is intuitively assumed to be fast and this would imply that the intracellular path-length, defined by RBC shape, is not a factor that could meaningfully compromise physiology. Here, we evaluated CO2 diffusivity (DCO2) in RBCs and related our results to cell shape. DCO2 inside RBCs was determined by fluorescence imaging of [H+] dynamics in cells under superfusion. This method is based on the principle that H+ diffusion is facilitated by CO2/HCO3− buffer and thus provides a read-out of DCO2. By imaging the spread of H+ ions from a photochemically-activated source (6-nitroveratraldehyde), DCO2 in human RBCs was calculated to be only 5% of the rate in water. Measurements on RBCs containing different hemoglobin concentrations demonstrated a halving of DCO2 with every 75 g/L increase in mean corpuscular hemoglobin concentration (MCHC). Thus, to compensate for highly-restricted cytoplasmic diffusion, RBC thickness must be reduced as appropriate for its MCHC. This can explain the inverse relationship between MCHC and RBC thickness determined from >250 animal species. PMID:27777410

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XVIII platypus (Ornithorhynchus anatinus) and saltwater crocodile (Crocodylus porosus).

PubMed

Benga, Gheorghe; Chapman, Bogdan E; Cox, Guy C; Kuchel, Philip W

2010-07-01

As part of a programme of comparative measurements of Pd (diffusional water permeability) the RBCs (red blood cells) from an aquatic monotreme, platypus (Ornithorhynchus anatinus), and an aquatic reptile, saltwater crocodile (Crocodylus porosus) were studied. The mean diameter of platypus RBCs was estimated by light microscopy and found to be approximately 6.3 microm. Pd was measured by using an Mn2+-doping 1H NMR (nuclear magnetic resonance) technique. The Pd (cm/s) values were relatively low: approximately 2.1 x 10(-3) at 25 degrees C, 2.5 x 10(-3) at 30 degrees C, 3.4 x 10(-3) at 37 degrees C and 4.5 at 42 degrees C for the platypus RBCs and approximately 2.8 x 10(-3) at 25 degrees C, 3.2 x 10(-3) at 30 degrees C, 4.5 x 10(-3) at 37 degrees C and 5.7 x 10(-3) at 42 degrees C for the crocodile RBCs. In parallel with the low water permeability, the Ea,d (activation energy of water diffusion) was relatively high, approximately 35 kJ/mol. These results suggest that "conventional" WCPs (water channel proteins), or AQPs (aquaporins), are probably absent from the plasma membranes of RBCs from both the platypus and the saltwater crocodile.

Red Blood Cell Dysfunction Induced by High-Fat Diet

PubMed Central

Unruh, Dusten; Srinivasan, Ramprasad; Benson, Tyler; Haigh, Stephen; Coyle, Danielle; Batra, Neil; Keil, Ryan; Sturm, Robert; Blanco, Victor; Palascak, Mary; Franco, Robert S.; Tong, Wilson; Chatterjee, Tapan; Hui, David Y.; Davidson, W. Sean; Aronow, Bruce J.; Kalfa, Theodosia; Manka, David; Peairs, Abigail; Blomkalns, Andra; Fulton, David J.; Brittain, Julia E.; Weintraub, Neal L.; Bogdanov, Vladimir Y.

2015-01-01

Background High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). Methods and Results A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC−/− mice. In RBCs from HFD-fed wild-type and DARC−/− mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. Conclusions RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic. PMID:26467254

Immune modulation and microchimerism after unmodified versus leukoreduced allogeneic red blood cell transfusion in cancer patients: results of a randomized study.

PubMed

Lapierre, Valérie; Aupérin, Anne; Robinet, Eric; Ferrand, Christophe; Oubouzar, Nadia; Tramalloni, Dominique; Saas, Philippe; Debaene, Bertrand; Lasser, Philippe; Tiberghien, Pierre

2007-09-01

Transfusion of red blood cells (RBCs) has been associated with immunomodulatory effects. Persistence of donor cells in the recipient may be contributive. A randomized single-center trial was conducted to compare microchimerism and immune responses in 35 patients undergoing cancer surgery and transfused perioperatively with either unmodified RBCs (UN-RBCs, n = 18) or leukoreduced RBCs (LR-RBCs, n = 17). Biologic parameters included microchimerism assessment peripheral blood mononuclear cell (PBMNC) phenotyping, cytokine production by stimulated PBMNCs, FoxP3 gene expression, and T-cell repertoire (TCR) analysis. Microchimerism was documented in 8 of 18 patients after UN-RBC transfusion while absent after LR-RBC transfusion (0/17; p = 0.001). After UN-RBC transfusion, microchimerism was associated with increased interleukin (IL)-10 production (p = 0.02), reduced TCR alteration (p = 0.04), and reduced CD56+ cell counts (p = 0.02) when compared to recipients without evidence for microchimerism. FoxP3 gene expression did not differ significantly between both treatment groups nor with the presence or absence of microchimerism in the UN-RBC group. Finally, after an initial early decrease after surgery and transfusion, IL-12 production increased and more significantly so after UN-RBC transfusion versus LR-RBC transfusion (p = 0.05). UN-RBC-induced microchimerism is associated with specific immunomodulatory effects in cancer patients who received transfusions during surgery.

Geranyl diphosphate synthase large subunit, and methods of use

DOEpatents

Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

2001-10-16

A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

Revisiting the phylogeny of Ocellularieae, the second largest tribe within Graphidaceae (lichenized Ascomycota: Ostropales)

Treesearch

Ekaphan Kraichak; Sittiporn Parnmen; Robert Lücking; Eimy Rivas Plata; Andre Aptroot; Marcela E.S. Caceres; Damien Ertz; Armin Mangold; Joel A. Mercado-Diaz; Khwanruan Papong; Dries Van der Broeck; Gothamie Weerakoon; H. Thorsten Lumbsch; NO-VALUE

2014-01-01

We present an updated 3-locus molecular phylogeny of tribe Ocellularieae, the second largest tribe within subfamily Graphidoideae in the Graphidaceae. Adding 165 newly generated sequences from the mitochondrial small subunit rDNA (mtSSU), the nuclear large subunit rDNA (nuLSU), and the second largest subunit of the DNA-directed RNA polymerase II (RPB2), we currently...

Identification of a small molecule that inhibits herpes simplex virus DNA Polymerase subunit interactions and viral replication.

PubMed

Pilger, Beatrice D; Cui, Can; Coen, Donald M

2004-05-01

The interaction between the catalytic subunit Pol and the processivity subunit UL42 of herpes simplex virus DNA polymerase has been characterized structurally and mutationally and is a potential target for novel antiviral drugs. We developed and validated an assay for small molecules that could disrupt the interaction of UL42 and a Pol-derived peptide and used it to screen approximately 16,000 compounds. Of 37 "hits" identified, four inhibited UL42-stimulated long-chain DNA synthesis by Pol in vitro, of which two exhibited little inhibition of polymerase activity by Pol alone. One of these specifically inhibited the physical interaction of Pol and UL42 and also inhibited viral replication at concentrations below those that caused cytotoxic effects. Thus, a small molecule can inhibit this protein-protein interaction, which provides a starting point for the discovery of new antiviral drugs.

Photocontrol of the expression of genes encoding chlorophyll a/b binding proteins and small subunit of ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (L. ) and Nicotiana tabacum (L. )

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wehmeyer, B.; Cashmore, A.R.; Schaefer, E.

Phytochrome and the blue ultraviolet-A photoreceptor control light-induced expression of genes encoding the chlorophyll a/b binding protein of photosystem II and photosystem I and the genes for the small subunit of the ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (tomato) and Nicotiana tabacum (tobacco). A high irradiance response also controls the induction of these genes. Genes encoding photosystem II- and I-associated chlorophyll a/b binding proteins both exhibit a transient rapid increase in expression in response to light pulse or to continuous irradiation. In contrast, genes encoding the small subunit exhibit a continuous increase in expression in response to light.more » These distinct expression characteristics are shown to reflect differences at the level of transcription.« less

Changes in hemoglobin-oxygen affinity with shape variations of red blood cells

NASA Astrophysics Data System (ADS)

Chowdhury, Aniket; Dasgupta, Raktim; Majumder, Shovan K.

2017-10-01

Shape variations of red blood cells (RBCs) are known to occur upon exposure to various drugs or under diseased conditions. The commonly observed discocytic RBCs can be transformed to echinocytic or stomatocytic shape under such conditions. Raman spectra of the three major shape variations, namely discocyte, echinocyte, and stomatocyte, of RBCs were studied while subjecting the cells to oxygenated and deoxygenated conditions. Analysis of the recorded spectra suggests an increased level of hemoglobin (Hb)-oxygen affinity for the echinocytes. Also, some level of Hb degradation could be noticed for the deoxygenated echinocytes. The effects may arise from a reduced level of intracellular adenosine triphosphate in echinocytic cells and an increased fraction of submembrane Hb.

Hemoglobin Dynamics in Red Blood Cells: Correlation to Body Temperature

PubMed Central

Stadler, A. M.; Digel, I.; Artmann, G. M.; Embs, J. P.; Zaccai, G.; Büldt, G.

2008-01-01

A transition in hemoglobin behavior at close to body temperature has been discovered recently by micropipette aspiration experiments on single red blood cells (RBCs) and circular dichroism spectroscopy on hemoglobin solutions. The transition temperature was directly correlated to the body temperatures of a variety of species. In an exploration of the molecular basis for the transition, we present neutron scattering measurements of the temperature dependence of hemoglobin dynamics in whole human RBCs in vivo. The data reveal a change in the geometry of internal protein motions at 36.9°C, at human body temperature. Above that temperature, amino acid side-chain motions occupy larger volumes than expected from normal temperature dependence, indicating partial unfolding of the protein. Global protein diffusion in RBCs was also measured and the findings compared favorably with theoretical predictions for short-time self-diffusion of noncharged hard-sphere colloids. The results demonstrated that changes in molecular dynamics in the picosecond time range and angstrom length scale might well be connected to a macroscopic effect on whole RBCs that occurs at body temperature. PMID:18708462

MiR-155 enhances phagocytic activity of β-thalassemia/HbE monocytes via targeting of BACH1.

PubMed

Srinoun, Kanitta; Nopparatana, Chamnong; Wongchanchailert, Malai; Fucharoen, Suthat

2017-11-01

Abnormal red blood cell (RBC) clearance in β-thalassemia is triggered by activated monocytes. Recent reports indicate that miRNA (miR-) plays a role in monocyte activation. To study phagocytic function, we co-cultured monocytes of normal, non-splenectomized and splenectomized β-thalassemia/HbE individuals with RBCs obtained from normal, non-splenectomized and splenectomized β-thalassemia/HbE individuals. The phagocytic activity of β-thalassemia/HbE monocytes co-cultured with β-thalassemia/HbE RBCs was significantly higher than that of normal monocytes co-cultured with normal RBCs. Upregulation of monocyte miR-155 was observed in β-thalassemia/HbE patients. Increased miR-155 was associated with reductions in BTB and CNC Homology1 (BACH1) target gene expression and increased phagocytic activity of β-thalassemia/HbE monocytes. Taken together, these findings suggested that increased miR-155 expression in activated monocytes leads to enhanced phagocytic activity via BACH-1 regulation in β-thalassemia/HbE. This provides novel insights into the phagocytic clearance of abnormal RBCs in β-thalassemia/HbE.

Dodecamer is required for agglutination of Litopenaeus vannamei hemocyanin with bacterial cells and red blood cells.

PubMed

Pan, Jian-yi; Zhang, Yue-ling; Wang, San-ying; Peng, Xuan-xian

2008-01-01

Hemocyanins are multi-functional proteins, although they are well known to be respiratory proteins of invertebrate to date. In the present study, the agglutination ability of two oligomers of hemocyanin, hexamer and dodecamer, with pathogenic bacteria and red blood cells (RBCs) is investigated in pacific white shrimp, Litopenaeus vannamei. Hexameric hemocyanin exhibits an extremely high stability even in the absence of Ca(2+) and in alkaline pH. Dodecamer (di-hexamer) is easily dissociated into hexamers in unphysiological conditions. Hexamer and dodecamer are interchanged reciprocally with environmental conditions. Both oligomers can bind to bacteria and RBCs, but agglutination is observed only using dodecamer but not using hexamer in agglutination assay. However, the agglutination is detected when hexamer is utilized in the presence of antiserum against hemocyanin. These results indicate that dodecamer of hemocyanin is required for agglutination with bacteria and RBCs. It can be logically inferred that there is only one carbohydrate-binding site to bacterial cells and RBCs in the hexamer, while at least two sites in the dodecamer. Our finding has provided new insights into structural-functional relationship of hemocyanin.

Measurement of RBC agglutination with microscopic cell image analysis in a microchannel chip.

PubMed

Cho, Chi Hyun; Kim, Ju Yeon; Nyeck, Agnes E; Lim, Chae Seung; Hur, Dae Sung; Chung, Chanil; Chang, Jun Keun; An, Seong Soo A; Shin, Sehyun

2014-01-01

Since Landsteiner's discovery of ABO blood groups, RBC agglutination has been one of the most important immunohematologic techniques for ABO and RhD blood groupings. The conventional RBC agglutination grading system for RhD blood typings relies on macroscopic reading, followed by the assignment of a grade ranging from (-) to (4+) to the degree of red blood cells clumping. However, with the new scoring method introduced in this report, microscopically captured cell images of agglutinated RBCs, placed in a microchannel chip, are used for analysis. Indeed, the cell images' pixel number first allows the differentiation of agglutinated and non-agglutinated red blood cells. Finally, the ratio of agglutinated RBCs per total RBC counts (CRAT) from 90 captured images is then calculated. During the trial, it was observed that the agglutinated group's CRAT was significantly higher (3.77-0.003) than that of the normal control (0). Based on these facts, it was established that the microchannel method was more suitable for the discrimination between agglutinated RBCs and non-agglutinated RhD negative, and thus more reliable for the grading of RBCs agglutination than the conventional method.

The role of facilitated diffusion in oxygen transport by cell-free hemoglobins: implications for the design of hemoglobin-based oxygen carriers.

PubMed

McCarthy, M R; Vandegriff, K D; Winslow, R M

2001-08-30

We compared rates of oxygen transport in an in vitro capillary system using red blood cells (RBCs) and cell-free hemoglobins. The axial PO(2) drop down the capillary was calculated using finite-element analysis. RBCs, unmodified hemoglobin (HbA(0)), cross-linked hemoglobin (alpha alpha-Hb) and hemoglobin conjugated to polyethylene-glycol (PEG-Hb) were evaluated. According to their fractional saturation curves, PEG-Hb showed the least desaturation down the capillary, which most closely matched the RBCs; HbA(0) and alpha alpha-Hb showed much greater desaturation. A lumped diffusion parameter, K*, was calculated based on the Fick diffusion equation with a term for facilitated diffusion. The overall rates of oxygen transfer are consistent with hemoglobin diffusion rates according to the Stokes-Einstein Law and with previously measured blood pressure responses in rats. This study provides a conceptual framework for the design of a 'blood substitute' based on mimicking O(2) transport by RBCs to prevent autoregulatory changes in blood flow and pressure.

Blood Perfusion in Microfluidic Models of Pulmonary Capillary Networks: Role of Geometry and Hematocrit

NASA Astrophysics Data System (ADS)

Stauber, Hagit; Waisman, Dan; Sznitman, Josue; Technion-IIT Team; Department of Neonatology Carmel Medical Center; Faculty of Medicine-Technion IIT Collaboration

2015-11-01

Microfluidic platforms are increasingly used to study blood microflows at true physiological scale due to their ability to overcome manufacturing obstacle of complex anatomical morphologies, such as the organ-specific architectures of the microcirculation. In the present work, we utilize microfluidic platforms to devise in vitro models of the underlying pulmonary capillary networks (PCN), where capillary lengths and diameters are similar to the size of RBCs (~ 5-10 μm). To better understand flow characteristics and dispersion of red blood cells (RBCs) in PCNs, we have designed microfluidic models of alveolar capillary beds inspired by the seminal ``sheet flow'' model of Fung and Sobin (1969). Our microfluidic PCNs feature confined arrays of staggered pillars with diameters of ~ 5,7 and 10 μm, mimicking the dense structure of pulmonary capillary meshes. The devices are perfused with suspensions of RBCs at varying hematocrit levels under different flow rates. Whole-field velocity patterns using micro-PIV and single-cell tracking using PTV are obtained with fluorescently-labelled RBCs and discussed. Our experiments deliver a real-scale quantitative description of RBC perfusion characteristics across the pulmonary capillary microcirculation.

Rapid computation of photoacoustic fields from normal and pathological red blood cells using a Green's function method

NASA Astrophysics Data System (ADS)

Saha, Ratan K.; Fadhel, Muhannad N.; Lawrence, Aamna; Karmakar, Subhajit; Adhikari, Arunabha; Kolios, Michael C.

2017-03-01

Photoacoustic (PA) field calculations using a Green's function approach is presented. The method has been applied to predict PA spectra generated by normal (discocyte) and pathological (stomatocyte) red blood cells (RBCs). The contours of normal and pathological RBCs were generated by employing a popular parametric model and accordingly, fitted with the Legendre polynomial expansions for surface parametrization. The first frequency minimum of theoretical PA spectrum approximately appears at 607 MHz for a discocyte and 410 MHz for a stomatocyte when computed from the direction of symmetry axis. The same feature occurs nearly at 247 and 331 MHz, respectively, for those particles when measured along the perpendicular direction. The average experimental spectrum for normal RBCs is found to be flat over a bandwidth of 150-500 MHz when measured along the direction of symmetry axis. For spherical RBCs, both the theoretical and experimental spectra demonstrate negative slope over a bandwidth of 250-500 MHz. Using the Green's function method discussed, it may be possible to rapidly characterize cellular morphology from single-particle PA spectra.

Optical phase nanoscopy in red blood cells using low-coherence spectroscopy.

PubMed

Shock, Itay; Barbul, Alexander; Girshovitz, Pinhas; Nevo, Uri; Korenstein, Rafi; Shaked, Natan T

2012-10-01

We propose a low-coherence spectral-domain phase microscopy (SDPM) system for accurate quantitative phase measurements in red blood cells (RBCs) for the prognosis and monitoring of disease conditions that affect the visco-elastic properties of RBCs. Using the system, we performed time-recordings of cell membrane fluctuations, and compared the nano-scale fluctuation dynamics of healthy and glutaraldehyde-treated RBCs. Glutaraldehyde-treated RBCs possess lower amplitudes of fluctuations, reflecting an increased membrane stiffness. To demonstrate the ability of our system to measure fluctuations of lower amplitudes than those measured by the commonly used holographic phase microscopy techniques, we also constructed wide-field digital interferometry (WFDI) system and compared the performances of both systems. Due to its common-path geometry, the optical-path-delay stability of SDPM was found to be less than 0.3 nm in liquid environment, at least three times better than WFDI under the same conditions. In addition, due to the compactness of SDPM and its inexpensive and robust design, the system possesses a high potential for clinical applications.

A hemolytic factor from Haemonchus contortus alters erythrocyte morphology.

PubMed

Fetterer, R H; Rhoads, M L

1998-12-15

A hemolytic factor from adult Haemonchus contortus caused distinct morphological changes in the surface of sheep red blood cells (RBCs). After a 15 min exposure to the hemolytic factor, hemolysis was not detected in incubation media, but RBCs were spherical in shape with numerous surface projections compared to control cells that were smooth-surfaced biconcave disks. After 30 min, a time at which significant hemolysis occurred, echinocytes were formed, and after 90 min, cells were severely disrupted with many visible holes in membranes. No RBC ghosts were observed. RBCs from four other mammalian species were lysed by the H. contortus hemolytic factor. However, the rate of hemolysis varied with a relative order of sheep approximately rabbit>goat>pig>calf. The morphology of RBCs from all four species was significantly altered after 30 min incubation with the degree of morphological changes related to the degree of hemolysis. These results support the hypothesis that the hemolytic factor acts as a pore-forming agent, although a phospholipase or other enzyme might play a role in solubilization of cell membranes.

Piezo1 links mechanical forces to red blood cell volume

PubMed Central

Cahalan, Stuart M; Lukacs, Viktor; Ranade, Sanjeev S; Chien, Shu; Bandell, Michael; Patapoutian, Ardem

2015-01-01

Red blood cells (RBCs) experience significant mechanical forces while recirculating, but the consequences of these forces are not fully understood. Recent work has shown that gain-of-function mutations in mechanically activated Piezo1 cation channels are associated with the dehydrating RBC disease xerocytosis, implicating a role of mechanotransduction in RBC volume regulation. However, the mechanisms by which these mutations result in RBC dehydration are unknown. In this study, we show that RBCs exhibit robust calcium entry in response to mechanical stretch and that this entry is dependent on Piezo1 expression. Furthermore, RBCs from blood-cell-specific Piezo1 conditional knockout mice are overhydrated and exhibit increased fragility both in vitro and in vivo. Finally, we show that Yoda1, a chemical activator of Piezo1, causes calcium influx and subsequent dehydration of RBCs via downstream activation of the KCa3.1 Gardos channel, directly implicating Piezo1 signaling in RBC volume control. Therefore, mechanically activated Piezo1 plays an essential role in RBC volume homeostasis. DOI: http://dx.doi.org/10.7554/eLife.07370.001 PMID:26001274

Vitamin E nanoemulsion activity on stored red blood cells.

PubMed

Silva, C A L; Azevedo Filho, C A; Pereira, G; Silva, D C N; Castro, M C A B; Almeida, A F; Lucena, S C A; Santos, B S; Barjas-Castro, M L; Fontes, A

2017-06-01

Stored red blood cells (RBCs) undergo numerous changes that have been termed RBC storage lesion, which can be related to oxidative damage. Vitamin E is an important antioxidant, acting on cell lipids. Thus, this study aimed to investigate vitamin E activity on stored RBCs. We prepared a vitamin E nanoemulsion that was added to RBC units and stored at 4 °C. Controls, without vitamin E, were kept under the same conditions. Reactive oxygen species (ROS) production was monitored for up to 35 days of storage. RBC elasticity was also evaluated using an optical tweezer system. Vitamin E-treated samples presented a significant decrease in ROS production. Additionally, the elastic constant for vitamin E-treated RBCs did not differ from the control. Vitamin E decreased the amount of ROS in stored RBCs. Because vitamin E acts on lipid oxidation, results suggest that protein oxidation should also be considered a key factor for erythrocyte elastic properties. Thus, further studies combining vitamin E with protein antioxidants deserve attention, aiming to better preserve overall stored RBC properties. © 2017 British Blood Transfusion Society.

Hemoglobin dynamics in red blood cells: correlation to body temperature.

PubMed

Stadler, A M; Digel, I; Artmann, G M; Embs, J P; Zaccai, G; Büldt, G

2008-12-01

A transition in hemoglobin behavior at close to body temperature has been discovered recently by micropipette aspiration experiments on single red blood cells (RBCs) and circular dichroism spectroscopy on hemoglobin solutions. The transition temperature was directly correlated to the body temperatures of a variety of species. In an exploration of the molecular basis for the transition, we present neutron scattering measurements of the temperature dependence of hemoglobin dynamics in whole human RBCs in vivo. The data reveal a change in the geometry of internal protein motions at 36.9 degrees C, at human body temperature. Above that temperature, amino acid side-chain motions occupy larger volumes than expected from normal temperature dependence, indicating partial unfolding of the protein. Global protein diffusion in RBCs was also measured and the findings compared favorably with theoretical predictions for short-time self-diffusion of noncharged hard-sphere colloids. The results demonstrated that changes in molecular dynamics in the picosecond time range and angstrom length scale might well be connected to a macroscopic effect on whole RBCs that occurs at body temperature.

Glucose-6-phosphate dehydrogenase deficiency in transfusion medicine: the unknown risks.

PubMed

Francis, R O; Jhang, J S; Pham, H P; Hod, E A; Zimring, J C; Spitalnik, S L

2013-11-01

The hallmark of glucose-6-phosphate dehydrogenase (G6PD) deficiency is red blood cell (RBC) destruction in response to oxidative stress. Patients requiring RBC transfusions may simultaneously receive oxidative medications or have concurrent infections, both of which can induce haemolysis in G6PD-deficient RBCs. Although it is not routine practice to screen healthy blood donors for G6PD deficiency, case reports identified transfusion of G6PD-deficient RBCs as causing haemolysis and other adverse events. In addition, some patient populations may be more at risk for complications associated with transfusions of G6PD-deficient RBCs because they receive RBCs from donors who are more likely to have G6PD deficiency. This review discusses G6PD deficiency, its importance in transfusion medicine, changes in the RBC antioxidant system (of which G6PD is essential) during refrigerated storage and mechanisms of haemolysis. In addition, as yet unanswered questions that could be addressed by translational and clinical studies are identified and discussed. © 2013 International Society of Blood Transfusion.

Quantification of Transthyretin Kinetic Stability in Human Plasma Using Subunit Exchange

PubMed Central

2015-01-01

The transthyretin (TTR) amyloidoses are a group of degenerative diseases caused by TTR aggregation, requiring rate-limiting tetramer dissociation. Kinetic stabilization of TTR, by preferential binding of a drug to the native tetramer over the dissociative transition state, dramatically slows the progression of familial amyloid polyneuropathy. An established method for quantifying the kinetic stability of recombinant TTR tetramers in buffer is subunit exchange, in which tagged TTR homotetramers are added to untagged homotetramers at equal concentrations to measure the rate at which the subunits exchange. Herein, we report a subunit exchange method for quantifying the kinetic stability of endogenous TTR in human plasma. The subunit exchange reaction is initiated by the addition of a substoichiometric quantity of FLAG-tagged TTR homotetramers to endogenous TTR in plasma. Aliquots of the subunit exchange reaction, taken as a function of time, are then added to an excess of a fluorogenic small molecule, which immediately arrests further subunit exchange. After binding, the small molecule reacts with the TTR tetramers, rendering them fluorescent and detectable in human plasma after subsequent ion exchange chromatography. The ability to report on the extent of TTR kinetic stabilization resulting from treatment with oral tafamidis is important, especially for selection of the appropriate dose for patients carrying rare mutations. This method could also serve as a surrogate biomarker for the prediction of the clinical outcome. Subunit exchange was used to quantify the stabilization of WT TTR from senile systemic amyloidosis patients currently being treated with tafamidis (20 mg orally, once daily). TTR kinetic stability correlated with the tafamidis plasma concentration. PMID:24661308

Effects of intravenous delivery systems on infused red blood cells.

PubMed

Gibson, J S; Leff, R D; Roberts, R J

1984-03-01

The effects of various intravenous delivery systems on the integrity of infused red blood cells (RBCs) were studied. Using a factorial design, whole blood and packed RBCs were infused through i.v. delivery systems employing various combinations of i.v. tubing diameter and length, needle gauge, infusion rate (5 and 50 ml/hr), type of infusion pump (piston, diaphragm, or peristaltic operation), and type of blood product. The age and temperature of the blood filter used were held constant. A 5-ml sample of the blood product obtained during each experimental run was analyzed for plasma free-hemoglobin to assess the degree of hemolysis. Osmotic fragility of the RBCs was evaluated by measuring the percentage of hemolysis in the blood products in various concentrations of sodium chloride solution. Type of blood product and i.v. pump were the only variables significantly influencing RBC hemolysis. In both blood products, a greater degree of hemolysis occurred with the peristaltic-type pump than with the other types of pumps. In packed RBCs, the diaphragm-type pump produced greater hemolysis than the piston-type pump, but hemolysis was similar in whole-blood samples. Regardless of the type of pump, more hemolysis occurred in whole blood at the 5-ml/hr infusion rate than at the 50-ml/hr rate, but the converse was true in packed RBCs. Samples of both blood products were less osmotically fragile than their respective controls at sodium chloride concentrations ranging from 0.30 to 0.50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic motion of red blood cells in simple shear flow

NASA Astrophysics Data System (ADS)

Sui, Y.; Chew, Y. T.; Roy, P.; Cheng, Y. P.; Low, H. T.

2008-11-01

A three-dimensional numerical model is proposed to simulate the dynamic motion of red blood cells (RBCs) in simple shear flow. The RBCs are approximated by ghost cells consisting of Newtonian liquid drops enclosed by Skalak membranes which take into account the membrane shear elasticity and the membrane area incompressibility. The RBCs have an initially biconcave discoid resting shape, and the internal liquid is assumed to have the same physical properties as the matrix fluid. The simulation is based on a hybrid method, in which the immersed boundary concept is introduced into the framework of the lattice Boltzmann method, and a finite element model is incorporated to obtain the forces acting on the nodes of the cell membrane which is discretized into flat triangular elements. The dynamic motion of RBCs is investigated in simple shear flow under a broad range of shear rates. At large shear rates, the cells are found to carry out a swinging motion, in which periodic inclination oscillation and shape deformation superimpose on the membrane tank treading motion. With the shear rate decreasing, the swinging amplitude of the cell increases, and finally triggers a transition to tumbling motion. This is the first direct numerical simulation that predicts both the swinging motion of the RBCs and the shear rate induced transition, which have been observed in a recent experiment. It is also found that as the mode changes from swinging to tumbling, the apparent viscosity of the suspension increases monotonically.

The dynamic behavior of chemically "stiffened" red blood cells in microchannel flows.

PubMed

Forsyth, Alison M; Wan, Jiandi; Ristenpart, William D; Stone, Howard A

2010-07-01

The rigidity of red blood cells (RBCs) plays an important role in whole blood viscosity and is correlated with several cardiovascular diseases. Two chemical agents that are commonly used to study cell deformation are diamide and glutaraldehyde. Despite diamide's common usage, there are discrepancies in the literature surrounding diamide's effect on the deformation of RBCs in shear and pressure-driven flows; in particular, shear flow experiments have shown that diamide stiffens cells, while pressure-driven flow in capillaries did not give this result. We performed pressure-driven flow experiments with RBCs in a microfluidic constriction and quantified the cell dynamics using high-speed imaging. Diamide, which affects RBCs by cross-linking spectrin skeletal membrane proteins, did not reduce deformation and showed an unchanged effective strain rate when compared to healthy cells. In contrast, glutaraldehyde, which is a non-specific fixative that acts on all components of the cell, did reduce deformation and showed increased instances of tumbling, both of which are characteristic features of stiffened, or rigidified, cells. Because glutaraldehyde increases the effective viscosity of the cytoplasm and lipid membrane while diamide does not, one possible explanation for our results is that viscous effects in the cytoplasm and/or lipid membrane are a dominant factor in dictating dynamic responses of RBCs in pressure-driven flows. Finally, literature on the use of diamide as a stiffening agent is summarized, and provides supporting evidence for our conclusions. Copyright 2010 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Glick, R.E.; Schlagnhaufer, C.D.; Arteca, R.N.

The relationships among O{sub 3}-induced accelerated senescence, induction of ethylene, and changes in specific mRNA and protein levels were investigated in potato (Solanum tuberosum L. cv Norland) plants. When plants were exposed to 0.08 {mu}L L{sup -1} O{sub 3} for 5 h d{sup -1}, steady-state levels of rbcS mRNA declined at least 5-fold in expanding leaves after 3 d of O{sub 3} exposure and ethylene levels increased 6- to 10-fold. The expression of OIP-1, a 1-aminocyclo-propane-1-carboxylate synthase cDNA from potato, correlated with increased production of ethylene and decreased levels of rbcS mRNA in foliage of plants treated with O{sub 3}.more » In plants exposed to 0.30 {mu}L L{sup -1} O{sub 3} for 4 h, rbcS transcript levels were reduced 4-fold, whereas nuclear run-on experiments revealed that rbcS mRNA may be due, in part, to posttranscriptional regulation. The levels of transcripts for other chloroplast proteins, glyceraldehyde-3-phosphate dehydrogenase, and a photosystem II chlorophyll a/b-binding protein decreased in O{sub 3}-treated plants, in parallel with the decrease in rbcS mRNA. The steady-state mRNA level of a cytosolic glyceraldehyde-3-phosphate dehydrogenase increased in O{sub 3}-treated plants. The induction of ethylene and changes in transcript levels preceded visible leaf damage and decreases in ribulose-1,5-biphosphate carboxylase/oxygenase protein levels. 40 refs., 6 figs.« less

Glucose 6-phosphate dehydrogenase deficient subjects may be better "storers" than donors of red blood cells.

PubMed

Tzounakas, Vassilis L; Kriebardis, Anastasios G; Georgatzakou, Hara T; Foudoulaki-Paparizos, Leontini E; Dzieciatkowska, Monika; Wither, Matthew J; Nemkov, Travis; Hansen, Kirk C; Papassideri, Issidora S; D'Alessandro, Angelo; Antonelou, Marianna H

2016-07-01

Storage of packed red blood cells (RBCs) is associated with progressive accumulation of lesions, mostly triggered by energy and oxidative stresses, which potentially compromise the effectiveness of the transfusion therapy. Concerns arise as to whether glucose 6-phosphate dehydrogenase deficient subjects (G6PD(-)), ~5% of the population in the Mediterranean area, should be accepted as routine donors in the light of the increased oxidative stress their RBCs suffer from. To address this question, we first performed morphology (scanning electron microscopy), physiology and omics (proteomics and metabolomics) analyses on stored RBCs from healthy or G6PD(-) donors. We then used an in vitro model of transfusion to simulate transfusion outcomes involving G6PD(-) donors or recipients, by reconstituting G6PD(-) stored or fresh blood with fresh or stored blood from healthy volunteers, respectively, at body temperature. We found that G6PD(-) cells store well in relation to energy, calcium and morphology related parameters, though at the expenses of a compromised anti-oxidant system. Additional stimuli, mimicking post-transfusion conditions (37°C, reconstitution with fresh healthy blood, incubation with oxidants) promoted hemolysis and oxidative lesions in stored G6PD(-) cells in comparison to controls. On the other hand, stored healthy RBC units showed better oxidative parameters and lower removal signaling when reconstituted with G6PD(-) fresh blood compared to control. Although the measured parameters of stored RBCs from the G6PD deficient donors appeared to be acceptable, the results from the in vitro model of transfusion suggest that G6PD(-) RBCs could be more susceptible to hemolysis and oxidative stresses post-transfusion. On the other hand, their chronic exposure to oxidative stress might make them good recipients, as they better tolerate exposure to oxidatively damaged long stored healthy RBCs. Copyright © 2016 Elsevier Inc. All rights reserved.

Red blood cells derived from whole blood treated with riboflavin and ultraviolet light maintain adequate survival in vivo after 21 days of storage.

PubMed

Cancelas, Jose A; Slichter, Sherrill J; Rugg, Neeta; Pratt, P Gayle; Nestheide, Shawnagay; Corson, Jill; Pellham, Esther; Huntington, Marty; Goodrich, Raymond P

2017-05-01

Pathogen reduction (PR) of whole blood (WB) may increase blood safety when applied before component separation. This study evaluates the in vivo performance of red blood cells (RBCs) derived from WB treated with the riboflavin and ultraviolet (UV) light PR (Mirasol) system. This was a prospective, two-center, single-blind, randomized, two-period, crossover clinical trial designed to evaluate autologous 51 Cr/ 99m Tc-radiolabeled recovery and survival of RBCs derived from Mirasol-treated WB compared to untreated WB. RBCs were stored in AS-3 for 21 days at 1 to 6°C. In vitro RBC variables were characterized. Frequency and severity of treatment-emergent adverse event (TEAE) and neoantigenicity were determined. Twenty-four healthy adult volunteers (n = 12 per site) were evaluated. The Mirasol 24-hr RBC recoveries were 82.5 ± 3.9% with one-sided 95% lower confidence limit of 80.9%, meeting US Food and Drug Administration acceptance criteria, albeit at lower level than controls (91.7 ± 6.8%, p < 0.001). Mean RBC survival and T 50 were reduced in the Mirasol group (61 and 23 days, respectively) versus controls (82 and 36 days, respectively; p < 0.001) with a mean area under the curve survival of treated RBCs of 83% of untreated controls. End-of-storage hemolysis in the Mirasol group was 0.22 ± 0.1% (control, 0.15 ± 0.1%; p < 0.001). No neoantigenicity or differences in TEAEs were found. RBCs derived from Mirasol WB and stored for up to 21 days in AS-3 maintained acceptable cell quality and recovery, albeit modestly reduced compared with untreated RBCs. Mirasol WB may represent a valid single WB PR platform that allows manufacture of RBC for storage for up to 21 days. © 2017 AABB.

The relationship between red blood cell deformability metrics and perfusion of an artificial microvascular network

PubMed Central

Sosa, Jose M.; Nielsen, Nathan D.; Vignes, Seth M.; Chen, Tanya G.; Shevkoplyas, Sergey S.

2013-01-01

The ability of red blood cells (RBC) to undergo a wide range of deformations while traversing the microvasculature is crucial for adequate perfusion. Interpretation of RBC deformability measurements performed in vitro in the context of microvascular perfusion has been notoriously difficult. This study compares the measurements of RBC deformability performed using micropore filtration and ektacytometry with the RBC ability to perfuse an artificial microvascular network (AMVN). Human RBCs were collected from healthy consenting volunteers, leukoreduced, washed and exposed to graded concentrations (0% – 0.08%) of glutaraldehyde (a non-specific protein cross-linker) and diamide (a spectrin-specific protein cross-linker) to impair the deformability of RBCs. Samples comprising cells with two different levels of deformability were created by adding non-deformable RBCs (hardened by exposure to 0.08% glutaraldehyde) to the sample of normal healthy RBCs. Ektacytometry indicated a nearly linear decline in RBC deformability with increasing glutaraldehyde concentration. Micropore filtration showed a significant reduction only for concentrations of glutaraldehyde higher than 0.04%. Neither micropore filtration nor ektacytometry measurements could accurately predict the AMVN perfusion. Treatment with diamide reduced RBC deformability as indicated by ektacytometry, but had no significant effect on either micropore filtration or the AMVN perfusion. Both micropore filtration and ektacytometry showed a linear decline in effective RBC deformability with increasing fraction of non-deformable RBCs in the sample. The corresponding decline in the AMVN perfusion plateaued above 50%, reflecting the innate ability of blood flow in the microvasculature to bypass occluded capillaries. Our results suggest that in vitro measurements of RBC deformability performed using either micropore filtration or ektacytometry may not represent the ability of same RBCs to perfuse microvascular networks. Further development of biomimetic tools for measuring RBC deformability (e.g. the AMVN) could enable a more functionally relevant testing of RBC mechanical properties. PMID:23603326

The relationship between red blood cell deformability metrics and perfusion of an artificial microvascular network.

PubMed

Sosa, Jose M; Nielsen, Nathan D; Vignes, Seth M; Chen, Tanya G; Shevkoplyas, Sergey S

2014-01-01

The ability of red blood cells (RBC) to undergo a wide range of deformations while traversing the microvasculature is crucial for adequate perfusion. Interpretation of RBC deformability measurements performed in vitro in the context of microvascular perfusion has been notoriously difficult. This study compares the measurements of RBC deformability performed using micropore filtration and ektacytometry with the RBC ability to perfuse an artificial microvascular network (AMVN). Human RBCs were collected from healthy consenting volunteers, leukoreduced, washed and exposed to graded concentrations (0-0.08%) of glutaraldehyde (a non-specific protein cross-linker) and diamide (a spectrin-specific protein cross-linker) to impair the deformability of RBCs. Samples comprising cells with two different levels of deformability were created by adding non-deformable RBCs (hardened by exposure to 0.08% glutaraldehyde) to the sample of normal healthy RBCs. Ektacytometry indicated a nearly linear decline in RBC deformability with increasing glutaraldehyde concentration. Micropore filtration showed a significant reduction only for concentrations of glutaraldehyde higher than 0.04%. Neither micropore filtration nor ektacytometry measurements could accurately predict the AMVN perfusion. Treatment with diamide reduced RBC deformability as indicated by ektacytometry, but had no significant effect on either micropore filtration or the AMVN perfusion. Both micropore filtration and ektacytometry showed a linear decline in effective RBC deformability with increasing fraction of non-deformable RBCs in the sample. The corresponding decline in the AMVN perfusion plateaued above 50%, reflecting the innate ability of blood flow in the microvasculature to bypass occluded capillaries. Our results suggest that in vitro measurements of RBC deformability performed using either micropore filtration or ektacytometry may not represent the ability of same RBCs to perfuse microvascular networks. Further development of biomimetic tools for measuring RBC deformability (e.g. the AMVN) could enable a more functionally relevant testing of RBC mechanical properties.

Estimation of Scatterer Diameter by Normalized Power Spectrum of High-Frequency Ultrasonic RF Echo for Assessment of Red Blood Cell Aggregation

NASA Astrophysics Data System (ADS)

Fukushima, Taku; Hasegawa, Hideyuki; Kanai, Hiroshi

2011-07-01

Red blood cell (RBC) aggregation, as one of the determinants of blood viscosity, plays an important role in blood rheology, including the condition of blood. RBC aggregation is induced by the adhesion of RBCs when the electrostatic repulsion between RBCs weakens owing to increases in protein and saturated fatty acid levels in blood, excessive RBC aggregation leads to various circulatory diseases. This study was conducted to establish a noninvasive quantitative method for assessment of RBC aggregation. The power spectrum of ultrasonic RF echoes from nonaggregating RBCs, which shows the frequency property of scattering, exhibits Rayleigh behavior. On the other hand, ultrasonic RF echoes from aggregating RBCs contain the components of reflection, which have no frequency dependence. By dividing the measured power spectrum of echoes from RBCs in the lumen by that of echoes from a posterior wall of the vein in the dorsum manus, the attenuation property of the propagating medium and the frequency responses of transmitting and receiving transducers are removed from the former spectrum. RBC aggregation was assessed by the diameter of a scatterer, which was estimated by minimizing the square difference between the measured normalized power spectrum and the theoretical power spectrum. In this study, spherical scatterers with diameters of 5, 11, 15, and 30 µm were measured in basic experiments. The estimated scatterer diameters were close to the actual diameters. Furthermore, the transient change of the scatterer diameters were measured in an in vivo experiment with respect to a 24-year-old healthy male during the avascularization using a cuff. The estimated diameters (12-22 µm) of RBCs during avascularization were larger than the diameters (4-8 µm) at rest and after recirculation. These results show the possibility of the use of the proposed method for noninvasive assessment of RBC aggregation.

A randomized controlled pilot study of VO2 max testing: a potential model for measuring relative in vivo efficacy of different red blood cell products.

PubMed

Bennett-Guerrero, Elliott; Lockhart, Evelyn L; Bandarenko, Nicholas; Campbell, Mary L; Natoli, Michael J; Jamnik, Veronika K; Carter, Timothy R; Moon, Richard E

2017-03-01

Randomized trials, for example, RECESS, comparing "young" (median, 7-day) versus "middle-aged" (median, 28-day) red blood cells (RBCs), showed no difference in outcome. These data are important; however, they do not inform us about the safety and effectiveness of the oldest RBCs, which some patients receive. It may not be feasible to conduct a clinical trial randomizing patients to receive the oldest blood. Therefore, we propose strenuous exercise (VO 2 max testing) as a model to study the relative efficacy to increase oxygen delivery to tissue of different RBC products, for example, extremes of storage duration. In this pilot study, eight healthy subjects had 2 units of leukoreduced RBCs collected by apheresis in AS-3 using standard methods. Subjects were randomized to receive both (2) units of their autologous RBCs at either 7 or 42 days after blood collection. VO 2 max testing on a cycle ergometer was performed 2 days before (Monday) and 2 days after (Friday) the transfusion visit (Wednesday). This design avoids confounding effects on intravascular volume from the 2-unit blood transfusion. The primary outcome was the difference in VO 2 max between Friday and Monday (delta VO 2 max). VO 2 max increased more in the 7-day RBC arm (8.7 ± 6.9% vs. 1.9 ± 6.5%, p = 0.202 for comparison between arms). Exercise duration (seconds) increased in the 7-day RBC arm (8.4 ± 1.7%) but actually decreased in the 42-day arm (-2.6 ± 3.6%, p = 0.002). This pilot study suggests that VO 2 max testing has potential as a rigorous and quantitative in vivo functional assay of RBC function. Our preliminary results suggest that 42-day RBCs are inferior to 7-day RBCs at delivering oxygen to tissues. © 2016 AABB.

Oxygen-sensitive regulatory volume increase and Na transport in red blood cells from the cane toad, Bufo marinus.

PubMed

Kristensen, Karina; Koldkjaer, Pia; Berenbrink, Michael; Wang, Tobias

2007-07-01

The red blood cells (RBCs) of cane toad, Bufo marinus, are only partially saturated with oxygen in most of the circulation due to cardiac shunts that cause desaturation of arterial blood. The present study examines the oxygen dependency of RBC ouabain-insensitive unidirectional Na transport, using 22Na, in control cells and in cells exposed to hyperosmotic shrinkage or the beta-adrenergic agonist isoproterenol. Deoxygenation per se induced a slow, but significant Na influx, which was paralleled by a slow increase in RBC volume. Hyperosmotic shrinkage by a calculated 25% activated a robust Na influx that in the first 30 min had a strong PO2 dependency with maximal activation at low PO2 values and a P50 of approximately 5.5 kPa. This activation was completely abolished by the Na/H exchanger (NHE) inhibitor EIPA (10(-4) mol l(-1)). Hyperosmotic shrinkage is particularly interesting in B. marinus as it withstands considerable elevation in extracellular osmolarity following dehydration. Parallel studies showed that deoxygenated B. marinus RBCs had a much faster regulatory volume increase (RVI) response than air-equilibrated RBCs, reflecting the difference in magnitude of Na influxes at the two PO2 values. The extent of RVI ( approximately 60%) after 90 min, however, was similar under the two conditions, reflecting a more prolonged elevation of the shrinkage-induced Na influx in air-equilibrated RBCs. There were no significant differences in the ability to perform RVI between whole blood cells at a PCO2 of 1 and 3 kPa or washed RBCs, and 10(-4) mol l(-1) amiloride reduced the RVI under all conditions, whereas 10(-5) mol l(-1) bumetanide had no effect. Isoproterenol (10(-5) mol l(-1)) induced a significant and prolonged increase in an EIPA-sensitive and bumetanide-insensitive Na influx at low PO2 under iso-osmotic conditions, whilst there was no stimulation by isoproterenol for up to 45 min in air-equilibrated RBCs. The prolonged beta-adrenergic activation of the Na influx at low PO2 is distinctly different from the rapid and transient stimulation in teleost RBCs, suggesting significant differences in the signal transduction pathways leading to transporter activation between vertebrate groups.

Anti-erythrocyte antibodies may contribute to anaemia in Plasmodium vivax malaria by decreasing red blood cell deformability and increasing erythrophagocytosis.

PubMed

Mourão, Luiza Carvalho; Roma, Paula Magda da Silva; Sultane Aboobacar, Jamila da Silva; Medeiros, Camila Maia Pantuzzo; de Almeida, Zélia Barbosa; Fontes, Cor Jesus Fernandes; Agero, Ubirajara; de Mesquita, Oscar Nassif; Bemquerer, Marcelo Porto; Braga, Érika Martins

2016-08-04

Plasmodium vivax accounts for the majority of human malaria infections outside Africa and is being increasingly associated in fatal outcomes with anaemia as one of the major complications. One of the causes of malarial anaemia is the augmented removal of circulating non-infected red blood cells (nRBCs), an issue not yet fully understood. High levels of auto-antibodies against RBCs have been associated with severe anaemia and reduced survival of nRBCs in patients with falciparum malaria. Since there are no substantial data about the role of those antibodies in vivax malaria, this study was designed to determine whether or not auto-antibodies against erythrocytes are involved in nRBC clearance. Moreover, the possible immune mechanisms elicited by them that may be associated to induce anaemia in P. vivax infection was investigated. Concentrations of total IgG were determined by sandwich ELISA in sera from clinically well-defined groups of P. vivax-infected patients with or without anaemia and in healthy controls never exposed to malaria, whereas the levels of specific IgG to nRBCs were determined by cell-ELISA. Erythrophagocytosis assay was used to investigate the ability of IgGs purified from each studied pooled sera in enhancing nRBC in vitro clearance by THP-1 macrophages. Defocusing microscopy was employed to measure the biomechanical modifications of individual nRBCs opsonized by IgGs purified from each group. Anaemic patients had higher levels of total and specific anti-RBC antibodies in comparison to the non-anaemic ones. Opsonization with purified IgG from anaemic patients significantly enhanced RBCs in vitro phagocytosis by THP-1 macrophages. Auto-antibodies purified from anaemic patients decreased the nRBC dynamic membrane fluctuations suggesting a possible participation of such antibodies in the perturbation of erythrocyte flexibility and morphology integrity maintenance. These findings revealed that vivax-infected patients with anaemia have increased levels of IgG auto-antibodies against nRBCs and that their deposition on the surface of non-infected erythrocytes decreases their deformability, which, in turn, may enhance nRBC clearance by phagocytes, contributing to the anaemic outcome. These data provide insights into the immune mechanisms associated with vivax malaria anaemia and may be important to the development of new therapy and vaccine strategies.

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XVI Dingo (Canis familiaris dingo) and dog (Canis familiaris).

PubMed

Benga, Gheorghe; Chapman, Bogdan E; Matei, Horea; Cox, Guy C; Romeo, Tony; Mironescu, Eugen; Kuchel, Philip W

2010-03-08

As part of a programme of comparative measurements of Pd (diffusional water permeability) the RBCs (red blood cells) from dingo (Canis familiaris dingo) and greyhound dog (Canis familiaris) were studied. The morphologies of the dingo and greyhound RBCs [examined by light and SEM (scanning electron microscopy)] were found to be very similar, with regard to aspect ratio and size; the mean diameters were estimated to be the same (approximately 7.2 microm) for both dingo and greyhound RBCs. The water diffusional permeability was monitored by using an Mn2+-doping 1H NMR technique at 400 MHz. The Pd (cm/s) values of dingo and greyhound RBCs were similar: 6.5 x 10(-3) at 25 degrees C, 7.5 x 10(-3) at 30 degrees C, 10 x 10(-3) at 37 degrees C and 11.5 x 10(-3) at 42 degrees C. The inhibitory effect of a mercury-containing SH (sulfhydryl)-modifying reagent PCMBS (p-chloromercuribenzene sulfonate) was investigated. The maximal inhibition of dingo and greyhound RBCs was reached in 15-30 min at 37 degrees C with 2 mmol/l PCMBS. The values of maximal inhibition were in the range 72-74% when measured at 25 degrees C and 30 degrees C, and approximately 66% at 37 degrees C. The lowest value of Pd (corresponding to the basal permeability to water) was approximately 2-3 x 10(-3) cm/s in the temperature range 25-37 degrees C. The Ea,d (activation energy of water diffusion) was 25 kJ/mol for dingo RBC and 23 kJ/mol for greyhound RBCs. After incubation with PCMBS, the values of Ea,d increased, reaching 46-48 kJ/mol in the condition of maximal inhibition of water exchange. The electrophoretograms of membrane polypeptides of the dingo and greyhound RBCs were compared and seen to be very similar. We postulate that the RBC parameters reported in the present study are characteristic of all canine species and, in particular in the two cases presented here, these parameters have not been changed by the peculiar Australian habitat over the millennia (as in the case of the dingo) or over shorter time periods, decades or centuries (as in the case of the domestic greyhound).

A study on the hemocompatibility of dendronized chitosan derivatives in red blood cells

PubMed Central

Zhou, Yanfang; Li, Jiemei; Lu, Fang; Deng, Junjie; Zhang, Jiahua; Fang, Peijie; Peng, Xinsheng; Zhou, Shu-Feng

2015-01-01

Dendrimers are hyperbranched macromolecules with well-defined topological structures and multivalent functionalization sites, but they may cause cytotoxicity due to the presence of cationic charge. Recently, we have introduced alkyne-terminated poly(amidoamine) (PAMAM) dendrons of different generations (G=2,3) into chitosan to obtain dendronized chitosan derivatives [Cs-g-PAMAM (G=2,3)], which exhibited a better water solubility and enhanced plasmid DNA transfection efficiency. In this study, we attempted to examine the impact of Cs-g-PAMAM (G=2,3) at different concentrations (25 μg/mL, 50 μg/mL, and 100 μg/mL) on the morphology, surface structure, and viability of rat red blood cells (RBCs). The results showed that treatment of RBCs with Cs-g-PAMAM (G=2,3) at 50 μg/mL and 100 μg/mL induced a slightly higher hemolysis than Cs, and Cs-g-PAMAM (G=3) caused a slightly higher hemolysis than Cs-g-PAMAM (G=2), but all values were <5.0%. Optical microscopic and atomic force microscopic examinations indicated that Cs-g-PAMAM (G=2,3) caused slight RBC aggregation and lysis. Treatment of RBCs with 100 μg/mL Cs-g-PAMAM (G=3) induced echinocytic transformation, and RBCs displayed characteristic irregular contour due to the folding of the periphery. Drephanocyte-like RBCs were observed when treated with 100 μg/mL Cs-g-PAMAM (G=3). Erythrocytes underwent similar shape transition upon treatment with Cs-g-PAMAM (G=2) or Cs. The roughness values (Rms) of RBCs incubated with Cs-g-PAMAM (G=2,3) were significantly larger than those for RBCs incubated with physiological saline (P<0.01), but the Rms showed no difference for Cs and Cs-g-PAMAM (G=2,3) (P>0.05). Furthermore, Cs-g-PAMAM (G=2,3) exhibited a lower cytotoxicity in human kidney 293T cells. These results indicate that Cs-g-PAMAM (G=2,3) are hemocompatible but may disturb membrane and lipid structures at higher concentrations. Further safety and biocompatibility evaluations are warranted for Cs-g-PAMAM. Our findings prove helpful for a better understanding of the advantages of combining PAMAM dendrimers and chitosan to design and develop new, safe, and effective drug delivery vehicles. PMID:25999697

Human-specific bacterial pore-forming toxins induce programmed necrosis in erythrocytes.

PubMed

LaRocca, Timothy J; Stivison, Elizabeth A; Hod, Eldad A; Spitalnik, Steven L; Cowan, Peter J; Randis, Tara M; Ratner, Adam J

2014-08-26

A subgroup of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins (PFTs) has an unusually narrow host range due to a requirement for binding to human CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-linked complement regulatory molecule. hCD59-specific CDCs are produced by several organisms that inhabit human mucosal surfaces and can act as pathogens, including Gardnerella vaginalis and Streptococcus intermedius. The consequences and potential selective advantages of such PFT host limitation have remained unknown. Here, we demonstrate that, in addition to species restriction, PFT ligation of hCD59 triggers a previously unrecognized pathway for programmed necrosis in primary erythrocytes (red blood cells [RBCs]) from humans and transgenic mice expressing hCD59. Because they lack nuclei and mitochondria, RBCs have typically been thought to possess limited capacity to undergo programmed cell death. RBC programmed necrosis shares key molecular factors with nucleated cell necroptosis, including dependence on Fas/FasL signaling and RIP1 phosphorylation, necrosome assembly, and restriction by caspase-8. Death due to programmed necrosis in RBCs is executed by acid sphingomyelinase-dependent ceramide formation, NADPH oxidase- and iron-dependent reactive oxygen species formation, and glycolytic formation of advanced glycation end products. Bacterial PFTs that are hCD59 independent do not induce RBC programmed necrosis. RBC programmed necrosis is biochemically distinct from eryptosis, the only other known programmed cell death pathway in mature RBCs. Importantly, RBC programmed necrosis enhances the growth of PFT-producing pathogens during exposure to primary RBCs, consistent with a role for such signaling in microbial growth and pathogenesis. In this work, we provide the first description of a new form of programmed cell death in erythrocytes (RBCs) that occurs as a consequence of cellular attack by human-specific bacterial toxins. By defining a new RBC death pathway that shares important components with necroptosis, a programmed necrosis module that occurs in nucleated cells, these findings expand our understanding of RBC biology and RBC-pathogen interactions. In addition, our work provides a link between cholesterol-dependent cytolysin (CDC) host restriction and promotion of bacterial growth in the presence of RBCs, which may provide a selective advantage to human-associated bacterial strains that elaborate such toxins and a potential explanation for the narrowing of host range observed in this toxin family. Copyright © 2014 LaRocca et al.

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XV. Agile wallaby (Macropus agilis), red-necked wallaby (Macropus rufogriseus) and Goodfellow's tree kangaroo (Dendrolagus goodfellowi).

PubMed

Benga, Gheorghe; Chapman, Bogdan E; Kuchel, Philip W

2009-09-01

The water diffusional permeability (P(d)) of red blood cells (RBC) from agile wallaby (Macropus agilis), red-necked wallaby (Macropus rufogriseus) and Goodfellow's tree kangaroo (Dendrolagus goodfellowi) was monitored using an Mn(2+)-doping (1)H nuclear magnetic resonance (NMR) technique at 400 MHz. The P(d) (cm s(-1)) values of agile wallaby RBCs were 7.5 x 10(-3) at 25 degrees C, 9 x 10(-3) at 30 degrees C, 11 x 10(-3) at 37 degrees C, and 13 x 10(-3) at 42 degrees C. The inhibitory effect of a mercury-containing sulfhydryl (SH)-modifying reagent p-chloromercuribenzoate (PCMB) on agile wallaby RBCs was investigated. The maximal inhibition was reached in 90 min at 37 degrees C with 2 mmol L(-1) PCMB. The value of maximal inhibition was approximately 63% when measured at 25 degrees C, approximately 52% at 37 degrees C and approximately 45% at 42 degrees C. The lowest value of P(d) (corresponding to the basal permeability to water) was approximately 3 x 10(-3) cm s(-1) at 25 degrees C. For the RBCs from red-necked wallaby (M. rufogriseus) the values of P(d) (cm s(-1)) were 7 x 10(-3) at 25 degrees C, 8 x 10(-3) at 30 degrees C, 10 x 10(-3) at 37 degrees C, and 12 x 10(-3) at 42 degrees C. Higher values of P(d) (cm s(-1)) were found for the RBCs from Goodfellow's tree kangaroo (D. goodfellowi): 8.5 x 10(-3) at 25 degrees C, 10 x 10(-3) at 30 degrees C, 13 x 10(-3) at 37 degrees C, and 15 x 10(-3) at 42 degrees C. The mean values of the activation energy of water diffusion (E(a,d)) were approximately 25 kJ mol(-1) for RBCs from the agile wallaby and tree kangaroo, respectively, and approximately 23 kJ mol(-1) for RBCs from red-necked wallaby. The values of E(a,d) increased after exposure of agile wallaby RBCs to PCMB, reaching a value of approximately 43-46 kJ mol(-1) when the maximal inhibition of P(d) was achieved.

Nuclear export of the small ribosomal subunit requires the Ran–GTPase cycle and certain nucleoporins

PubMed Central

Moy, Terence I.; Silver, Pamela A.

1999-01-01

After their assembly in the nucleolus, ribosomal subunits are exported from the nucleus to the cytoplasm. After export, the 20S rRNA in the small ribosomal subunit is cleaved to yield 18S rRNA and the small 5′ ITS1 fragment. The 5′ ITS1 RNA is normally degraded by the cytoplasmic Xrn1 exonuclease, but in strains lacking XRN1, the 5′ ITS1 fragment accumulates in the cytoplasm. Using the cytoplasmic localization of the 5′ ITS1 fragment as an indicator for the export of the small ribosomal subunit, we have identified genes that are required for ribosome export. Ribosome export is dependent on the Ran–GTPase as mutations in Ran or its regulators caused 5′ ITS1 to accumulate in the nucleoplasm. Mutations in the genes encoding the nucleoporin Nup82 and in the NES exporter Xpo1/Crm1 also caused the nucleoplasmic accumulation of 5′ ITS1. Mutants in a subset of nucleoporins and in the nuclear transport factors Srp1, Kap95, Pse1, Cse1, and Mtr10 accumulate the 5′ ITS1 in the nucleolus and affect ribosome assembly. In contrast, we did not detect nuclear accumulation of 5′ ITS1 in 28 yeast strains that have mutations in other genes affecting nuclear trafficking. PMID:10465789

The roles of RIIbeta linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of Type IIbeta Protein Kinase A. A small angle X-ray and neutron scattering study

DOE PAGES

Blumenthal, Donald K.; Copps, Jeffrey; Smith-Nguyen, Eric V.; ...

2014-08-11

Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. Moreover, the PKA holoenzyme is a tetramer (R 2:C 2), with a regulatory subunit homodimer (R 2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the typemore » IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1–280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. These results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA.« less

Isolation and characterization of the stage-specific cytochrome b small subunit (CybS) of Ascaris suum complex II from the aerobic respiratory chain of larval mitochondria.

PubMed

Amino, Hisako; Osanai, Arihiro; Miyadera, Hiroko; Shinjyo, Noriko; Tomitsuka, Eriko; Taka, Hikari; Mineki, Reiko; Murayama, Kimie; Takamiya, Shinzaburo; Aoki, Takashi; Miyoshi, Hideto; Sakamoto, Kimitoshi; Kojima, Somei; Kita, Kiyoshi

2003-05-01

We recently reported that Ascaris suum mitochondria express stage-specific isoforms of complex II: the flavoprotein subunit and the small subunit of cytochrome b (CybS) of the larval complex II differ from those of adult enzyme, while two complex IIs share a common iron-sulfur cluster subunit (Ip). In the present study, A. suum larval complex II was highly purified to characterize the larval cytochrome b subunits in more detail. Peptide mass fingerprinting and N-terminal amino acid sequencing showed that the larval and adult cytochrome b (CybL) proteins are identical. In contrast, cDNA sequences revealed that the small subunit of larval cytochrome b (CybS(L)) is distinct from the adult CybS (CybS(A)). Furthermore, Northern analysis and immunoblotting showed stage-specific expression of CybS(L) and CybS(A) in larval and adult mitochondria, respectively. Enzymatic assays revealed that the ratio of rhodoquinol-fumarate reductase (RQFR) to succinate-ubiquinone reductase (SQR) activities and the K(m) values for quinones are almost identical for the adult and larval complex IIs, but that the fumarate reductase (FRD) activity is higher for the adult form than for the larval form. These results indicate that the adult and larval A. suum complex IIs have different properties than the complex II of the mammalian host and that the larval complex II is able to function as a RQFR. Such RQFR activity of the larval complex II would be essential for rapid adaptation to the dramatic change of oxygen availability during infection of the host.

The roles of the RIIβ linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of the type IIβ protein kinase A: a small angle x-ray and neutron scattering study.

PubMed

Blumenthal, Donald K; Copps, Jeffrey; Smith-Nguyen, Eric V; Zhang, Ping; Heller, William T; Taylor, Susan S

2014-10-10

Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Velocity measurements of heterogeneous RBC flow in capillary vessels using dynamic laser speckle signal.

PubMed

Li, Chenxi; Wang, Ruikang

2017-04-01

We propose an approach to measure heterogeneous velocities of red blood cells (RBCs) in capillary vessels using full-field time-varying dynamic speckle signals. The approach utilizes a low coherent laser speckle imaging system to record the instantaneous speckle pattern, followed by an eigen-decomposition-based filtering algorithm to extract dynamic speckle signal due to the moving RBCs. The velocity of heterogeneous RBC flows is determined by cross-correlating the temporal dynamic speckle signals obtained at adjacent locations. We verify the approach by imaging mouse pinna in vivo, demonstrating its capability for full-field RBC flow mapping and quantifying flow pattern with high resolution. It is expected to investigate the dynamic action of RBCs flow in capillaries under physiological changes.

The linkage between ribosomal crystallography, metal ions, heteropolytungstates and functional flexibility

PubMed Central

Bashan, Anat; Yonath, Ada

2009-01-01

Crystallography of ribosomes, the universal cell nucleoprotein assemblies facilitating the translation of the genetic-code into proteins, met with severe problems owing to their large size, complex structure, inherent flexibility and high conformational variability. For the case of the small ribosomal subunit, which caused extreme difficulties, post crystallization treatment by minute amounts of a heteropolytungstate cluster allowed structure determination at atomic resolution. This cluster played a dual role in ribosomal crystallography: providing anomalous phasing power and dramatically increased the resolution, by stabilization of a selected functional conformation. Thus, four out of the fourteen clusters that bind to each of the crystallized small subunits are attached to a specific ribosomal protein in a fashion that may control a significant component of the subunit internal flexibility, by “gluing” symmetrical related subunits. Here we highlight basic issues in the relationship between metal ions and macromolecules and present common traits controlling in the interactions between polymetalates and various macromolecules, which may be extended towards the exploitation of polymetalates for therapeutical treatment. PMID:19915655

Decreased surface expression of the δ subunit of the GABAA receptor contributes to reduced tonic inhibition in dentate granule cells in a mouse model of fragile X syndrome.

PubMed

Zhang, Nianhui; Peng, Zechun; Tong, Xiaoping; Lindemeyer, A Kerstin; Cetina, Yliana; Huang, Christine S; Olsen, Richard W; Otis, Thomas S; Houser, Carolyn R

2017-11-01

While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABA A receptors (GABA A Rs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABA A R, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABA A Rs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with immunolabeling at perisynaptic locations in Fmr1 KO mice. While α4 immunogold particles were also reduced at perisynaptic locations in the Fmr1 KO mice, the labeling was increased at synaptic sites. Together these findings suggest that, in the dentate gyrus, altered surface expression of the δ subunit, rather than a decrease in δ subunit expression alone, could be limiting δ subunit-mediated tonic inhibition in this model of FXS. Finding ways to increase surface expression of the δ subunit of the GABA A R could be a novel approach to treatment of hyperexcitability-related alterations in FXS. Copyright © 2017 Elsevier Inc. All rights reserved.

Cytochrome b in human complex II (succinate-ubiquinone oxidoreductase): cDNA cloning of the components in liver mitochondria and chromosome assignment of the genes for the large (SDHC) and small (SDHD) subunits to 1q21 and 11q23.

PubMed

Hirawake, H; Taniwaki, M; Tamura, A; Kojima, S; Kita, K

1997-01-01

Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex in both the tricarboxylic acid cycle and the aerobic respiratory chains of mitochondria in eukaryotic cells and prokaryotic organisms. In this study, the amino acid sequences of the large (cybL) and small (cybS) subunits of cytochrome b in human liver complex II were deduced from cDNAs isolated by homology probing with mixed primers for the polymerase chain reaction. The mature cybL and cybS contain 140 and 103 amino acids, respectively, and show little similarity to the amino acid sequences of the subunits from other species in contrast to the highly conserved features of the flavoprotein (Fp) subunit and iron-sulfur protein (Ip) subunit. From hydrophobicity analysis, both cybL and cybS appear to have three transmembrane segments, indicating their role as membrane-anchors for the enzyme complex. Histidine residues, which are possible heme axial ligands in cytochrome b of complex II, were found in the second transmembrane segment of each subunit. The genes for cybL (SDHC) and cybS (SDHD) were mapped to chromosome 1q21 and 11q23, respectively by fluorescent in situ hybridization (FISH).

Assessing subunit dependency of the Plasmodium proteasome using small molecule inhibitors and active site probes.

PubMed

Li, Hao; van der Linden, Wouter A; Verdoes, Martijn; Florea, Bogdan I; McAllister, Fiona E; Govindaswamy, Kavitha; Elias, Joshua E; Bhanot, Purnima; Overkleeft, Herman S; Bogyo, Matthew

2014-08-15

The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the β5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the β2 subunit did not affect viability. Interestingly, coinhibition of both the β2 and β5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.

Human-Specific Bacterial Pore-Forming Toxins Induce Programmed Necrosis in Erythrocytes

PubMed Central

LaRocca, Timothy J.; Stivison, Elizabeth A.; Hod, Eldad A.; Spitalnik, Steven L.; Cowan, Peter J.; Randis, Tara M.

2014-01-01

ABSTRACT A subgroup of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins (PFTs) has an unusually narrow host range due to a requirement for binding to human CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-linked complement regulatory molecule. hCD59-specific CDCs are produced by several organisms that inhabit human mucosal surfaces and can act as pathogens, including Gardnerella vaginalis and Streptococcus intermedius. The consequences and potential selective advantages of such PFT host limitation have remained unknown. Here, we demonstrate that, in addition to species restriction, PFT ligation of hCD59 triggers a previously unrecognized pathway for programmed necrosis in primary erythrocytes (red blood cells [RBCs]) from humans and transgenic mice expressing hCD59. Because they lack nuclei and mitochondria, RBCs have typically been thought to possess limited capacity to undergo programmed cell death. RBC programmed necrosis shares key molecular factors with nucleated cell necroptosis, including dependence on Fas/FasL signaling and RIP1 phosphorylation, necrosome assembly, and restriction by caspase-8. Death due to programmed necrosis in RBCs is executed by acid sphingomyelinase-dependent ceramide formation, NADPH oxidase- and iron-dependent reactive oxygen species formation, and glycolytic formation of advanced glycation end products. Bacterial PFTs that are hCD59 independent do not induce RBC programmed necrosis. RBC programmed necrosis is biochemically distinct from eryptosis, the only other known programmed cell death pathway in mature RBCs. Importantly, RBC programmed necrosis enhances the growth of PFT-producing pathogens during exposure to primary RBCs, consistent with a role for such signaling in microbial growth and pathogenesis. PMID:25161188

Investigation of the quality of stored red blood cells after simulated air drop in the maritime environment.

PubMed

Meli, Athinoula; Hancock, Vicky; Doughty, Heidi; Smedley, Steve; Cardigan, Rebecca; Wiltshire, Michael

2018-02-01

Maritime medical capability may be compromised by blood resupply. Air-dropped red blood cells (RBCs) is a possible mitigation factor. This study set out to evaluate RBC storage variables after a simulated parachute air drop into the sea, as limited data exist. The air load construction for the air drop of blood was subject to static drop assessment to simulate a worst-case parachute drop scenario. One control and two test Golden Hour shipping containers were each packaged with 10 RBC units. The control box was not dropped; Test Boxes 1 and 2 were further reinforced with waterproof boxes and underwent a simulated air drop on Day 7 or Day 8 postdonation, respectively. One day after the drop and once a week thereafter until Day 43 of storage, RBCs from each box were sampled and tested for full blood counts, hemolysis, adenosine triphosphate, 2,3-diphosphoglycerate, pH, extracellular potassium, glucose, lactate, deformability, and RBC microvesicles. The packaging configuration completed the air drop with no water ingress or physical damage. All units met UK specifications for volume, hemoglobin, and hemolysis. There were no significant differences for any of the variables studied between RBCs in the control box compared to RBCs in Test Boxes 1 and 2 combined over storage. The test proved that the packaging solution and the impact of a maritime air drop as performed in this study, on Day 7 or Day 8 postdonation, did not affect the in vitro quality of RBCs in SAGM over storage for 35 days. © 2017 AABB.

Excessive quantities of red blood cells are issued to the operating room.

PubMed

Collins, R A; Wisniewski, M K; Waters, J H; Triulzi, D J; Alarcon, L H; Yazer, M H

2015-12-01

To determine what percentage of red blood cell (RBC) units that were issued to the operating room (OR) were returned unused, and to determine how often all of the RBCs that were issued for a patient were returned unused using the institution's maximum surgical blood ordering schedule (MSBOS) as a guide. The MSBOS provides guidelines for blood ordering, but is merely a suggestion for the ordering clinicians. This study examined how closely ordering practices followed the MSBOS, and how often ordered RBCs were actually transfused. For a 4-week period, RBC issue and utilization data were collected on elective surgery patients who were eligible for electronic cross-match at a tertiary care hospital. These data were compared to the MSBOS. There were 1350 surgical procedures performed. Of these cases, 439 patients had a type and screen (T&S) performed, and 215/439 (49%) patients had at least 1 RBC issued during their case. To these 215 patients, 742 RBC units were issued and 537/742 (72%) of these units were returned to the blood bank unused. In 152/215 (71%) cases with issued RBCs, all of the RBCs were returned to the blood bank unused. Amongst the surgical categories in this study, the percentage of cases where none of the issued RBCs were transfused ranged from 38 to 93%. Significant numbers of RBC units are issued but not transfused during surgery. Involving the surgical team in the blood issuing process and using a data-driven MSBOS may reduce the number of unused units. © 2015 British Blood Transfusion Society.

Klebsiella pneumoniae type 3 fimbriae agglutinate yeast in a mannose-resistant manner.

PubMed

Stahlhut, Steen G; Struve, Carsten; Krogfelt, Karen A

2012-03-01

The ability of bacterial pathogens to express different fimbrial adhesins plays a significant role in virulence. Thus, specific detection of fimbrial expression is an important task in virulence characterization and epidemiological studies. Most clinical Klebsiella pneumoniae isolates express type 1 and type 3 fimbriae, which are characterized by mediation of mannose-sensitive agglutination of yeast cells and agglutination of tannic acid-treated ox red blood cells (RBCs), respectively. It has been observed that K. pneumoniae isolates agglutinate yeast cells and commercially available sheep RBCs in a mannose-resistant manner. Thus, this study was initiated to identify the adhesin involved. Screening of a mutant library surprisingly revealed that the mannose-resistant agglutination of yeast and sheep RBCs was mediated by type 3 fimbriae. Specific detection of type 1 fimbriae expression in K. pneumoniae was feasible only by the use of guinea pig RBCs. This was further verified by the use of isogenic fimbriae mutants and by cloning and expressing K. pneumoniae fimbrial gene clusters in Escherichia coli. Yeast agglutination assays are commonly used to detect type 1 fimbriae expression but should not be used for bacterial species able to express type 3 fimbriae. For these species, the use of guinea pig blood for specific type 1 fimbriae detection is essential. The use of commercially available sheep RBCs or yeast is an easy alternative to traditional methods to detect type 3 fimbriae expression. Easy and specific detection of expression of type 1 and type 3 fimbriae is essential in the continuous characterization of these important adhesive virulence factors present in members of the Enterobacteriaceae.

The value of automated gel column agglutination technology in the identification of true inherited D blood types in massively transfused patients.

PubMed

Summers, Thomas; Johnson, Viviana V; Stephan, John P; Johnson, Gloria J; Leonard, George

2009-08-01

Massive transfusion of D- trauma patients in the combat setting involves the use of D+ red blood cells (RBCs) or whole blood along with suboptimal pretransfusion test result documentation. This presents challenges to the transfusion service of tertiary care military hospitals who ultimately receive these casualties because initial D typing results may only reflect the transfused RBCs. After patients are stabilized, mixed-field reaction results on D typing indicate the patient's true inherited D phenotype. This case series illustrates the utility of automated gel column agglutination in detecting mixed-field reactions in these patients. The transfusion service test results, including the automated gel column agglutination D typing results, of four massively transfused D- patients transfused D+ RBCs is presented. To test the sensitivity of the automated gel column agglutination method in detecting mixed-field agglutination reactions, a comparative analysis of three automated technologies using predetermined mixtures of D+ and D- RBCs is also presented. The automated gel column agglutination method detected mixed-field agglutination in D typing in all four patients and in the three prepared control specimens. The automated microwell tube method identified one of the three prepared control specimens as indeterminate, which was subsequently manually confirmed as a mixed-field reaction. The automated solid-phase method was unable to detect any mixed fields. The automated gel column agglutination method provides a sensitive means for detecting mixed-field agglutination reactions in the determination of the true inherited D phenotype of combat casualties transfused massive amounts of D+ RBCs.

A novel flow cytometry-based method of analyzing Heinz bodies.

PubMed

Palasuwan, D; Palasuwan, A; Charoensappakit, A; Noulsri, E

2017-02-01

Heinz bodies are important to diagnosing and managing patients. However, microscopic examination of Heinz bodies has several disadvantages, demonstrating the need for a better method. We explored the potential use of flow cytometry to examine Heinz bodies. Whole-blood samples were collected from patients deficient in G6PD and healthy volunteers. Acetylphenylhydrazine was used to induce formation of Heinz bodies in red blood cells (RBCs). Then, RBCs positive for Heinz bodies were examined using a FACSCanto II cytometer. RBCs treated with acetylphenylhydrazine formed Heinz bodies and emitted a broad spectrum of fluorescence that could be detected by flow cytometry. The maximum emission of fluorescence was observed at 45 min after the incubation with acetylphenylhydrazine. In addition, the fluorescence emitted was stable for at least 72 h. The flow cytometer could detect the RBCs positive for Heinz bodies even if they made up as little as 0.1% of the total RBC population. Furthermore, the percentage and number, respectively, of RBCs positive for Heinz bodies in G6PD-deficient patients and normal donors exhibited a mean ± standard deviation (SD) of 68.9 ± 27.5 vs. 50.9 ± 28.6 and 96 014 ±35 732 cells/μL vs. 74 688 ± 36 514 cells/μL. Heinz bodies induced by acetylphenylhydrazine emit fluorescence, and this fluorescence could be examined using flow cytometry. Our study suggests the potential use of the developed method to investigate the formation of Heinz bodies in clinical samples. © 2016 John Wiley & Sons Ltd.

Liposomes composed of unsaturated lipids for membrane modification of human erythrocytes.

PubMed

Stoll, Christoph; Holovati, Jelena L; Acker, Jason P; Wolkers, Willem F

2011-01-01

Previous studies have shown that certain saturated lipids protect red blood cells (RBCs) during hypothermic storage but provide little protection during freezing or freeze-drying, whereas various unsaturated lipids destabilize RBCs during hypothermic storage but protect during freezing and freeze-drying. The protective effect of liposomes has been attributed to membrane modifications. We have previously shown that cholesterol exchange and lipid transfer between liposomes composed of saturated lipids and RBCs critically depends on the length of the lipid acyl chains. In this study the effect of unsaturated lipids with differences in their number of unsaturated bonds (18:0/18:1, 18:1/18:1, 18:2/18:2) on RBC membrane properties has been studied. RBCs were incubated in the presence of liposomes and both the liposomal and RBC fraction were analyzed by Fourier transform infrared spectroscopy (FTIR) after incubation. The liposomes caused an increase in RBC membrane conformational disorder at suprazero temperatures. The fluidizing effect of the liposomes on the RBC membranes, however, was found to be similar for the different lipids irrespective of their unsaturation level. The gel to liquid crystalline phase transition temperature of the liposomes increased after incubation with RBCs. RBC membrane fluidity increased linearly during the first 8 hours of incubation in the presence of liposomes. The increase in RBC membrane fluidity was found to be temperature dependent and displayed Arrhenius behaviour between 20 and 40°C, with an activation energy of 88 kJ mol⁻¹. Taken together, liposomes composed of unsaturated lipids increase RBC membrane conformational disorder, which could explain their cryoprotective action.

Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin.

PubMed

Maciaszek, Jamie L; Partola, Kostyantyn; Zhang, Jing; Andemariam, Biree; Lykotrafitis, George

2014-12-18

Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell-cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm(2) areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding. Copyright © 2014 Elsevier Ltd. All rights reserved.

Shared epitopes of glycoprotein A and protein 4.1 defined by antibody NaM10-3C10.

PubMed

Rasamoelisolo, M; Czerwinski, M; Willem, C; Blanchard, D

1998-06-01

We have produced the murine monoclonal antibody (MAb) NaM70-3C10 (IgM) from splenocytes of mice immunized with human red blood cells (RBCs). The MAb agglutinated untreated as well as trypsin, chymotrypsin, neuraminidase, or ficin-treated RBCs from controls. In contrast, control RBCs treated with papaine or bromelaine were not agglutinated. On immunoblots, the MAb bound to glycophorin A (GPA) and to a 80 kDa protein identified as protein 4.1. Analysis by agglutination of variant RBCs carrying hybrid glycophorins made of the N-terminus (amino acids 1-58) of GPA and of the C-terminus (amino acids 27-72) of glycophorin B (GPB) and competition-inhibition test using purified GPA and a synthetic peptide corresponding to the amino acid sequence 48-58 of GPA demonstrated that the epitope is located within residues 48-58 of GPA. Epitope analysis with immobilized peptides showed that the MAb recognizes the sequence 53Pro-Pro-Glu-Glu-GIu58 of GPA. A homologous sequence is also present within amino acids 395 to 405 of protein 4.1. Finally, the MAb bound to 16 kDa chymotryptic peptide of protein 4.1, which carries the above amino acid sequence. In conclusion, it may be assumed that NaM70-3C10 specifically recognizes a common epitope on the extracellular domain of GPA and on the intracellular protein 4.1; this specificity explains the persistence of the 80 kDa band on blots when RBCs are treated with papain.

Effects of 5-hydroxymethyl-2-furfural on the volume and membrane permeability of red blood cells from patients with sickle cell disease

PubMed Central

Hannemann, Anke; Cytlak, Urszula M; Rees, David C; Tewari, Sanjay; Gibson, John S

2014-01-01

The heterocyclic aldehyde 5-hydroxymethyl-2-furfural (5HMF) interacts allosterically with the abnormal form of haemoglobin (Hb), HbS, in red blood cells (RBCs) from patients with sickle cell disease (SCD), thereby increasing oxygen affinity and decreasing HbS polymerization and RBC sickling during hypoxia. We hypothesized that should 5HMF also inhibit the main cation pathways implicated in the dehydration of RBCs from SCD patients – the deoxygenation-induced cation pathway (Psickle), the Ca2+-activated K+ channel (the Gardos channel) and the K+–Cl− cotransporter (KCC) – it would have a synergistic effect in protection against sickling, directly through interacting with HbS, and indirectly through maintaining hydration and reducing [HbS]. This study was therefore designed to investigate the effects of 5HMF on RBC volume and K+ permeability in vitro. 5HMF markedly reduced the deoxygenation-induced dehydration of RBCs whether in response to maintained deoxygenation or to cyclical deoxygenation/re-oxygenation. 5HMF was found to inhibit Psickle, an effect which correlated with its effects on sickling. Deoxygenation-induced activation of the Gardos channel and exposure of phosphatidylserine were also inhibited, probably indirectly via reduced entry of Ca2+ through the Psickle pathway. Effects of 5HMF on KCC were more modest with a slight inhibition in N-ethylmaleimide (NEM, 1 mm)-treated RBCs and stimulation in RBCs untreated with NEM. These findings support the hypothesis that 5HMF may also be beneficial through effects on RBC ion and water homeostasis. PMID:25015917

High-Dose Vitamin C Injection to Cancer Patients May Promote Thrombosis Through Procoagulant Activation of Erythrocytes.

PubMed

Kim, Keunyoung; Bae, Ok-Nam; Koh, Sung-Hee; Kang, Seojin; Lim, Kyung-Min; Noh, Ji-Yoon; Shin, Sue; Kim, Inho; Chung, Jin-Ho

2015-10-01

Potential risk of high-dose vitamin C consumption is often ignored. Recently, gram-dose vitamin C is being intravenously injected for the treatment of cancer, which can expose circulating blood cells to extremely high concentrations of vitamin C. As well as platelets, red blood cells (RBCs) can actively participate in thrombosis through procoagulant activation. Here, we examined the procoagulant and prothrombotic risks associated with the intravenous injection of gram-dose vitamin C. Vitamin C (0.5-5 mM) increased procoagulant activity of freshly isolated human RBCs via the externalization of phosphatidylserine (PS) to outer cellular membrane and the formation of PS-bearing microvesicles. PS exposure was induced by the dysregulation of key enzymes for the maintenance of membrane phospholipid asymmetry, which was from vitamin C-induced oxidative stress, and resultant disruption of calcium and thiol homeostasis. Indeed, the intravenous injection of vitamin C (0.5-1.0 g/kg) in rats in vivo significantly increased thrombosis. Notably, the prothrombotic effects of vitamin C were more prominent in RBCs isolated from cancer patients, who are at increased risks of thrombotic events. Vitamin C-induced procoagulant and prothrombotic activation of RBCs, and increased thrombosis in vivo. RBCs from cancer patients exhibited increased sensitivity to the prothrombotic effects of vitamin C, reflecting that intravenous gram-dose vitamin C therapy needs to be carefully revisited. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Timing of gamma irradiation and blood donor sex influences in vitro characteristics of red blood cells.

PubMed

de Korte, Dirk; Thibault, Louis; Handke, Wiebke; Harm, Sarah K; Morrison, Alex; Fitzpatrick, Aine; Marks, Denese C; Yi, Qi-Long; Acker, Jason P

2018-04-01

There are few studies investigating the effect of irradiation on red blood cells (RBCs) during storage. This study analyzed changes in in vitro quality of RBCs irradiated at several points during storage with the aim of providing evidence to support current maximum pre- and postirradiation storage limits. Each of seven participating centers produced four pools of 7 standard RBC units (SAGM, AS-3, or PAGGSM), which were then split back into 7 units. All units in a pool were from sex-matched blood donors. Every week during 6 weeks of refrigerated storage, 1 unit was irradiated, while 1 unit was not irradiated (control). Units were tested weekly for biochemical variables, morphology, and mechanical fragility. The earlier during storage that units were irradiated, the higher the hemolysis and K + at end of storage. Irrespective of the timing of irradiation, there was a rapid increase in extracellular K + , followed by a more gradual increase in hemolysis. ATP levels decreased faster in irradiated units and were reduced below accepted values if irradiated early. Irradiated female RBCs had an absolute lower hemolysis and K + level compared to male RBCs at all time points. The method of blood component manufacturing determined the absolute levels of hemolysis and potassium in irradiated and nonirradiated units, but did not influence the effect that timing of irradiation had on the in vitro quality characteristics. This study provides support for the current Council of Europe guidelines on the time limitations for the irradiation of RBCs. © 2017 AABB.

Safe transition to surgery: working differently to make blood transfusion process safer for elective surgery.

PubMed

Badjie, Karafa S W; Rogers, James C; Jenkins, Sarah M; Bundy, Kevin L; Stubbs, James R; Cima, Robert R

2015-09-01

Our institutional policy allows patients who are scheduled for elective surgery with no history of a pregnancy or blood transfusion in the preceding 3 months to have a presurgical sample (PSS) collected and tested up to 56 days before their scheduled surgery; however, our PSS TS completion rate in eligible patients before the morning of surgery was 83%. In 2011, a team was charged to develop a standardized process along with other process improvements while ensuring no increase in transfusion-related events. The team followed the DMAIC framework in appraising the effectiveness and efficiency of the current state process including baseline data collection such as PSS TS completion rate, number of eligible patients needing a PSS TS on the day of surgery, benchmarking, SSBO utilization, and future state mapping. First quarter (Q1) 2011 versus Q1 2012 postimplementation results showed significant improvements of the process including a 53% decrease in PSS TS on the day of surgery; a 13% increase in PSS TS completion before the morning of surgery; a 26% reduction in total XM RBCs; and a 58.8% reduction in XM RBCs not issued, plus a 47% decrease in RBC wastage. Q1 2011 versus Q1 2013 showed a 41% reduction in total XM RBCs and an 88.4% reduction in XM RBCs not issued but overall RBCs issued versus returned increased slightly and represents a future opportunity for improvement. The redesigned, transformational process eliminated SSBO and improved ordering process and PSS TS completion rate as well as blood product ordering and utilization. © 2015 AABB.

Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability.

PubMed

Smith, Alyson S; Nowak, Roberta B; Zhou, Sitong; Giannetto, Michael; Gokhin, David S; Papoin, Julien; Ghiran, Ionita C; Blanc, Lionel; Wan, Jiandi; Fowler, Velia M

2018-05-08

The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.

The study on RBC characteristic in paroxysmal nocturnal hemoglobinuria (PNH) patients using common path interferometric quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Park, Byung Jun; Won, Youngjae; Kim, Byungyeon; Lee, Seungrag

2016-03-01

We have studied the RBC membrane properties between a normal RBC and a RBC in Paroxysrnal nocturnal hemoglobinuria (PNH) patient using common path interferometric quantitative phase microscopy (CPIQPM). CPIQPM system has provided the subnanometer optical path length sensitivity on a millisecond. We have measured the dynamic thickness fluctuations of a normal RBC membrane and a RBC membrane in PNH patient over the whole cell surface with CPIQPM. PNH is a rare and serious disease of blood featured by destruction of red blood cells (RBCs). This destruction happens since RBCs show the defect of protein which protects RBCs from the immune system. We have applied CPIQPM to study the characteristic of RBC membrane in PNH patient. We have shown the morphological shape, volume, and projected surface for both different RBC types. The results have showed both RBCs had the similar shape with donut, but membrane fluctuations in PNH patient was shown to reveal the difference of temporal properties compared with a normal RBC. In order to demonstrate the practical tool of the CPIQPM technique, we have also obtained the time series thickness fluctuation outside a cell.

High ferritin levels have major effects on the morphology of erythrocytes in Alzheimer's disease.

PubMed

Bester, Janette; Buys, Antoinette V; Lipinski, Boguslaw; Kell, Douglas B; Pretorius, Etheresia

2013-01-01

Unliganded iron both contributes to the pathology of Alzheimer's disease (AD) and also changes the morphology of erythrocytes (RBCs). We tested the hypothesis that these two facts might be linked, i.e., that the RBCs of AD individuals have a variant morphology, that might have diagnostic or prognostic value. We included a literature survey of AD and its relationships to the vascular system, followed by a laboratory study. Four different microscopy techniques were used and results statistically compared to analyze trends between high and normal serum ferritin (SF) AD individuals. Light and scanning electron microscopies showed little difference between the morphologies of RBCs taken from healthy individuals and from normal SF AD individuals. By contrast, there were substantial changes in the morphology of RBCs taken from high SF AD individuals. These differences were also observed using confocal microscopy and as a significantly greater membrane stiffness (measured using force-distance curves). We argue that high ferritin levels may contribute to an accelerated pathology in AD. Our findings reinforce the importance of (unliganded) iron in AD, and suggest the possibility both of an early diagnosis and some means of treating or slowing down the progress of this disease.

Normalized levels of red blood cells expressing phosphatidylserine, their microparticles, and activated platelets in young patients with β-thalassemia following bone marrow transplantation.

PubMed

Klaihmon, Phatchanat; Vimonpatranon, Sinmanus; Noulsri, Egarit; Lertthammakiat, Surapong; Anurathapan, Usanarat; Sirachainan, Nongnuch; Hongeng, Suradej; Pattanapanyasat, Kovit

2017-10-01

Bone marrow transplantation (BMT) serves as the only curative treatment for patients with β-thalassemia major; however, hemostatic changes have been observed in these BMT patients. Aggregability of thalassemic red blood cells (RBCs) and increased red blood cell-derived microparticles (RMPs) expressing phosphatidylserine (PS) are thought to participate in thromboembolic events by initially triggering platelet activation. To our knowledge, there has been no report providing quantitation of these circulating PS-expressing RBCs and RMPs in young β-thalassemia patients after BMT. Whole blood from each subject was fluorescently labeled to detect RBC markers (CD235a) and annexin-V together with the known number TruCount™ beads. PS-expressing RBCs, RMPs, and activated platelets were identified by flow cytometry. In our randomized study, we found the decreased levels of three aforementioned factors compared to levels in patients receiving regular blood transfusion (RT). This study showed that BMT in β-thalassemia patients decreases the levels of circulating PS-expressing RBCs, their MPs, and procoagulant platelets when compared to patients who received RT. Normalized levels of these coagulation markers may provide the supportive evidence of the effectiveness of BMT for curing thalassemia.

A critical review of published methods for analysis of red cell antigen-antibody reactions by flow cytometry, and approaches for resolving problems with red cell agglutination.

PubMed

Arndt, Patricia A; Garratty, George

2010-07-01

Flow cytometry operators often apply familiar white blood cell (WBC) methods when studying red blood cell (RBC) antigens and antibodies. Some WBC methods are not appropriate for RBCs, as the analysis of RBCs requires special considerations, for example, avoidance of agglutination. One hundred seventy-six published articles from 88 groups studying RBC interactions were reviewed. Three fourths of groups used at least one unnecessary WBC procedure for RBCs, and about one fourth did not use any method to prevent/disperse RBC agglutination. Flow cytometric studies were performed to determine the effect of RBC agglutination on results and compare different methods of preventing and/or dispersing agglutination. The presence of RBC agglutinates have been shown to be affected by the type of pipette tip used for mixing RBC suspensions, the number of antigen sites/RBC, the type and concentration of primary antibody, and the type of secondary antibody. For quantitation methods, for example, fetal maternal hemorrhage, the presence of agglutinates have been shown to adversely affect results (fewer fetal D+ RBCs detected). Copyright 2010 Elsevier Inc. All rights reserved.

Newly developed photon-cell interactive Monte Carlo (pciMC) simulation for non-invasive and continuous diagnosis of blood during extracorporeal circulation support

NASA Astrophysics Data System (ADS)

Sakota, Daisuke; Takatani, Setsuo

2011-07-01

We have sought for non-invasive diagnosis of blood during the extracorporeal circulation support. To achieve the goal, we have newly developed a photon-cell interactive Monte Carlo (pciMC) model for optical propagation through blood. The pciMC actually describes the interaction of photons with 3-dimentional biconcave RBCs. The scattering is described by micro-scopical RBC boundary condition based on geometric optics. By using pciMC, we modeled the RBCs inside the extracorporeal circuit will be oriented by the blood flow. The RBCs' orientation was defined as their long axis being directed to the center of the circulation tube. Simultaneously the RBCs were allowed to randomly rotate about the long axis direction. As a result, as flow rate increased, the orientation rate increased and converged to approximately 22% at 0.5 L/min flow rate and above. And finally, by using this model, the pciMC non-invasively and absolutely predicted Hct and hemoglobin with the accuracies of 0.84+/-0.82 [HCT%] and 0.42+/-0.28 [g/dL] respectively against measurements by a blood gas analyzer.

Aged erythrocytes: a fine wine or sour grapes?

PubMed

Cohen, B; Matot, I

2013-12-01

Blood transfusion saves many lives but carries significant risk of injury. Currently, red blood cell (RBC) concentrates can be stored up to 42 days. Concerns have recently been raised about the safety and efficacy of transfusing stored RBCs. Refrigerated storage results in a 'storage lesion' that is reflected by metabolic derangements, RBC shape modification, rheological changes, oxidative injury to lipids and proteins, alterations in oxygen affinity and delivery, increased adhesion of RBCs to endothelial cells, and accumulation of bioactive substances in storage media. In animal models, transfusion of aged, but not fresh, RBCs induces organ injury, inflammation, coagulopathy, and impaired oxygen delivery. A number of clinical studies, mostly observational or retrospective and from a single centre, have reported an association between transfusion of older RBCs and increased clinically significant outcomes, such as increased morbidity and mortality in certain patient populations, including trauma, critical care, and cardiac surgery. Others, however, have failed to indicate an influence of RBC age on outcome. The quality of evidence is currently too poor to make recommendations to change current transfusion practice; however, the transfusion community looks forward to the results of randomized trials currently addressing the long-standing question regarding the effects of RBC storage on clinically significant outcomes.

Normal saline is associated with increased sickle red cell stiffness and prolonged transit times in a microfluidic model of the capillary system.

PubMed

Carden, Marcus A; Fay, Meredith; Sakurai, Yumiko; McFarland, Brynn; Blanche, Sydney; DiPrete, Caleb; Joiner, Clinton H; Sulchek, Todd; Lam, Wilbur A

2017-07-01

Vaso-occlusive crisis (VOC) is a complex process that occurs in patients with sickle cell disease (SCD) and is often associated with pain and urgent hospitalization. A major instigator of VOC is microvascular obstruction by pathologically stiffened sickle red blood cells (RBCs), and thus, therapy relies heavily on optimizing intravenous fluid (IVF) hydration to increase RBC deformability. However, no evidence-based guidelines regarding the choice of IVF currently exist. We therefore analyzed alterations in biomechanical properties of sickle RBCs isolated from patients with homozygous SCD (hemoglobin SS) after exposure to different osmolarities of clinical IVF formulations. Atomic force microscopy (AFM) was used to assess stiffness of RBCs after exposure to different IVFs. A microfluidic model of the human capillary system was used to assess transit time (TT) and propensity to occlusion after exposure to the different IVF formulations. Sickle RBCs exposed to normal saline (NS) had increased stiffness, TTs, and propensity to microchannel occlusion compared to other osmolarities. NS, an IVF formulation often used to treat patients with SCD during VOC, may induce localized microvascular obstruction due to alterations of sickle RBC biomechanical properties. © 2017 John Wiley & Sons Ltd.

Age-related defects in erythrocyte 2,3-diphosphoglycerate metabolism in dementia.

PubMed

Kaminsky, Yury G; Reddy, V Prakash; Ashraf, Ghulam Md; Ahmad, Ausaf; Benberin, Valery V; Kosenko, Elena A; Aliev, Gjumrakch

2013-01-01

Alzheimer disease (AD) is the most common dementing illness. Metabolic defects in the brain with aging contribute to the pathogenesis of AD. These changes can be found systematically and thus can be used as potential biomarkers. Erythrocytes (RBCs) are passive "reporter cells" that are not well studied in AD. In the present study, we analyzed an array of glycolytic and related enzymes and intermediates in RBCs from patients with AD and non-Alzheimer dementia (NA), age-matched controls (AC) and young adult controls (YC). AD is characterized by higher activities of hexokinase, phosphofructokinase, and bisphosphoglycerate mutase and bisphosphoglycerate phosphatase in RBCs. In our study, we observed that glycolytic and related enzymes displayed significantly lower activities in AC. However, similar or significantly higher activities were observed in AD and NA groups as compared to YC group. 2,3-diphosphoglycerate (2,3-DPG) levels were significantly decreased in AD and NA patients. The pattern of changes between groups in the above indices strongly correlates with each other. Collectively, our data suggested that AD and NA patients are associated with chronic disturbance of 2,3-DPG metabolism in RBCs. These defects may play a pivotal role in physiological processes, which predispose elderly subjects to AD and NA.

The Age of BLood Evaluation (ABLE) randomised controlled trial: description of the UK-funded arm of the international trial, the UK cost-utility analysis and secondary analyses exploring factors associated with health-related quality of life and health-care costs during the 12-month follow-up.

PubMed

Walsh, Timothy S; Stanworth, Simon; Boyd, Julia; Hope, David; Hemmatapour, Sue; Burrows, Helen; Campbell, Helen; Pizzo, Elena; Swart, Nicholas; Morris, Stephen

2017-10-01

At present, red blood cells (RBCs) are stored for up to 42 days prior to transfusion. The relative effectiveness and safety of different RBC storage times prior to transfusion is uncertain. To assess the clinical effectiveness and cost-effectiveness of transfusing fresher RBCs (stored for ≤ 7 days) compared with current standard-aged RBCs in critically ill patients requiring blood transfusions. The international Age of BLood Evaluation (ABLE) trial was a multicentre, randomised, blinded trial undertaken in Canada, the UK, the Netherlands and France. The UK trial was funded to contribute patients to the international trial and undertake a UK-specific health economic evaluation. Twenty intensive care units (ICUs) in the UK, as part of 64 international centres. Critically ill patients aged ≥ 18 years (≥ 16 years in Scotland) expected to require mechanical ventilation for ≥ 48 hours and requiring a first RBC transfusion during the first 7 days in the ICU. All decisions to transfuse RBCs were made by clinicians. One patient group received exclusively fresh RBCs stored for ≤ 7 days whenever transfusion was required from randomisation until hospital discharge. The other group received standard-issue RBCs throughout their hospital stay. The primary outcome was 90-day mortality. Secondary outcomes included development of organ dysfunction, new thrombosis, infections and transfusion reactions. The primary economic evaluation was a cost-utility analysis. The international trial took place between March 2009 and October 2014 (UK recruitment took place between January 2012 and October 2014). In total, 1211 patients were assigned to receive fresh blood and 1219 patients to receive standard-aged blood. RBCs were stored for a mean of 6.1 days [standard deviation (SD) ± 4.9 days] in the group allocated to receive fresh blood and 22.0 days (SD ± 8.4 days) in the group allocated to receive standard-aged blood. Patients received a mean of 4.3 RBC units (SD ± 5.2 RBC units) and 4.3 RBC units (SD ± 5.5 RBC units) in the groups receiving fresh blood and standard-aged blood, respectively. At 90 days, 37.0% of patients in the group allocated to receive fresh blood and 35.3% of patients in the group allocated to receive standard-aged blood had died {absolute risk difference 1.7% [95% confidence interval (CI) -2.1% to 5.5%]}. There were no between-group differences in any secondary outcomes. The UK cohort comprised 359 patients randomised and followed up for 12 months for the cost-utility analysis. UK patients had similar characteristics and outcomes to the international cohort. Mean total costs per patient were £32,346 (95% CI £29,306 to £35,385) in the group allocated to receive fresh blood and £33,353 (95% CI £29,729 to £36,978) in the group allocated to receive standard-aged blood. Approximately 85% of the total costs were incurred during the index hospital admission. There were no significant cost differences between the two groups [mean incremental costs for those receiving fresh vs. standard-aged blood: -£231 (95% CI -£4876 to £4415)], nor were there significant differences in outcomes (mean difference in quality-adjusted life-years -0.010, 95% CI -0.078 to 0.057). Adverse effects from the exclusive use of older RBCs compared with standard or fresh RBCs cannot be excluded. The use of RBCs aged ≤ 7 days confers no clinical or economic benefit in critically ill patients compared with standard-aged RBCs. Future studies should address the safety of RBCs near the end of the current permitted storage age. Current Controlled Trials ISRCTN44878718. This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment ; Vol. 21, No. 62. See the NIHR Journals Library website for further project information. The international ABLE trial was also supported by peer-reviewed grants from the Canadian Institutes of Health Research (177453), Fonds de Recherche du Québec - Santé (24460), the French Ministry of Health Programme Hospitalier de Recherche Clinique (12.07, 2011) and by funding from Établissement Français du Sang and Sanquin Blood Supply.

Development of Small-molecule HIV Entry Inhibitors Specifically Targeting gp120 or gp41

PubMed Central

Lu, Lu; Yu, Fei; Cai, Lifeng; Debnath, Asim K.; Jiang, Shibo

2015-01-01

Human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein surface subunit gp120 and transmembrane subunit gp41 play important roles in HIV-1 entry, thus serving as key targets for the development of HIV-1 entry inhibitors. T20 peptide (enfuvirtide) is the first U.S. FDA-approved HIV entry inhibitor; however, its clinical application is limited by the lack of oral availability. Here, we have described the structure and function of the HIV-1 gp120 and gp41 subunits and reviewed advancements in the development of small-molecule HIV entry inhibitors specifically targeting these two Env glycoproteins. We then compared the advantages and disadvantages of different categories of HIV entry inhibitor candidates and further predicted the future trend of HIV entry inhibitor development. PMID:26324044

Implications of High Molecular Divergence of Nuclear rRNA and Phylogenetic Structure for the Dinoflagellate Prorocentrum (Dinophyceae, Prorocentrales).

PubMed

Boopathi, Thangavelu; Faria, Daphne Georgina; Cheon, Ju-Yong; Youn, Seok Hyun; Ki, Jang-Seu

2015-01-01

The small and large nuclear subunit molecular phylogeny of the genus Prorocentrum demonstrated that the species are dichotomized into two clades. These two clades were significantly different (one-factor ANOVA, p < 0.01) with patterns compatible for both small and large subunit Bayesian phylogenetic trees, and for a larger taxon sampled dinoflagellate phylogeny. Evaluation of the molecular divergence levels showed that intraspecies genetic variations were significantly low (t-test, p < 0.05), than those for interspecies variations (> 2.9% and > 26.8% dissimilarity in the small and large subunit [D1/D2], respectively). Based on the calculated molecular divergence, the genus comprises two genetically distinct groups that should be considered as two separate genera, thereby setting the pace for major systematic changes for the genus Prorocentrum sensu Dodge. Moreover, the information presented in this study would be useful for improving species identification, detection of novel clades from environmental samples. © 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.

PCR amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues.

PubMed

Villand, P; Aalen, R; Olsen, O A; Lüthi, E; Lönneborg, A; Kleczkowski, L A

1992-06-01

Several cDNAs encoding the small and large subunit of ADP-glucose pyrophosphorylase (AGP) were isolated from total RNA of the starchy endosperm, roots and leaves of barley by polymerase chain reaction (PCR). Sets of degenerate oligonucleotide primers, based on previously published conserved amino acid sequences of plant AGP, were used for synthesis and amplification of the cDNAs. For either the endosperm, roots and leaves, the restriction analysis of PCR products (ca. 550 nucleotides each) has revealed heterogeneity, suggesting presence of three transcripts for AGP in the endosperm and roots, and up to two AGP transcripts in the leaf tissue. Based on the derived amino acid sequences, two clones from the endosperm, beps and bepl, were identified as coding for the small and large subunit of AGP, respectively, while a leaf transcript (blpl) encoded the putative large subunit of AGP. There was about 50% identity between the endosperm clones, and both of them were about 60% identical to the leaf cDNA. Northern blot analysis has indicated that beps and bepl are expressed in both the endosperm and roots, while blpl is detectable only in leaves. Application of the PCR technique in studies on gene structure and gene expression of plant AGP is discussed.

Velocity measurements of heterogeneous RBC flow in capillary vessels using dynamic laser speckle signal

PubMed Central

Li, Chenxi; Wang, Ruikang

2017-01-01

Abstract. We propose an approach to measure heterogeneous velocities of red blood cells (RBCs) in capillary vessels using full-field time-varying dynamic speckle signals. The approach utilizes a low coherent laser speckle imaging system to record the instantaneous speckle pattern, followed by an eigen-decomposition-based filtering algorithm to extract dynamic speckle signal due to the moving RBCs. The velocity of heterogeneous RBC flows is determined by cross-correlating the temporal dynamic speckle signals obtained at adjacent locations. We verify the approach by imaging mouse pinna in vivo, demonstrating its capability for full-field RBC flow mapping and quantifying flow pattern with high resolution. It is expected to investigate the dynamic action of RBCs flow in capillaries under physiological changes. PMID:28384709

Stretching of red blood cells using an electro-optics trap.

PubMed

Haque, Md Mozzammel; Moisescu, Mihaela G; Valkai, Sándor; Dér, András; Savopol, Tudor

2015-01-01

The stretching stiffness of Red Blood Cells (RBCs) was investigated using a combination of an AC dielectrophoretic apparatus and a single-beam optical tweezer. The experiments were performed at 10 MHz, a frequency high enough to avoid conductivity losses, but below the second turnover point between positive and negative dielectrophoresis. By measuring the geometrical parameters of single healthy human RBCs as a function of the applied voltage, the elastic modulus of RBCs was determined (µ = 1.80 ± 0.5 µN/m) and compared with similar values of the literature got by other techniques. The method is expected to be an easy-to-use, alternative tool to determine the mechano-elastic properties of living cells, and, on this basis, to distinguish healthy and diseased cells.

Functional conservation of RNA polymerase II in fission and budding yeasts.

PubMed

Shpakovski, G V; Gadal, O; Labarre-Mariotte, S; Lebedenko, E N; Miklos, I; Sakurai, H; Proshkin, S A; Van Mullem, V; Ishihama, A; Thuriaux, P

2000-02-04

The complementary DNAs of the 12 subunits of fission yeast (Schizosaccharomyces pombe) RNA polymerase II were expressed from strong promoters in Saccharomyces cerevisiae and tested for heterospecific complementation by monitoring their ability to replace in vivo the null mutants of the corresponding host genes. Rpb1 and Rpb2, the two largest subunits and Rpb8, a small subunit shared by all three polymerases, failed to support growth in S. cerevisiae. The remaining nine subunits were all proficient for heterospecific complementation and led in most cases to a wild-type level of growth. The two alpha-like subunits (Rpb3 and Rpb11), however, did not support growth at high (37 degrees C) or low (25 degrees C) temperatures. In the case of Rpb3, growth was restored by increasing the gene dosage of the host Rpb11 or Rpb10 subunits, confirming previous evidence of a close genetic interaction between these three subunits. Copyright 2000 Academic Press.

Inhibition of herpesvirus and influenza virus replication by blocking polymerase subunit interactions.

PubMed

Palù, Giorgio; Loregian, Arianna

2013-09-01

Protein-protein interactions (PPIs) play a key role in many biological processes, including virus replication in the host cell. Since most of the PPIs are functionally essential, a possible strategy to inhibit virus replication is based on the disruption of viral protein complexes by peptides or small molecules that interfere with subunit interactions. In particular, an attractive target for antiviral drugs is the binding between the subunits of essential viral enzymes. This review describes the development of new antiviral compounds that inhibit herpesvirus and influenza virus replication by blocking interactions between subunit proteins of their polymerase complexes. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular phylogeny of Laetiporus and other brown rot polypore genera in North America

Treesearch

Daniel L. Lindner; Mark T. Banik

2008-01-01

Phylogenetic relationships were investigated among North American species of Laetiporus, Leptoporus, Phaeolus, Pycnoporellus, and Wolfiporia using ITS, nuclear large subunit and mitochondrial small subunit rDNA sequences. Members of these genera have poroid hymenophores, simple septate hyphae and cause brown rots in a variety of...

Sequence and properties of HMW subunit 1Bx20 from pasta wheat (Triticum durum) which is associated with poor end use properties.

PubMed

Shewry, P R; Gilbert, S M; Savage, A W J; Tatham, A S; Wan, Y-F; Belton, P S; Wellner, N; D'Ovidio, R; Békés, F; Halford, N G

2003-02-01

The gene encoding high-molecular-weight (HMW) subunit 1Bx20 was isolated from durum wheat cv. Lira. It encodes a mature protein of 774 amino acid residues with an M(r) of 83,913. Comparison with the sequence of subunit 1Bx7 showed over 96% identity, the main difference being the substitution of two cysteine residues in the N-terminal domain of subunit 1Bx7 with tyrosine residues in 1Bx20. Comparison of the structures and stabilities of the two subunits purified from wheat using Fourier-transform infra-red and circular dichroism spectroscopy showed no significant differences. However, incorporation of subunit 1Bx7 into a base flour gave increased dough strength and stability measured by Mixograph analysis, while incorporation of subunit 1Bx20 resulted in small positive or negative effects on the parameters measured. It is concluded that the different effects of the two subunits could relate to the differences in their cysteine contents, thereby affecting the cross-linking and hence properties of the glutenin polymers.

Function of Protein Phosphatase 2A in Control of Proliferation: Isolation and Analysis of Dominant-Defective Mutants

DTIC Science & Technology

1999-06-01

subunits are expressed ubiquitously and appear to be encoded by small and quite homogeneous gene families. In plants , however, A and C subunit gene...1996). In both plants and animals, different B subunit isoforms are encoded by two or more unrelated gene families, some of which are expressed in a...PP2A functions in whole plants and in mammalian tissue culture cells. This genetic system may also prove useful for analyzing interactions between

Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells.

PubMed

Akide-Ndunge, Oscar Bate; Tambini, Elisa; Giribaldi, Giuliana; McMillan, Paul J; Müller, Sylke; Arese, Paolo; Turrini, Francesco

2009-05-29

Plasmodium falciparum-parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy. Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70-2/70-3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane. In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs. Protein expression of antioxidant enzymes was also increased in oxidatively-stressed trophozoites. Results indicated that mRNA expression of parasite antioxidant enzymes and HSPs was co-ordinated and stage-dependent. Secondly, both systems were redox-responsive and showed remarkably increased and co-ordinated expression in oxidatively-stressed parasites and in parasites growing in antioxidant blunted G6PD-deficient RBCs. Lastly, as important anti-malarials either increase oxidant stress or impair antioxidant defense, results may encourage the inclusion of anti-HSP molecules in anti-malarial combined drugs.

Subunit mass fingerprinting of mitochondrial complex I.

PubMed

Morgner, Nina; Zickermann, Volker; Kerscher, Stefan; Wittig, Ilka; Abdrakhmanova, Albina; Barth, Hans-Dieter; Brutschy, Bernhard; Brandt, Ulrich

2008-10-01

We have employed laser induced liquid bead ion desorption (LILBID) mass spectrometry to determine the total mass and to study the subunit composition of respiratory chain complex I from Yarrowia lipolytica. Using 5-10 pmol of purified complex I, we could assign all 40 known subunits of this membrane bound multiprotein complex to peaks in LILBID subunit fingerprint spectra by comparing predicted protein masses to observed ion masses. Notably, even the highly hydrophobic subunits encoded by the mitochondrial genome were easily detectable. Moreover, the LILBID approach allowed us to spot and correct several errors in the genome-derived protein sequences of complex I subunits. Typically, the masses of the individual subunits as determined by LILBID mass spectrometry were within 100 Da of the predicted values. For the first time, we demonstrate that LILBID spectrometry can be successfully applied to a complex I band eluted from a blue-native polyacrylamide gel, making small amounts of large multiprotein complexes accessible for subunit mass fingerprint analysis even if they are membrane bound. Thus, the LILBID subunit mass fingerprint method will be of great value for efficient proteomic analysis of complex I and its assembly intermediates, as well as of other water soluble and membrane bound multiprotein complexes.

Structural and functional aspects of hetero-oligomers formed by the small heat shock proteins αB-crystallin and HSP27.

PubMed

Aquilina, J Andrew; Shrestha, Sudichhya; Morris, Amie M; Ecroyd, Heath

2013-05-10

αB-crystallin and HSP27 are mammalian intracellular small heat shock proteins. These proteins exchange subunits in a rapid and temperature-dependent manner. This facile subunit exchange suggests that differential expression could be used by the cell to regulate the response to stress. A robust technique defines parameters for the dynamic interaction between the major mammalian small heat shock proteins. Small heat shock proteins (sHSPs) exist as large polydisperse species in which there is constant dynamic subunit exchange between oligomeric and dissociated forms. Their primary role in vivo is to bind destabilized proteins and prevent their misfolding and aggregation. αB-Crystallin (αB) and HSP27 are the two most widely distributed and most studied sHSPs in the human body. They are coexpressed in different tissues, where they are known to associate with each other to form hetero-oligomeric complexes. In this study, we aimed to determine how these two sHSPs interact to form hetero-oligomers in vitro and whether, by doing so, there is an increase in their chaperone activity and stability compared with their homo-oligomeric forms. Our results demonstrate that HSP27 and αB formed polydisperse hetero-oligomers in vitro, which had an average molecular mass that was intermediate of each of the homo-oligomers and which were more thermostable than αB, but less so than HSP27. The hetero-oligomer chaperone function was found to be equivalent to that of αB, with each being significantly better in preventing the amorphous aggregation of α-lactalbumin and the amyloid fibril formation of α-synuclein in comparison with HSP27. Using mass spectrometry to monitor subunit exchange over time, we found that HSP27 and αB exchanged subunits 23% faster than the reported rate for HSP27 and αA and almost twice that for αA and αB. This represents the first quantitative evaluation of αB/HSP27 subunit exchange, and the results are discussed in the broader context of regulation of function and cellular proteostasis.

The c.64_80del SMIM1 allele is segregating in the Hutterite population.

PubMed

Coghlan, Gail; Zelinski, Teresa

2016-04-01

The high-incidence red blood cell (RBC) antigen Vel is coded by SMIM1 (small-membrane molecule 1 gene), where a homozygous 17 base pair deletion underlies the majority of Vel- phenotypes. Because anti-Vel has been reported to cause severe hemolytic transfusion reactions and periodically hemolytic disease of the newborn and fetus, identification of individuals negative for Vel is clinically important. RBCs from the members of a large three-generation Hutterite family were serologically determined to be Vel+(w) . Genomic DNA from these family members was polymerase chain reaction amplified and analyzed for SMIM1 polymorphisms by either Sanger sequencing or restriction fragment length polymorphisms. SMIM1 genotyping was also conducted on DNA from an additional 104 Hutterites. All family members whose RBCs weakly expressed the Vel antigen were found to be heterozygous for the c.64_80del mutation in SMIM1. Of the 104 additional Hutterite samples, four were found to be heterozygous for the same SMIM1 mutation. After emigrating to the United States and Canada, the Hutterite population has expanded dramatically. Alleles that initially entered the population have been maintained within the population. The c.64_80del null allele of SMIM1 is one such allele, thus having implications for transfusion medicine and child or maternal health. © 2015 AABB.

Capillary red blood cell velocimetry by phase-resolved optical coherence tomography

NASA Astrophysics Data System (ADS)

Tang, Jianbo; Erdener, Sefik Evren; Fu, Buyin; Boas, David A.

2018-02-01

Quantitative measurement of blood flow velocity in capillaries is challenging due to their small size (around 5-10 μm), and the discontinuity and single-file feature of RBCs flowing in a capillary. In this work, we present a phase-resolved Optical Coherence Tomography (OCT) method for accurate measurement of the red blood cell (RBC) speed in cerebral capillaries. To account for the discontinuity of RBCs flowing in capillaries, we applied an M-mode scanning strategy that repeated A-scans at each scanning position for an extended time. As the capillary size is comparable to the OCT resolution size (3.5×3.5×3.5μm), we applied a high pass filter to remove the stationary signal component so that the phase information of the dynamic component (i.e. from the moving RBC) could be enhanced to provide an accurate estimate of the RBC axial speed. The phase-resolved OCT method accurately quantifies the axial velocity of RBC's from the phase shift of the dynamic component of the signal. We validated our measurements by RBC passage velocimetry using the signal magnitude of the same OCT time series data. These proposed method of capillary velocimetry proved to be a robust method of mapping capillary RBC speeds across the micro-vascular network.

Monitoring humans for somatic mutation in the endogenous PIG-a gene using red blood cells.

PubMed

Dobrovolsky, Vasily N; Elespuru, Rosalie K; Bigger, C Anita H; Robison, Timothy W; Heflich, Robert H

2011-12-01

The endogenous X-linked PIG-A gene is involved in the synthesis of glycosyl phosphatidyl inositol (GPI) anchors that tether specific protein markers to the exterior of mammalian cell cytoplasmic membranes. Earlier studies in rodent models indicate that Pig-a mutant red blood cells (RBCs) can be induced in animals treated with genotoxic agents, and that flow cytometry can be used to identify rare RBCs deficient in the GPI-anchored protein, CD59, as a marker of Pig-a gene mutation. We investigated if a similar approach could be used for detecting gene mutation in humans. We first determined the frequency of spontaneous CD59-deficient RBCs (presumed PIG-A mutants) in 97 self-identified healthy volunteers. For most subjects, the frequency of CD59-deficient RBCs was low (average of 5.1 ± 4.9 × 10(-6) ; median of 3.8 × 10(-6) and mutant frequency less than 8 × 10(-6) for 75% of subjects), with a statistically significant difference in median mutant frequencies between males and females. PIG-A RBC mutant frequency displayed poor correlation with the age and no correlation with the smoking status of the subjects. Also, two individuals had markedly increased CD59-deficient RBC frequencies of ∼300 × 10(-6) and ∼100 × 10(-6) . We then monitored PIG-A mutation in 10 newly diagnosed cancer patients undergoing chemotherapy with known genotoxic drugs. The frequency of CD59-deficient RBCs in the blood of the patients was measured before the start of chemotherapy and three times over a period of ∼6 months while on/after chemotherapy. Responses were generally weak, most observations being less than the median mutant frequency for both males and females; the greatest response was an approximate three-fold increase in the frequency of CD59-deficient RBCs in one patient treated with a combination of cisplatin and etoposide. These results suggest that the RBC PIG-A assay can be adopted to measuring somatic cell mutation in humans. Further research is necessary to determine the assay's sensitivity in detecting mutations induced by genotoxic agents acting via different mechanisms. Copyright © 2011 Wiley-Liss, Inc.

Deleterious Effects of Intra-arterial Administration of Particulate Steroids on Microvascular Perfusion in a Mouse Model.

PubMed

Laemmel, Elisabeth; Segal, Nicolas; Mirshahi, Massoud; Azzazene, Dalel; Le Marchand, Sylvie; Wybier, Marc; Vicaut, Eric; Laredo, Jean-Denis

2016-06-01

Purpose To determine the in vivo effects of several particulate steroids on microvascular perfusion by using intravital microscopy in a mice model and to investigate the in vitro interactions between these particulate steroids and red blood cells (RBCs). Materials and Methods The study was conducted in agreement with the guidelines of the National Committee of Ethic Reflection on Animal Experimentation. By using intravital microscopy of mouse cremaster muscle, the in vivo effects of several particulate steroids on microvascular perfusion were assessed. Four to five mice were allocated to each of the following treatment groups: saline solution, dexamethasone sodium phosphate, a nonparticulate steroid, and the particulate steroids cortivazol, methylprednisolone, triamcinolone, and prednisolone. By using in vitro blood microcinematography and electron microscopy, the interactions between these steroids and human RBCs were studied. All results were analyzed by using nonparametric tests. Results With prednisolone, methylprednisolone, or triamcinolone, blood flow was rapidly and completely stopped in all the arterioles and venules (median RBC velocity in first-order arterioles, 5 minutes after administration was zero for these three groups) compared with a limited effect in mice treated with saline, dexamethasone, and cortivazol (20.3, 21.3, and 27.5 mm/sec, respectively; P < .003). This effect was associated with a large decrease in the functional capillary density (4.21, 0, and 0 capillaries per millimeter for methylprednisolone, triamcinolone, or prednisolone, respectively, vs 21.0, 21.4, and 19.1 capillaries per millimeter in mice treated with saline, dexamethasone, and cortivazol, respectively; P < .003). This was because of the rapid formation of RBC aggregates. However, no change in microvascular perfusion was associated with administration of cortivazol or dexamethasone. In vitro experiments confirmed the formation of RBC aggregates associated with the transformation of RBCs into spiculated RBCs with the same steroids. Conclusion Several particulate steroids have an immediate and massive effect on microvascular perfusion because of formation of RBC aggregates associated with the transformation of RBCs into spiculated RBCs. (©) RSNA, 2016 Online supplemental material is available for this article.

Anti-U-like as an alloantibody in S-s-U- and S-s-U+(var) black people.

PubMed

Peyrard, Thierry; Lam, Yin; Saison, Carole; Arnaud, Lionel; Babinet, Jérôme; Rouger, Philippe; Bierling, Philippe; Janvier, Daniel

2012-03-01

S, s, and U antigens belong to the MNS system. They are carried by glycophorin B (GPB), encoded by GYPB. Black people with the low-prevalence S-s- phenotype, either U- or U+(var), can make a clinically significant anti-U. Anti-U-like, a cold immunoglobulin G autoantibody quite commonly observed in S-s+U+ black persons, was previously described to be nonreactive with ficin-, α-chymotrypsin-, and pronase-treated red blood cells (RBCs); nonreactive or weakly reactive with papain-treated RBCs; and reactive with trypsin-treated RBCs. Here we describe, in S-s- people from different molecular backgrounds, an alloantibody to a high-prevalence GPB antigen, which presents the same pattern of reactivity with proteases as autoanti-U-like. Four S-s- patients with an alloantibody to a high-prevalence GPB antigen were investigated by serologic and molecular methods. An alloantibody was observed in two S-s-U-/Del GYPB, one S-s-U+(var)/GYPB(P2), and one S-s-U+(var)/GYPB(NY) patients. As this alloantibody showed the same pattern of reactivity with proteases as autoanti-U-like, we decided to name it "anti-U-like." Anti-U-like made by the two S-s-U- patients was reactive with the S-s-U+(var) RBCs of the two other patients. S-s-U-/Del GYPB, S-s-U+(var)/GYPB(P2), and S-s-U+(var)/GYPB(NY) patients can make an alloanti-U-like. Anti-U-like made by S-s-U- people appears reactive with GYPB(P2) and GYPB(NY) RBCs, which both express a weak and partial U-like reactivity. We recommend transfusing S-s-U- RBCs in S-s-U- patients showing alloanti-U-like. Our study contributes to a better understanding of alloimmunization to GPB in black people and confirms importance of genotyping in S-s- patients, especially those with sickle cell disease to be frequently transfused. © 2011 American Association of Blood Banks.

Prehospital plasma resuscitation associated with improved neurologic outcomes after traumatic brain injury.

PubMed

Hernandez, Matthew C; Thiels, Cornelius A; Aho, Johnathon M; Habermann, Elizabeth B; Zielinski, Martin D; Stubbs, James A; Jenkins, Donald H; Zietlow, Scott P

2017-09-01

Trauma-related hypotension and coagulopathy worsen secondary brain injury in patients with traumatic brain injuries (TBIs). Early damage control resuscitation with blood products may mitigate hypotension and coagulopathy. Preliminary data suggest resuscitation with plasma in large animals improves neurologic function after TBI; however, data in humans are lacking. We retrospectively identified all patients with multiple injuries age >15 years with head injuries undergoing prehospital resuscitation with blood products at a single Level I trauma center from January 2002 to December 2013. Inclusion criteria were prehospital resuscitation with either packed red blood cells (pRBCs) or thawed plasma as sole colloid resuscitation. Patients who died in hospital and those using anticoagulants were excluded. Primary outcomes were Glasgow Outcomes Score Extended (GOSE) and Disability Rating Score (DRS) at dismissal and during follow-up. Of 76 patients meeting inclusion criteria, 53% (n = 40) received prehospital pRBCs and 47% (n = 36) received thawed plasma. Age, gender, injury severity or TBI severity, arrival laboratory values, and number of prehospital units were similar (all p > 0.05). Patients who received thawed plasma had an improved neurologic outcome compared to those receiving pRBCs (median GOSE 7 [7-8] vs. 5.5 [3-7], p < 0.001). Additionally, patients who received thawed plasma had improved functionality compared to pRBCs (median DRS 2 [1-3.5] vs. 9 [3-13], p < 0.001). Calculated GOSE and DRS scores during follow-up, median 6 [5-7] months, demonstrated increased function in those resuscitated with thawed plasma compared to pRBCs by both median GOSE (8 [7-8] vs. 6 [6-7], p < 0.001) and DRS (0 [0-1] vs. 4 [2-8], p < 0.001). In critically injured trauma patients with TBI, early resuscitation with thawed plasma is associated with improved neurologic and functional outcomes at discharge and during follow-up compared to pRBCs alone. These preliminary data support the further investigation and use of plasma in the resuscitation of critically injured TBI patients. Therapeutic, level V.

Three-dimensional counting of morphologically normal human red blood cells via digital holographic microscopy

NASA Astrophysics Data System (ADS)

Yi, Faliu; Moon, Inkyu; Lee, Yeon H.

2015-01-01

Counting morphologically normal cells in human red blood cells (RBCs) is extremely beneficial in the health care field. We propose a three-dimensional (3-D) classification method of automatically determining the morphologically normal RBCs in the phase image of multiple human RBCs that are obtained by off-axis digital holographic microscopy (DHM). The RBC holograms are first recorded by DHM, and then the phase images of multiple RBCs are reconstructed by a computational numerical algorithm. To design the classifier, the three typical RBC shapes, which are stomatocyte, discocyte, and echinocyte, are used for training and testing. Nonmain or abnormal RBC shapes different from the three normal shapes are defined as the fourth category. Ten features, including projected surface area, average phase value, mean corpuscular hemoglobin, perimeter, mean corpuscular hemoglobin surface density, circularity, mean phase of center part, sphericity coefficient, elongation, and pallor, are extracted from each RBC after segmenting the reconstructed phase images by using a watershed transform algorithm. Moreover, four additional properties, such as projected surface area, perimeter, average phase value, and elongation, are measured from the inner part of each cell, which can give significant information beyond the previous 10 features for the separation of the RBC groups; these are verified in the experiment by the statistical method of Hotelling's T-square test. We also apply the principal component analysis algorithm to reduce the dimension number of variables and establish the Gaussian mixture densities using the projected data with the first eight principal components. Consequently, the Gaussian mixtures are used to design the discriminant functions based on Bayesian decision theory. To improve the performance of the Bayes classifier and the accuracy of estimation of its error rate, the leaving-one-out technique is applied. Experimental results show that the proposed method can yield good results for calculating the percentage of each typical normal RBC shape in a reconstructed phase image of multiple RBCs that will be favorable to the analysis of RBC-related diseases. In addition, we show that the discrimination performance for the counting of normal shapes of RBCs can be improved by using 3-D features of an RBC.

Non-polar lipids accumulate during storage of transfusion products and do not contribute to the onset of transfusion-related acute lung injury.

PubMed

Peters, A L; Vervaart, M A T; van Bruggen, R; de Korte, D; Nieuwland, R; Kulik, W; Vlaar, A P J

2017-01-01

The accumulation of non-polar lipids arachidonic acid, 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE and 15-HETE during storage of transfusion products may play a role in the onset of transfusion-related acute lung injury (TRALI), a syndrome of respiratory distress after transfusion. We investigated non-polar lipid accumulation in red blood cells (RBCs) stored for 42 days, plasma stored for 7 days at either 4 or 20°C and platelet (PLT) transfusion products stored for 7 days. Furthermore, we investigated whether transfusion of RBCs with increased levels of non-polar lipids induces TRALI in a 'two-hit' human volunteer model. All products were produced following Dutch Blood Bank protocols and are according to European standards. Non-polar lipids were measured with high-performance liquid chromotography followed by mass spectrometry. All non-polar lipids increased in RBCs after 21 days of storage compared to baseline. The non-polar lipid concentration in plasma increased significantly, and the increase was even more pronounced in products stored at 20°C. In platelets, baseline levels of 5-HETE and 15-HETE were higher than in RBCs or plasma. However, the non-polar lipids did not change significantly during storage of PLT products. Infusion of RBCs with increased levels of non-polar lipids did not induce TRALI in LPS-primed human volunteers. We conclude that non-polar lipids accumulate in RBC and plasma transfusion products and that accumulation is temperature dependent. Accumulation of non-polar lipids does not appear to explain the onset of TRALI (Dutch Trial Register - NTR4455). © 2016 International Society of Blood Transfusion.

Effect of light curing units on the polymerization of bulk fill resin-based composites.

PubMed

Shimokawa, Carlos Alberto Kenji; Turbino, Míriam Lacalle; Giannini, Marcelo; Braga, Roberto Ruggiero; Price, Richard Bengt

2018-05-22

To determine the potential effect of four different light curing units (LCUs) on the curing profile of two bulk fill resin-based composites (RBCs). Four LCUs (Bluephase 20i, Celalux 3, Elipar DeepCure-S and Valo Grand) were used to light cure two RBCs (Filtek Bulk Fill Posterior Restorative and Tetric EvoCeram Bulk Fill). The effective tip diameter, radiant power, radiant emittance, emission spectrum and light beam profile of the LCUs were measured. Knoop microhardness was measured at the top and bottom surfaces of RBC specimens that were 12-mm in diameter and 4-mm deep (n=5). The distribution of the spectral radiant power that was delivered to the surface of the specimen and the light transmission through the 4-mm thick specimens was measured using an integrating sphere. Two-way ANOVA and Tukey tests (α=0.05) were applied. The Valo Grand produced the most homogeneous microhardness across the surfaces of the RBCs (p>0.05). When the Celalux 3, Bluephase 20i and Elipar DeepCure-S lights were used, the center of the specimens achieved greater hardness values compared to their outer regions (p<0.05). Approximately 10% of the radiant power delivered to the top reached the bottom of the specimen, although almost no violet light passed through 4mm of either RBC. A positive correlation was observed between the radiant exposure and microhardness. The characteristics of the LCUs influenced the photoactivation of the RBCs. The use of a wide tip with a homogeneous light distribution is preferred when light curing RBCs using a bulk curing technique. Copyright © 2018. Published by Elsevier Inc.

Sepsis does not alter red blood cell glucose metabolism or Na+ concentration: A 2H-, 23Na-NMR study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hotchkiss, R.S.; Song, S.K.; Ling, C.S.

The effects of sepsis on intracellular Na+ concentration ((Na+)i) and glucose metabolism were examined in rat red blood cells (RBCs) by using 23Na- and 2H-nuclear magnetic resonance (NMR) spectroscopy. Sepsis was induced in 15 halothane-anesthetized female Sprague-Dawley rats by using the cecal ligation and perforation technique; 14 control rats underwent cecal manipulation without ligation. The animals were fasted for 36 h, but allowed free access to water. At 36 h postsurgery, RBCs were examined by 23Na-NMR by using dysprosium tripolyphosphate as a chemical shift reagent. Human RBCs from 17 critically ill nonseptic patients and from 7 patients who were diagnosedmore » as septic were also examined for (Na+)i. Five rat RBC specimens had (Na+)i determined by both 23Na-NMR and inductively coupled plasma-atomic emission spectroscopy (ICP-AES). For glucose metabolism studies, RBCs from septic and control rats were suspended in modified Krebs-Henseleit buffer containing (6,6-2H2)glucose and examined by 2H-NMR. No significant differences in (Na+)i or glucose utilization were found in RBCs from control or septic rats. There were no differences in (Na+)i in the two groups of patients. The (Na+)i determined by NMR spectroscopy agreed closely with measurements using ICP-AES and establish that 100% of the (Na+)i of the RBC is visible by NMR. Glucose measurements determined by 2H-NMR correlated closely (correlation coefficient = 0.93) with enzymatic analysis. These studies showed no evidence that sepsis disturbed RBC membrane function or metabolism.« less

RHCE*ceCF encodes partial c and partial e but not CELO an antigen antithetical to Crawford

PubMed Central

Hipsky, Christine Halter; Lomas-Francis, Christine; Fuchisawa, Akiko; Reid, Marion E.; Moulds, Marilyn; Christensen, JoAnn; Nickle, Pam; Vege, Sunitha; Westhoff, Connie

2010-01-01

Background RH43 (Crawford) is encoded by RHCE*ce with nucleotide changes 48G>C, 697C>G, and 733C>G (RHCE*ceCF). We investigated the Rh antigen expression and antibody specificities in four patients with this allele. Methods Hemagglutination tests, DNA extraction, PCR-RFLP, AS-PCR, reticulocyte RNA isolation, RT-PCR cDNA analyses, cloning, and sequencing were performed by standard procedures. Results RBCs from two patients typed D+C−E−c+e+/−, hrS−/+W, hrB− and their serum was reactive (3+) with all RBC samples of common Rh phenotype tested, but non reactive with Rhnull or D- - RBCs (apparent alloanti-Rh17). At the RHCE locus, Patient 1 was homozygous for RHCE*ceCF, and Patient 2 inherited RHCE*ceCF in trans to a silenced RHCE*cE. Cross testing of serum and RBCs from these two samples showed mutual compatibility, indicating that both antibodies define the same novel high prevalence antigen on Rhce. Two additional patients, one whose serum contained alloanti-c but the RBCs typed C+c+, and one whose serum contained anti-e but the RBCs typed E+e+, also had RHCE*ceCF. RHCE*Ce was present in trans in the former and RHCE*cE in the latter patient. Conclusion We report that amino acid changes on RhceCF (Trp16Cys, Gln233Glu, and Leu245Val) alter the protein to the extent that c and e antigens are partial, and a high prevalence antigen, we have named CELO (provisional ISBT number 004058; RH58) is not expressed. CELO is antithetical to RH43 (Crawford). PMID:20609196

The in vitro wear behavior of experimental resin-based composites derived from a commercial formulation.

PubMed

Finlay, Nessa; Hahnel, Sebastian; Dowling, Adam H; Fleming, Garry J P

2013-04-01

To investigate the short- and long-term in vitro wear resistance of experimental resin-based composites (RBCs) derived from a commercial formulation. Six experimental RBCs were manufactured by manipulating the monomeric resin composition and the filler characteristics of Grandio (Voco GmbH, Cuxhaven, Germany). The Oregon Health Sciences University (OHSU) oral wear simulator was used in the presence of a food-like slurry to simulate three-body abrasion and attrition wear for 50,000, 150,000 and 300,000 cycles. A three-dimensional image of each wear facet was created and the total volumetric wear (mm(3)) and maximum wear depth (μm) were quantified for the RBC and antagonist. Statistical analyses of the total volumetric wear and maximum wear depth data (two- and one-way analyses of variance (ANOVA), with Tukey's post hoc tests where required) and regression analyses, were conducted at p=0.05. Two-way ANOVAs identified a significant effect of RBC material×wear cycles, RBC material and wear cycles (all p<0.0001). Regression analyses showed significant increases in the total volumetric wear (p≤0.001) and maximum wear depth data (p≤0.004) for all RBCs with increasing wear cycles. Differences between all RBC materials were evident after ≥150,000 wear cycles and antagonist wear provided valuable information to support the experimental findings. Wear simulating machines can provide an indication of the clinical performance but clinical performance is multi-factorial and wear is only a single facet. Employing experimental RBCs provided by a dental manufacturer rather than using self-manufactured RBCs or dental products provides increased experimental control by limiting the variables involved. Copyright © 2013 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Inverse relationship between erythrocyte size and platelet reactivity in elderly.

PubMed

Milovanovic, M; Nilsson, S; Winblad, B; Jelic, V; Behbahani, H; Shahnaz, T; Oweling, M; Järemo, P

2017-03-01

Previous work indicates that erythrocytes (RBCs) accumulate β-amyloid X-40 (Aβ 40 ) in individuals with Alzheimer disease (AD) and to a lesser extent in healthy elderly. The toxin damages RBCs and increases their mean corpuscular volume (MCV). Furthermore, AD platelets demonstrate lower reactivity. This study investigated interactions between RBCs and platelets. Older individuals with moderate hypertension (n = 57) were classified into two groups, depending on MCV in whole blood: The MCV high group comprised individuals with higher MCV (n = 27; 97 ± 3(SD) fl) and MCV low group had relatively lower MCV (n = 30; 90 ± 3(SD) fl). Flow cytometry was used to determine platelet reactivity, i.e., the surface binding of fibrinogen after provocation. Adenosine diphosphate (ADP) and a thrombin receptor-activating protein (TRAP-6) were used as agonists. Subsequently, blood cells were divided according to density into 17 subfractions. Intra-RBC Aβ 40 content was analyzed and in all platelet populations surface-bound fibrinogen was determined to estimate platelet in vivo activity. We found Aβ 40 inside RBCs of approximately 50% of participants, but the toxin did not affect MCV and platelet reactivity. In contrast, MCV associated inversely with platelet reactivity as judged from surface-attached fibrinogen after ADP (1.7 μmol/L) (p < 0.05) and TRAP-6 provocation (57 μmol/L (p = 0.01) and 74 μmol/L (p < 0.05)). In several density fractions (nos. 3, 4, 8, 11-13 (p < 0.05) and nos. 5-7 (p < 0.01)) MCV linked inversely with platelet-attached fibrinogen. In our community-dwelling sample, enhanced MCV associated with decreased platelet reactivity and lower in vivo platelet activity. It resembles RBCs and platelet behavior in AD-type dementia.

Age of red blood cells and outcome in acute kidney injury

PubMed Central

2013-01-01

Introduction Transfusion of red blood cells (RBCs) and, in particular, older RBCs has been associated with increased short-term mortality in critically ill patients. We evaluated the association between age of transfused RBCs and acute kidney injury (AKI), hospital, and 90-day mortality in critically ill patients. Methods We conducted a prospective, observational, predefined sub-study within the FINNish Acute Kidney Injury (FINNAKI) study. This study included all elective ICU admissions with expected ICU stay of more than 24 hours and all emergency admissions from September to November 2011. To study the age of RBCs, we classified transfused patients into quartiles according to the age of oldest transfused RBC unit in the ICU. AKI was defined according to KDIGO (Kidney Disease: Improving Global Outcomes) criteria. Results Out of 1798 patients, 652 received at least one RBC unit. The median [interquartile range] age of the oldest RBC unit transfused was 12 [11-13] days in the freshest quartile and 21 [17-27] days in the quartiles 2 to 4. On logistic regression, RBC age was not associated with the development of KDIGO stage 3 AKI. Patients in the quartile of freshest RBCs had lower crude hospital and 90-day mortality rates compared to those in the quartiles of older blood. After adjustments, older RBC age was associated with significantly increased risk for hospital mortality. Age, Simplified Acute Physiology Score II (SAPS II)-score without age points, maximum Sequental Organ Failure Assessment (SOFA) score and the total number of transfused RBC units were independently associated with 90-day mortality. Conclusions The age of transfused RBC units was independently associated with hospital mortality but not with 90-day mortality or KDIGO stage 3 AKI. The number of transfused RBC units was an independent risk factor for 90-day mortality. PMID:24093554

Effects of sevuparin on rosette formation and cytoadherence of Plasmodium falciparum infected erythrocytes

PubMed Central

Saiwaew, Somporn; Sritabal, Juntima; Piaraksa, Nattaporn; Keayarsa, Srisuda; Ruengweerayut, Ronnatrai; Utaisin, Chirapong; Sila, Patima; Niramis, Rangsan; Udomsangpetch, Rachanee; Charunwatthana, Prakaykaew; Pongponratn, Emsri; Pukrittayakamee, Sasithon; Leitgeb, Anna M.; Wahlgren, Mats; Lee, Sue J.; Day, Nicholas P. J.; White, Nicholas J.; Dondorp, Arjen M.; Chotivanich, Kesinee

2017-01-01

In severe falciparum malaria cytoadherence of parasitised red blood cells (PRBCs) to vascular endothelium (causing sequestration) and to uninfected red cells (causing rosette formation) contribute to microcirculatory flow obstruction in vital organs. Heparin can reverse the underlying ligand-receptor interactions, but may increase the bleeding risks. As a heparin-derived polysaccharide, sevuparin has been designed to retain anti-adhesive properties, while the antithrombin-binding domains have been eliminated, substantially diminishing its anticoagulant activity. Sevuparin has been evaluated recently in patients with uncomplicated falciparum malaria, and is currently investigated in a clinical trial for sickle cell disease. The effects of sevuparin on rosette formation and cytoadherence of Plasmodium falciparum isolates from Thailand were investigated. Trophozoite stages of P. falciparum-infected RBCs (Pf-iRBCs) were cultured from 49 patients with malaria. Pf-iRBCs were treated with sevuparin at 37°C and assessed in rosetting and in cytoadhesion assays with human dermal microvascular endothelial cells (HDMECs) under static and flow conditions. The proportion of Pf-iRBCs forming rosettes ranged from 6.5% to 26.0% (median = 12.2%). Rosetting was dose dependently disrupted by sevuparin (50% disruption by 250 μg/mL). Overall 57% of P. falciparum isolates bound to HDMECs under static conditions; median (interquartile range) Pf-iRBC binding was 8.5 (3.0–38.0) Pf-iRBCs/1000 HDMECs. Sevuparin in concentrations ≥ 100 μg/mL inhibited cytoadherence. Sevuparin disrupts P. falciparum rosette formation in a dose dependent manner and inhibits cytoadherence to endothelial cells. The data support assessment of sevuparin as an adjunctive treatment to the standard therapy in severe falciparum malaria. PMID:28249043

The Effect of PKCα on the Light Response of Rod Bipolar Cells in the Mouse Retina

PubMed Central

Xiong, Wei-Hong; Pang, Ji-Jie; Pennesi, Mark E.; Duvoisin, Robert M.; Wu, Samuel M.; Morgans, Catherine W.

2015-01-01

Purpose Protein kinase C α (PKCα) is abundantly expressed in rod bipolar cells (RBCs) in the retina, yet the physiological function of PKCα in these cells is not well understood. To elucidate the role of PKCα in visual processing in the eye, we examined the effect of genetic deletion of PKCα on the ERG and on RBC light responses in the mouse. Methods Immunofluorescent labeling was performed on wild-type (WT), TRPM1 knockout, and PKCα knockout (PKC-KO) retina. Scotopic and photopic ERGs were recorded from WT and PKC-KO mice. Light responses of RBCs were measured using whole-cell recordings in retinal slices from WT and PKC-KO mice. Results Protein kinase C alpha expression in RBCs is correlated with the activity state of the cell. Rod bipolar cells dendrites are a major site of PKCα phosphorylation. Electroretinogram recordings indicated that loss of PKCα affects the scotopic b-wave, including a larger peak amplitude, longer implicit time, and broader width of the b-wave. There were no differences in the ERG a- or c-wave between PKCα KO and WT mice, indicating no measurable effect of PKCα in photoreceptors or the RPE. The photopic ERG was unaffected consistent with the lack of detectable PKCα in cone bipolar cells. Whole-cell recordings from RBCs in PKC-KO retinal slices revealed that, compared with WT, RBC light responses in the PKC-KO retina are delayed and of longer duration. Conclusions Protein kinase C alpha plays an important modulatory role in RBCs, regulating both the peak amplitude and temporal properties of the RBC light response in the rod visual pathway. PMID:26230760

Effects of 5-hydroxymethyl-2-furfural on the volume and membrane permeability of red blood cells from patients with sickle cell disease.

PubMed

Hannemann, Anke; Cytlak, Urszula M; Rees, David C; Tewari, Sanjay; Gibson, John S

2014-09-15

The heterocyclic aldehyde 5-hydroxymethyl-2-furfural (5HMF) interacts allosterically with the abnormal form of haemoglobin (Hb), HbS, in red blood cells (RBCs) from patients with sickle cell disease (SCD), thereby increasing oxygen affinity and decreasing HbS polymerization and RBC sickling during hypoxia. We hypothesized that should 5HMF also inhibit the main cation pathways implicated in the dehydration of RBCs from SCD patients - the deoxygenation-induced cation pathway (Psickle), the Ca(2+)-activated K(+) channel (the Gardos channel) and the K(+)-Cl(-) cotransporter (KCC) - it would have a synergistic effect in protection against sickling, directly through interacting with HbS, and indirectly through maintaining hydration and reducing [HbS]. This study was therefore designed to investigate the effects of 5HMF on RBC volume and K(+) permeability in vitro. 5HMF markedly reduced the deoxygenation-induced dehydration of RBCs whether in response to maintained deoxygenation or to cyclical deoxygenation/re-oxygenation. 5HMF was found to inhibit Psickle, an effect which correlated with its effects on sickling. Deoxygenation-induced activation of the Gardos channel and exposure of phosphatidylserine were also inhibited, probably indirectly via reduced entry of Ca(2+) through the Psickle pathway. Effects of 5HMF on KCC were more modest with a slight inhibition in N-ethylmaleimide (NEM, 1 mm)-treated RBCs and stimulation in RBCs untreated with NEM. These findings support the hypothesis that 5HMF may also be beneficial through effects on RBC ion and water homeostasis. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

Inhibitory effects of retigabine, a Kv7 channel activator, on mechanosensitive primary bladder afferent activities and nociceptive behaviors in rats.

PubMed

Aizawa, Naoki; Wakamatsu, Daisuke; Kida, Jun; Otsuki, Takeya; Saito, Yasuho; Matsuya, Hidekazu; Homma, Yukio; Igawa, Yasuhiko

2017-02-01

Kv7 voltage-gated potassium channels have been suggested to modulate mechano-afferent transduction and nociception in the bladder. We investigated the effects of retigabine, a Kv7 channel activator, on rhythmic bladder contractions (RBCs), and single-unit afferent activities (SAAs) of the primary bladder mechanosensitive afferent nerve fibers in urethane-anesthetized rats. In addition, the effects of pretreatment with retigabine on the nociceptive behaviors provoked by an intravesical instillation of resiniferatoxin (RTX) were evaluated in the conscious condition. Female Sprague-Dawley rats were used. Under urethane anesthesia, saline was instilled into the bladder until RBCs were induced reproducibly. Then, the effects of intravenous, cumulative administrations of retigabine (0.1-3 mg/kg) or vehicle (saline) on RBCs were assessed. In separate animals, SAAs of Aδ- and C-fibers were identified by electrical stimulation of the pelvic nerve and by bladder distention with saline. After baseline recording, vehicle or retigabine (0.01-1 mg/kg) was administered intravenously and further recordings were performed. Under pretreatment with vehicle or retigabine (3 mg/kg intraperitoneally), the frequencies of lower abdominal licking and freezing were counted and scored as the bladder nociceptive behaviors induced by intravesical RTX instillation (3 µM, 0.3 ml). Retigabine dose-dependently decreased both the frequency and the amplitude of RBCs and SAAs of both Aδ- and C-fibers. The effect on RBCs was more potent on the frequency than the amplitude. Retigabine inhibited the RTX-induced abdominal licking, but not freezing. Kv7 channels are likely to be implicated in inhibition of bladder mechano- and nociceptive sensory transduction. Neurourol. Urodynam. 36:280-285, 2017. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Modulation of Starch Digestion for Slow Glucose Release through “Toggling” of Activities of Mucosal α-Glucosidases*

PubMed Central

Lee, Byung-Hoo; Eskandari, Razieh; Jones, Kyra; Reddy, Kongara Ravinder; Quezada-Calvillo, Roberto; Nichols, Buford L.; Rose, David R.; Hamaker, Bruce R.; Pinto, B. Mario

2012-01-01

Starch digestion involves the breakdown by α-amylase to small linear and branched malto-oligosaccharides, which are in turn hydrolyzed to glucose by the mucosal α-glucosidases, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI). MGAM and SI are anchored to the small intestinal brush-border epithelial cells, and each contains a catalytic N- and C-terminal subunit. All four subunits have α-1,4-exohydrolytic glucosidase activity, and the SI N-terminal subunit has an additional exo-debranching activity on the α-1,6-linkage. Inhibition of α-amylase and/or α-glucosidases is a strategy for treatment of type 2 diabetes. We illustrate here the concept of “toggling”: differential inhibition of subunits to examine more refined control of glucogenesis of the α-amylolyzed starch malto-oligosaccharides with the aim of slow glucose delivery. Recombinant MGAM and SI subunits were individually assayed with α-amylolyzed waxy corn starch, consisting mainly of maltose, maltotriose, and branched α-limit dextrins, as substrate in the presence of four different inhibitors: acarbose and three sulfonium ion compounds. The IC50 values show that the four α-glucosidase subunits could be differentially inhibited. The results support the prospect of controlling starch digestion rates to induce slow glucose release through the toggling of activities of the mucosal α-glucosidases by selective enzyme inhibition. This approach could also be used to probe associated metabolic diseases. PMID:22851177

Unexpected high digestion rate of cooked starch by the Ct-Maltase-Glucoamylase small intestine mucosal alpha-glucosidase subunit

USDA-ARS?s Scientific Manuscript database

For starch digestion to glucose, two luminal alpha-amylases and four gut mucosal alpha-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal alpha-glucosidases on cooked (gelatinized) starch. Gelatinized ...

Automated high resolution full-field spatial coherence tomography for quantitative phase imaging of human red blood cells

NASA Astrophysics Data System (ADS)

Singla, Neeru; Dubey, Kavita; Srivastava, Vishal; Ahmad, Azeem; Mehta, D. S.

2018-02-01

We developed an automated high-resolution full-field spatial coherence tomography (FF-SCT) microscope for quantitative phase imaging that is based on the spatial, rather than the temporal, coherence gating. The Red and Green color laser light was used for finding the quantitative phase images of unstained human red blood cells (RBCs). This study uses morphological parameters of unstained RBCs phase images to distinguish between normal and infected cells. We recorded the single interferogram by a FF-SCT microscope for red and green color wavelength and average the two phase images to further reduced the noise artifacts. In order to characterize anemia infected from normal cells different morphological features were extracted and these features were used to train machine learning ensemble model to classify RBCs with high accuracy.

Phylogeny, divergence time and historical biogeography of Laetiporus (Basidiomycota, Polyporales).

PubMed

Song, Jie; Cui, Bao-Kai

2017-04-20

The aim of this study was to characterize the molecular relationship, origin and historical biogeography of the species in important brown rot fungal genus Laetiporus from East Asia, Europe, Pan-America, Hawaii and South Africa. We used six genetic markers to estimate a genus-level phylogeny including (1) the internal transcribed spacer (ITS), (2) nuclear large subunit rDNA (nrLSU), (3) nuclear small subunit rDNA (nrSSU), (4) translation elongation factor 1-α (EF-1α), (5) DNA-directed RNA polymerase II subunit 2 (RPB2), and (6) mitochondrial small subunit rDNA (mtSSU). Results of multi-locus phylogenetic analyses show clade support for at least seventeen species-level lineages including two new Laetiporus in China. Molecular dating using BEAST estimated the present crown group diverged approximately 20.16 million years ago (Mya) in the early Miocene. Biogeographic analyses using RASP indicated that Laetiporus most likely originated in temperate zones with East Asia and North America having the highest probability (48%) of being the ancestral area. Four intercontinental dispersal routes and a possible concealed dispersal route were established for the first time.

RT-qPCR Demonstrates Light-Dependent AtRBCS1A and AtRBCS3B mRNA Expressions in "Arabidopsis thaliana" Leaves

ERIC Educational Resources Information Center

Chang, Ming-Mei; Li, Anna; Feissner, Robert; Ahmad, Talal

2016-01-01

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is widely used in diagnosis and research to determine specific mRNA expressions in cells. As RT-qPCR applications increase, it is necessary to provide undergraduates hands-on experience of this modern technique. Here, we report a 3-week laboratory exercise using RT-qPCR to…

Enhancing the Detection of Dysmorphic Red Blood Cells and Renal Tubular Epithelial Cells with a Modified Urinalysis Protocol.

PubMed

Chu-Su, Yu; Shukuya, Kenichi; Yokoyama, Takashi; Lin, Wei-Chou; Chiang, Chih-Kang; Lin, Chii-Wann

2017-01-11

Urinary sediment is used to evaluate patients with possible urinary tract diseases. Currently, numerous protocols are applied to detect dysmorphic red blood cells (RBCs) and renal tubular epithelial cells (RTECs) in urinary sediment. However, distinct protocols are used by nephrologists and medical technologists for specimen concentration and observation, which leads to major discrepancies in the differential counts of formed elements such as dysmorphic RBCs and RTECs and might interfere with an accurate clinical diagnosis. To resolve these problems, we first tested a modified urinalysis protocol with an increased relative centrifuge force and concentration factor in 20 biopsy-confirmed glomerulonephritis patients with haematuria. We successfully improved the recovery ratio of dysmorphic RBCs in clinical specimens from 34.7% to 42.0% (P < 0.001). Furthermore, we confirmed the correlation between counts by the modified urinary protocol and Sysmex UF-1000i urinary flow cytometer (r ≥ 0.898, P < 0.001). A total of 28 types of isomorphic and dysmorphic RBCs were detected using a bright field microscope, with results comparable to those using a standard phase contrast microscope. Finally, we applied Sternheimer stain to enhance the contrast of RTECs in the urinary sediments. We concluded that this modified urinalysis protocol significantly enhanced the quality of urinalysis.

Enhancing the Detection of Dysmorphic Red Blood Cells and Renal Tubular Epithelial Cells with a Modified Urinalysis Protocol

PubMed Central

Chu-Su, Yu; Shukuya, Kenichi; Yokoyama, Takashi; Lin, Wei-Chou; Chiang, Chih-Kang; Lin, Chii-Wann

2017-01-01

Urinary sediment is used to evaluate patients with possible urinary tract diseases. Currently, numerous protocols are applied to detect dysmorphic red blood cells (RBCs) and renal tubular epithelial cells (RTECs) in urinary sediment. However, distinct protocols are used by nephrologists and medical technologists for specimen concentration and observation, which leads to major discrepancies in the differential counts of formed elements such as dysmorphic RBCs and RTECs and might interfere with an accurate clinical diagnosis. To resolve these problems, we first tested a modified urinalysis protocol with an increased relative centrifuge force and concentration factor in 20 biopsy-confirmed glomerulonephritis patients with haematuria. We successfully improved the recovery ratio of dysmorphic RBCs in clinical specimens from 34.7% to 42.0% (P < 0.001). Furthermore, we confirmed the correlation between counts by the modified urinary protocol and Sysmex UF-1000i urinary flow cytometer (r ≥ 0.898, P < 0.001). A total of 28 types of isomorphic and dysmorphic RBCs were detected using a bright field microscope, with results comparable to those using a standard phase contrast microscope. Finally, we applied Sternheimer stain to enhance the contrast of RTECs in the urinary sediments. We concluded that this modified urinalysis protocol significantly enhanced the quality of urinalysis. PMID:28074941

Rapid, controllable growth of silver nanostructured surface-enhanced Raman scattering substrates for red blood cell detection

NASA Astrophysics Data System (ADS)

Zhang, Shu; Tian, Xueli; Yin, Jun; Liu, Yu; Dong, Zhanmin; Sun, Jia-Lin; Ma, Wanyun

2016-04-01

Silver nanostructured films suitable for use as surface-enhanced Raman scattering (SERS) substrates are prepared in just 2 hours by the solid-state ionics method. By changing the intensity of the external direct current, we can readily control the surface morphology and growth rate of the silver nanostructured films. A detailed investigation of the surface enhancement of the silver nanostructured films using Rhodamine 6G (R6G) as a molecular probe revealed that the enhancement factor of the films was up to 1011. We used the silver nanostructured films as substrates in SERS detection of human red blood cells (RBCs). The SERS spectra of RBCs on the silver nanostructured film could be clearly detected at a laser power of just 0.05 mW. Comparison of the SERS spectra of RBCs obtained from younger and older donors showed that the SERS spectra depended on donor age. A greater proportion of the haemoglobin in the RBCs of older donors was in the deoxygenated state than that of the younger donors. This implies that haemoglobin of older people has lower oxygen-carrying capacity than that of younger people. Overall, the fabricated silver substrates show promise in biomedical SERS spectral detection.

Age-Related Defects in Erythrocyte 2,3-Diphosphoglycerate Metabolism in Dementia

PubMed Central

Kaminsky, Yury G.; Reddy, V. Prakash; Ashraf, Ghulam Md; Ahmad, Ausaf; Benberin, Valery V.; Kosenko, Elena A.; Aliev, Gjumrakch

2013-01-01

Alzheimer disease (AD) is the most common dementing illness. Metabolic defects in the brain with aging contribute to the pathogenesis of AD. These changes can be found systematically and thus can be used as potential biomarkers. Erythrocytes (RBCs) are passive “reporter cells” that are not well studied in AD. In the present study, we analyzed an array of glycolytic and related enzymes and intermediates in RBCs from patients with AD and non-Alzheimer dementia (NA), age-matched controls (AC) and young adult controls (YC). AD is characterized by higher activities of hexokinase, phosphofructokinase, and bisphosphoglycerate mutase and bisphosphoglycerate phosphatase in RBCs. In our study, we observed that glycolytic and related enzymes displayed significantly lower activities in AC. However, similar or significantly higher activities were observed in AD and NA groups as compared to YC group. 2,3-diphosphoglycerate (2,3-DPG) levels were significantly decreased in AD and NA patients. The pattern of changes between groups in the above indices strongly correlates with each other. Collectively, our data suggested that AD and NA patients are associated with chronic disturbance of 2,3-DPG metabolism in RBCs. These defects may play a pivotal role in physiological processes, which predispose elderly subjects to AD and NA. PMID:24124630

Erythrocyte shape abnormalities, membrane oxidative damage, and β-actin alterations: an unrecognized triad in classical autism.

PubMed

Ciccoli, Lucia; De Felice, Claudio; Paccagnini, Eugenio; Leoncini, Silvia; Pecorelli, Alessandra; Signorini, Cinzia; Belmonte, Giuseppe; Guerranti, Roberto; Cortelazzo, Alessio; Gentile, Mariangela; Zollo, Gloria; Durand, Thierry; Valacchi, Giuseppe; Rossi, Marcello; Hayek, Joussef

2013-01-01

Autism spectrum disorders (ASDs) are a complex group of neurodevelopment disorders steadily rising in frequency and treatment refractory, where the search for biological markers is of paramount importance. Although red blood cells (RBCs) membrane lipidomics and rheological variables have been reported to be altered, with some suggestions indicating an increased lipid peroxidation in the erythrocyte membrane, to date no information exists on how the oxidative membrane damage may affect cytoskeletal membrane proteins and, ultimately, RBCs shape in autism. Here, we investigated RBC morphology by scanning electron microscopy in patients with classical autism, that is, the predominant ASDs phenotype (age range: 6-26 years), nonautistic neurodevelopmental disorders (i.e., "positive controls"), and healthy controls (i.e., "negative controls"). A high percentage of altered RBCs shapes, predominantly elliptocytes, was observed in autistic patients, but not in both control groups. The RBCs altered morphology in autistic subjects was related to increased erythrocyte membrane F2-isoprostanes and 4-hydroxynonenal protein adducts. In addition, an oxidative damage of the erythrocyte membrane β-actin protein was evidenced. Therefore, the combination of erythrocyte shape abnormalities, erythrocyte membrane oxidative damage, and β-actin alterations constitutes a previously unrecognized triad in classical autism and provides new biological markers in the diagnostic workup of ASDs.

Erythrocyte Shape Abnormalities, Membrane Oxidative Damage, and β-Actin Alterations: An Unrecognized Triad in Classical Autism

PubMed Central

Ciccoli, Lucia; De Felice, Claudio; Pecorelli, Alessandra; Belmonte, Giuseppe; Guerranti, Roberto; Cortelazzo, Alessio; Durand, Thierry; Valacchi, Giuseppe; Rossi, Marcello; Hayek, Joussef

2013-01-01

Autism spectrum disorders (ASDs) are a complex group of neurodevelopment disorders steadily rising in frequency and treatment refractory, where the search for biological markers is of paramount importance. Although red blood cells (RBCs) membrane lipidomics and rheological variables have been reported to be altered, with some suggestions indicating an increased lipid peroxidation in the erythrocyte membrane, to date no information exists on how the oxidative membrane damage may affect cytoskeletal membrane proteins and, ultimately, RBCs shape in autism. Here, we investigated RBC morphology by scanning electron microscopy in patients with classical autism, that is, the predominant ASDs phenotype (age range: 6–26 years), nonautistic neurodevelopmental disorders (i.e., “positive controls”), and healthy controls (i.e., “negative controls”). A high percentage of altered RBCs shapes, predominantly elliptocytes, was observed in autistic patients, but not in both control groups. The RBCs altered morphology in autistic subjects was related to increased erythrocyte membrane F2-isoprostanes and 4-hydroxynonenal protein adducts. In addition, an oxidative damage of the erythrocyte membrane β-actin protein was evidenced. Therefore, the combination of erythrocyte shape abnormalities, erythrocyte membrane oxidative damage, and β-actin alterations constitutes a previously unrecognized triad in classical autism and provides new biological markers in the diagnostic workup of ASDs. PMID:24453417

RBCs as microlenses: wavefront analysis and applications

NASA Astrophysics Data System (ADS)

Merola, Francesco; Barroso, Álvaro; Miccio, Lisa; Memmolo, Pasquale; Mugnano, Martina; Ferraro, Pietro; Denz, Cornelia

2017-06-01

Developing the recently discovered concept of RBCs as microlenses, we demonstrate further applications in wavefront analysis and diagnostics. Correlation between RBC's morphology and its behavior as a refractive optical element has been established. In fact, any deviation from the healthy RBC morphology can be seen as additional aberration in the optical wavefront passing through the cell. By this concept, accurate localization of focal spots of RBCs can become very useful in blood disorders identification. Moreover, By modelling RBC as bio-lenses through Zernike polynomials it is possible to identify a series of orthogonal parameters able to recognise RBC shapes. The main improvement concerns the possibility to combine such parameters because of their independence conversely to standard image-based analysis where morphological factors are dependent each-others. We investigate the three-dimensional positioning of such focal spots over time for samples with two different osmolarity conditions, i.e. discocytes and spherocytes. Finally, Zernike polynomials wavefront analysis allows us to study the optical behavior of RBCs under an optically-induced mechanical stress. Detailed wavefront analysis provides comprehensive information about the aberrations induced by the deformation obtained using optical tweezers. This could open new routes for analyzing cell elasticity by examining optical parameters instead of direct but with low resolution strain analysis, thanks to the high sensitivity of the interferometric tool.

Identification of malaria parasite-infected red blood cell surface aptamers by inertial microfluidic SELEX (I-SELEX)

NASA Astrophysics Data System (ADS)

Birch, Christina M.; Hou, Han Wei; Han, Jongyoon; Niles, Jacquin C.

2015-07-01

Plasmodium falciparum malaria parasites invade and remodel human red blood cells (RBCs) by trafficking parasite-synthesized proteins to the RBC surface. While these proteins mediate interactions with host cells that contribute to disease pathogenesis, the infected RBC surface proteome remains poorly characterized. Here we use a novel strategy (I-SELEX) to discover high affinity aptamers that selectively recognize distinct epitopes uniquely present on parasite-infected RBCs. Based on inertial focusing in spiral microfluidic channels, I-SELEX enables stringent partitioning of cells (efficiency ≥ 106) from unbound oligonucleotides at high volume throughput (~2 × 106 cells min-1). Using an RBC model displaying a single, non-native antigen and live malaria parasite-infected RBCs as targets, we establish suitability of this strategy for de novo aptamer selections. We demonstrate recovery of a diverse set of aptamers that recognize distinct, surface-displayed epitopes on parasite-infected RBCs with nanomolar affinity, including an aptamer against the protein responsible for placental sequestration, var2CSA. These findings validate I-SELEX as a broadly applicable aptamer discovery platform that enables identification of new reagents for mapping the parasite-infected RBC surface proteome at higher molecular resolution to potentially contribute to malaria diagnostics, therapeutics and vaccine efforts.

New prodrugs of metformin do not influence the overall haemostasis potential and integrity of the erythrocyte membrane.

PubMed

Markowicz-Piasecka, Magdalena; Sikora, Joanna; Mateusiak, Łukasz; Mikiciuk-Olasik, Elżbieta; Huttunen, Kristiina M

2017-09-15

Although metformin, an oral anti-diabetic drug, has been found to have multidirectional effects over the past decade, it is characterised by unfavourable pharmacokinetic properties. This study discusses the effects of metformin, phenformin and three prodrugs of metformin on the haemostasis and integrity of Red Blood Cells (RBCs). The influence of examined biguanide derivatives on haemostasis was evaluated spectrophotometrically by clot formation and lysis test (CL-test) at 405nm. The extrinsic and intrinsic coagulation pathway were examined by measuring the PT (Prothrombin Time) and aPTT (Activated Partial Tromboplastin Time). Haemolysis assay, microscopy and flow cytometry studies were used to assess the effect of the tested compounds on RBCs. Although none of the tested biguanide derivatives significantly influenced the overall potential of clot formation and fibrinolysis (CL AUC constants), statistically significant changes were seen in the values of the kinetic parameters of fibrinolysis. Furthermore, only prodrug 2, with an 8-carbon alkyl chain, unfavourably affected RBCs by interaction with the erythrocyte membrane leading to significant haemolysis. Our results provide a further insight into the effects of metformin and its prodrugs on haemostasis and RBCs and underscore the necessity for further research. Copyright © 2017 Elsevier B.V. All rights reserved.

Hemoglobin consumption by P. falciparum in individual erythrocytes imaged via quantitative phase spectroscopy

NASA Astrophysics Data System (ADS)

Rinehart, Matthew T.; Park, Han Sang; Walzer, Katelyn A.; Chi, Jen-Tsan Ashley; Wax, Adam

2016-04-01

Plasmodium falciparum infection causes structural and biochemical changes in red blood cells (RBCs). To quantify these changes, we apply a novel optical technique, quantitative phase spectroscopy (QPS) to characterize individual red blood cells (RBCs) during the intraerythrocytic life cycle of P. falciparum. QPS captures hyperspectral holograms of individual RBCs to measure spectroscopic changes across the visible wavelength range (475-700 nm), providing complex information, i.e. amplitude and phase, about the light field which has interacted with the cell. The complex field provides complimentary information on hemoglobin content and cell mass, which are both found to dramatically change upon infection by P. falciparum. Hb content progressively decreases with parasite life cycle, with an average 72.2% reduction observed for RBCs infected by schizont-stage P. falciparum compared to uninfected cells. Infection also resulted in a 33.1% reduction in RBC’s optical volume, a measure of the cells’ non-aqueous components. Notably, optical volume is only partially correlated with hemoglobin content, suggesting that changes in other dry mass components such as parasite mass may also be assessed using this technique. The unique ability of QPS to discriminate individual healthy and infected cells using spectroscopic changes indicates that the approach can be used to detect disease.

A system for counting fetal and maternal red blood cells.

PubMed

Ge, Ji; Gong, Zheng; Chen, Jun; Liu, Jun; Nguyen, John; Yang, Zongyi; Wang, Chen; Sun, Yu

2014-12-01

The Kleihauer-Betke (KB) test is the standard method for quantitating fetal-maternal hemorrhage in maternal care. In hospitals, the KB test is performed by a certified technologist to count a minimum of 2000 fetal and maternal red blood cells (RBCs) on a blood smear. Manual counting suffers from inherent inconsistency and unreliability. This paper describes a system for automated counting and distinguishing fetal and maternal RBCs on clinical KB slides. A custom-adapted hardware platform is used for KB slide scanning and image capturing. Spatial-color pixel classification with spectral clustering is proposed to separate overlapping cells. Optimal clustering number and total cell number are obtained through maximizing cluster validity index. To accurately identify fetal RBCs from maternal RBCs, multiple features including cell size, roundness, gradient, and saturation difference between cell and whole slide are used in supervised learning to generate feature vectors, to tackle cell color, shape, and contrast variations across clinical KB slides. The results show that the automated system is capable of completing the counting of over 60,000 cells (versus ∼2000 by technologists) within 5 min (versus ∼15 min by technologists). The throughput is improved by approximately 90 times compared to manual reading by technologists. The counting results are highly accurate and correlate strongly with those from benchmarking flow cytometry measurement.

Self-separation of blood plasma from whole blood during the capillary flow in microchannel

NASA Astrophysics Data System (ADS)

Nunna, Bharath Babu; Zhuang, Shiqiang; Lee, Eon Soo

2017-11-01

Self-separation of blood plasma from whole blood in microchannels is of great importance due to the enormous range of applications in healthcare and diagnostics. Blood is a multiphase complex fluid, composed of cells suspended in blood plasma. RBCs are the suspended particles whose shape changes during the flow of blood. The primary constituents of blood are erythrocytes or red blood cells (RBCs), leukocytes or white blood cells (WBCs), thrombocytes or platelets and blood plasma. The existence of RBCs in blood makes the blood a non-Newtonian fluid. The current study of separation of blood plasma from whole blood during self-driven flows in a single microchannel without bifurcation, by enhancing the capillary effects. The change in the capillary effect results in a change in contact angle which directly influences the capillary flow. The flow velocity directly influences the net force acting on the RBCs and influence the separation process. The experiments are performed on the PDMS microchannels with different contact angles by altering the surface characteristics using plasma treatment. The change in the separation length is studied during the capillary flow of blood in microchannel. Bharath Babu Nunna is a researcher in mechanical engineering and implementing the novel and innovative technologies in the biomedical devices to enhance the sensitivity of the disease diagnosis.

Ex vivo perfusion of human spleens maintains clearing and processing functions.

PubMed

Buffet, Pierre A; Milon, Geneviève; Brousse, Valentine; Correas, Jean-Michel; Dousset, Bertrand; Couvelard, Anne; Kianmanesh, Reza; Farges, Olivier; Sauvanet, Alain; Paye, François; Ungeheuer, Marie-Noëlle; Ottone, Catherine; Khun, Huot; Fiette, Laurence; Guigon, Ghislaine; Huerre, Michel; Mercereau-Puijalon, Odile; David, Peter H

2006-05-01

The spleen plays a central role in the pathophysiology of several potentially severe diseases such as inherited red cell membrane disorders, hemolytic anemias, and malaria. Research on these diseases is hampered by ethical constraints that limit human spleen tissue explorations. We identified a surgical situation--left splenopancreatectomy for benign pancreas tumors--allowing spleen retrieval at no risk for patients. Ex vivo perfusion of retrieved intact spleens for 4 to 6 hours maintained a preserved parenchymal structure, vascular flow, and metabolic activity. Function preservation was assessed by testing the ability of isolated-perfused spleens to retain Plasmodium falciparum-infected erythrocytes preexposed to the antimalarial drug artesunate (Art-iRBCs). More than 95% of Art-iRBCs were cleared from the perfusate in 2 hours. At each transit through isolated-perfused spleens, parasite remnants were removed from 0.2% to 0.23% of Art-iRBCs, a proportion consistent with the 0.02% to 1% pitting rate previously established in artesunate-treated patients. Histologic analysis showed that more than 90% of Art-iRBCs were retained and processed in the red pulp, providing the first direct evidence of a zone-dependent parasite clearance by the human spleen. Human-specific physiologic or pathophysiologic mechanisms involving clearing or processing functions of the spleen can now be experimentally explored in a human tissue context.

Distribution of dehydration rates generated by maximal Gardos-channel activation in normal and sickle red blood cells.

PubMed

Lew, Virgilio L; Tiffert, Teresa; Etzion, Zipora; Perdomo, Deisy; Daw, Nuala; Macdonald, Lynn; Bookchin, Robert M

2005-01-01

The Ca(2+)-activated K+ channels of human red blood cells (RBCs) (Gardos channels, hIK1, hSK4) can mediate rapid cell dehydration, of particular relevance to the pathophysiology of sickle cell disease. Previous investigations gave widely discrepant estimates of the number of Gardos channels per RBC, from as few as 1 to 3 to as many as 300, with large cell-to-cell differences, suggesting that RBCs could differ extensively in their susceptibility to dehydration by elevated Ca2+. Here we investigated the distribution of dehydration rates induced by maximal and uniform Ca2+ loads in normal (AA) and sickle (SS) RBCs by measuring the time-dependent changes in osmotic fragility and RBC volume distributions. We found a remarkable conservation of osmotic lysis and volume distribution profiles during Ca(2+)-induced dehydration, indicating overall uniformity of dehydration rates among AA and SS RBCs. In light of these results, alternative interpretations were suggested for the previously proposed low estimates and heterogeneity of channel numbers per cell. The results support the view that stochastic Ca2+ permeabilization rather than Gardos-channel variation is the main determinant selecting which SS cells dehydrate through Gardos channels in each sickling episode.

Docosahexaenoic acid is an independent predictor of all-cause mortality in hemodialysis patients.

PubMed

Hamazaki, Kei; Terashima, Yoshihiro; Itomura, Miho; Sawazaki, Shigeki; Inagaki, Hitoshi; Kuroda, Masahiro; Tomita, Shin; Hirata, Hitoshi; Inadera, Hidekuni; Hamazaki, Tomohito

2011-01-01

Dietary n-3 polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA) and eicosapentaenoic acid have been shown to reduce cardiovascular mortality. Patients on hemodialysis (HD) have a very high mortality from cardiovascular disease. Fish consumption reduces all-cause mortality in patients on HD. Moreover, n-3 PUFAs, especially DHA levels in red blood cells (RBCs), are associated with arteriosclerosis in patients on HD. The aim of this study was to determine whether DHA levels in RBCs predict the mortality of patients on HD in a prospective cohort study. A cohort of 176 patients (64.1 ± 12.0 (mean ± SD) years of age, 96 men and 80 women) under HD treatment was studied. The fatty acid composition of their RBCs was analyzed by gas chromatography. During the study period of 5 years, 54 deaths occurred. After adjustment for 10 confounding factors, the Cox hazard ratio of all-cause mortality of the patients on HD in the highest DHA tertile (>8.1%, 15 deaths) was 0.43 (95% CI 0.21-0.88) compared with those patients in the lowest DHA tertile (<7.2%, 21 deaths). The findings suggest that the level of DHA in RBCs could be an independent predictor of all-cause mortality in patients on HD. Copyright © 2010 S. Karger AG, Basel.

An Fe-S cluster in the conserved Cys-rich region in the catalytic subunit of FAD-dependent dehydrogenase complexes.

PubMed

Shiota, Masaki; Yamazaki, Tomohiko; Yoshimatsu, Keiichi; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

2016-12-01

Several bacterial flavin adenine dinucleotide (FAD)-harboring dehydrogenase complexes comprise three distinct subunits: a catalytic subunit with FAD, a cytochrome c subunit containing three hemes, and a small subunit. Owing to the cytochrome c subunit, these dehydrogenase complexes have the potential to transfer electrons directly to an electrode. Despite various electrochemical applications and engineering studies of FAD-dependent dehydrogenase complexes, the intra/inter-molecular electron transfer pathway has not yet been revealed. In this study, we focused on the conserved Cys-rich region in the catalytic subunits using the catalytic subunit of FAD dependent glucose dehydrogenase complex (FADGDH) as a model, and site-directed mutagenesis and electron paramagnetic resonance (EPR) were performed. By co-expressing a hitch-hiker protein (γ-subunit) and a catalytic subunit (α-subunit), FADGDH γα complexes were prepared, and the properties of the catalytic subunit of both wild type and mutant FADGDHs were investigated. Substitution of the conserved Cys residues with Ser resulted in the loss of dye-mediated glucose dehydrogenase activity. ICP-AEM and EPR analyses of the wild-type FADGDH catalytic subunit revealed the presence of a 3Fe-4S-type iron-sulfur cluster, whereas none of the Ser-substituted mutants showed the EPR spectrum characteristic for this cluster. The results suggested that three Cys residues in the Cys-rich region constitute an iron-sulfur cluster that may play an important role in the electron transfer from FAD (intra-molecular) to the multi-heme cytochrome c subunit (inter-molecular) electron transfer pathway. These features appear to be conserved in the other three-subunit dehydrogenases having an FAD cofactor. Copyright © 2016 Elsevier B.V. All rights reserved.

Kv channel subunits that contribute to voltage-gated K+ current in renal vascular smooth muscle.

PubMed

Fergus, Daniel J; Martens, Jeffrey R; England, Sarah K

2003-03-01

The rat renal arterial vasculature displays differences in K(+) channel current phenotypes along its length. Small arcuate to cortical radial arteries express a delayed rectifier phenotype, while the predominant Kv current in larger arcuate and interlobar arteries is composed of both transient and sustained components. We sought to determine whether Kvalpha subunits in the rat renal interlobar and arcuate arteries form heterotetramers, which may account for the unique currents, and whether modulatory Kvbeta subunits are present in renal vascular smooth muscle cells. RT-PCR indicated the presence of several different Kvalpha subunit isoform transcripts. Co-immunoprecipitation with immunoblotting and immunohistochemical evidence suggests that a portion of the K(+) current phenotype is a heteromultimer containing delayed-rectifier Kv1.2 and A-type Kv1.4 channel subunits. RT-PCR and immunoblot analyses also demonstrated the presence of both Kvbeta1.2 and Kvbeta1.3 in renal arteries. These results suggest that heteromultimeric formation of Kvalpha subunits and the presence of modulatory Kvbeta subunits are important factors in mediating Kv currents in the renal microvasculature and suggest a potentially critical role for these channel subunits in blood pressure regulation.

Modify washing solutions in the process of deglycerolization in ACP 215 and storage at 4°C in 0.9% sodium chloride in 24h.

PubMed

Zhao, Yang; Luo, Guangping; Luo, Hong; Ye, Xin; Rong, Xia; Huang, Kejun

2010-10-01

The ACP 215 was a functional closed system for preparing glycerolized and deglycerolized RBCs, CSBT had approved the technique of long term storage glycerolized rare blood lower than -65°C, and then deglycerolized by this machine. From the manual method to use ACP 215, Chinese blood banks chose 9% sodium chloride and 0.9% sodium chloride in deglycerolization process, while the AABB guideline prescribed that 12% sodium chloride and 0.9% sodium chloride-0.2% glucose were acceptable in washing step of ACP 215. In addition, 0.9% sodium chloride was the only solution which was permitted by CSBT to be added into postwash RBCs, while in America many kinds of additive solutions like AS-3 could be added into postwash RBCs and stored at 4°C for 14 days. Changes of washing solutions and preservation solution were much different from the original procedure of ACP 215 approved by the FDA. It was necessary to assess the quality of deglyceroled and postwash RBCs by this modified process in ACP 215 in China. Two-unit whole bloods were collected from each volunteer and preserved in CP2D for anticoagulant. It was then centrifuged to separate the plasma, and suspending RBCs were stored at 4°C in MAP for 6 days. Each unit of RBC was transferred to a 1000-ml PVC plastic bag, an improved procedure including the single-disposable glycerolization set in an automated, functionally closed system (ACP 215, Haemonetics) was used to glycerolize RBC with 40% (wt/vol) glycerol, then frozen at -80°C. Two modified washing solutions of 9% sodium chloride and 0.9% sodium chloride were used to deglycerolize the same RBCs with single disposable deglycerolization set in ACP 215. The deglycerolized RBCs were stored at 4°C in 0.9% sodium chloride for 24h. The freeze-thaw recovery value was 95.3±1.8% (mean±SD); the freeze-thaw-wash recovery value was 82.3±5.94% (mean±SD); the residure glycerol was 6.1±1.66 mg/dl (mean±SD), storage at 4°C in 0.9% sodium chloride within 24h after deglycerolization the supernatant Hb was 43±12 mg/dl (mean±SD); the hemolysis rate was 0.2±0.1% (mean±SD); the supernatant potassium level was 4.03±0.81 mM (mean±SD); and the postwash units were negative for both aerobic and anaerobic bacteria. The postwash of deglycerolized RBC had an acceptable FTW recovery value, and stored in 0.9% sodium chloride for 24h at 4°C, had an acceptable hemolysis. Modified washing solutions of 9% sodium chloride and 0.9% sodium chloride and without any of preservation had not affected the quality of frozen RBCs prepared in ACP 215 and postwash stored at 4°C in 0.9% sodium chloride within 24h. Copyright © 2010 Elsevier Ltd. All rights reserved.

Cloning and characterization of two novel zebrafish P2X receptor subunits.

PubMed

Diaz-Hernandez, Miguel; Cox, Jane A; Migita, Keisuke; Haines, William; Egan, Terrance M; Voigt, Mark M

2002-07-26

In this report we describe the cloning and characterization of two P2X receptor subunits cloned from the zebrafish (Danio rerio). Primary sequence analysis suggests that one cDNA encodes an ortholog of the mammalian P2X(4) subunit and the second cDNA encodes the ortholog of the mammalian P2X(5) subunit. The zP2X(4) subunit forms a homo-oligomeric receptor that displays a low affinity for ATP (EC(50)=274+/-48 microM) and very low affinity (EC(50)>500 microM) for other purinergic ligands such as alphabetameATP, suramin, and PPADS. As seen with the mammalian orthologs, the zP2X(5) subunit forms a homo-oligomeric receptor that yields very small whole-cell currents (<20pA), making determination of an EC(50) problematic. Both subunit genes were physically mapped onto the zebrafish genome using radiation hybrid analysis of the T51 panel, with the zp2x4 localized to LG21 and zp2x5 to LG5.

Similarities in transcription factor IIIC subunits that bind to the posterior regions of internal promoters for RNA polymerase III.

PubMed

Matsutani, Sachiko

2004-08-09

In eukaryotes, RNA polymerase III (RNAP III) transcribes the genes for small RNAs like tRNAs, 5S rRNA, and several viral RNAs, and short interspersed repetitive elements (SINEs). The genes for these RNAs and SINEs have internal promoters that consist of two regions. These two regions are called the A and B blocks. The multisubunit transcription factor TFIIIC is required for transcription initiation of RNAP III; in transcription of tRNAs, the B-block binding subunit of TFIIIC recognizes a promoter. Although internal promoter sequences are conserved in eukaryotes, no evidence of homology between the B-block binding subunits of vertebrates and yeasts has been reported previously. Here, I reported the results of PSI-BLAST searches using the B-block binding subunits of human and Shizosacchromyces pombe as queries, showing that the same Arabidopsis proteins were hit with low E-values in both searches. Comparison of the convergent iterative alignments obtained by these PSI-BLAST searches revealed that the vertebrate, yeast, and Arabidopsis proteins have similarities in their N-terminal one-third regions. In these regions, there were three domains with conserved sequence similarities, one located in the N-terminal end region. The N-terminal end region of the B-block binding subunit of Saccharomyces cerevisiae is tentatively identified as a HMG box, which is the DNA binding motif. Although I compared the alignment of the N-terminal end regions of the B-block binding subunits, and their homologs, with that of the HMG boxes, it is not clear whether they are related. Molecular phylogenetic analyses using the small subunit rRNA and ubiquitous proteins like actin and alpha-tubulin, show that fungi are more closely related to animals than either is to plants. Interestingly, the results obtained in this study show that, with respect to the B-block binding subunits of TFIIICs, animals appear to be evolutionarily closer to plants than to fungi.

A saposin-like domain influences the intracellular localization, stability, and catalytic activity of human acyloxyacyl hydrolase.

PubMed

Staab, J F; Ginkel, D L; Rosenberg, G B; Munford, R S

1994-09-23

Acyloxyacyl hydrolase, a leukocyte enzyme that acts on bacterial lipopolysaccharides (LPSs) and many glycerolipids, is synthesized as a precursor polypeptide that undergoes internal disulfide linkage before being proteolytically processed into two subunits. The larger subunit contains an amino acid sequence (Gly-X-Ser-X-Gly) that is found at the active sites of many lipases, while the smaller subunit has amino acid sequence similarity to saposins (sphingolipid activator proteins), cofactors for sphingolipid glycohydrolases. We show here that both acyloxyacyl hydrolase subunits are required for catalytic activity toward LPS and glycerophosphatidylcholine. In addition, mutations that truncate or delete the small subunit have profound effects on the intracellular localization, proteolytic processing, and stability of the enzyme in baby hamster kidney cells. Remarkably, proteolytic cleavage of the precursor protein increases the activity of the enzyme toward LPS by 10-20-fold without altering its activity toward glycerophosphatidylcholine. Proper orientation of the two subunits thus seems very important for the substrate specificity of this unusual enzyme.

Genome-Wide Identification and Comprehensive Expression Profiling of Ribosomal Protein Small Subunit (RPS) Genes and their Comparative Analysis with the Large Subunit (RPL) Genes in Rice

PubMed Central

Saha, Anusree; Das, Shubhajit; Moin, Mazahar; Dutta, Mouboni; Bakshi, Achala; Madhav, M. S.; Kirti, P. B.

2017-01-01

Ribosomal proteins (RPs) are indispensable in ribosome biogenesis and protein synthesis, and play a crucial role in diverse developmental processes. Our previous studies on Ribosomal Protein Large subunit (RPL) genes provided insights into their stress responsive roles in rice. In the present study, we have explored the developmental and stress regulated expression patterns of Ribosomal Protein Small (RPS) subunit genes for their differential expression in a spatiotemporal and stress dependent manner. We have also performed an in silico analysis of gene structure, cis-elements in upstream regulatory regions, protein properties and phylogeny. Expression studies of the 34 RPS genes in 13 different tissues of rice covering major growth and developmental stages revealed that their expression was substantially elevated, mostly in shoots and leaves indicating their possible involvement in the development of vegetative organs. The majority of the RPS genes have manifested significant expression under all abiotic stress treatments with ABA, PEG, NaCl, and H2O2. Infection with important rice pathogens, Xanthomonas oryzae pv. oryzae (Xoo) and Rhizoctonia solani also induced the up-regulation of several of the RPS genes. RPS4, 13a, 18a, and 4a have shown higher transcript levels under all the abiotic stresses, whereas, RPS4 is up-regulated in both the biotic stress treatments. The information obtained from the present investigation would be useful in appreciating the possible stress-regulatory attributes of the genes coding for rice ribosomal small subunit proteins apart from their functions as house-keeping proteins. A detailed functional analysis of independent genes is required to study their roles in stress tolerance and generating stress- tolerant crops. PMID:28966624

Dietary phenolic compounds selectively inhibit the individual subunits of maltase-glucoamylase and sucrase-isomaltase with the potential of modulating glucose release

USDA-ARS?s Scientific Manuscript database

In this study, it was hypothesized that dietary phenolic compounds selectively inhibit the individual C- and N-terminal (Ct, Nt) subunits of the two small intestinal alpha-glucosidases, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI), for a modulated glycemic carbohydrate digestion. The inhi...

Three new anascosporic genera of the Saccharomycotina: Danielozyma gen. nov., Deakozyma gen. nov. and Middelhovenomyces gen. nov.

USDA-ARS?s Scientific Manuscript database

Three new non-ascosporic, ascomycetous yeast genera are proposed based on their isolation from currently described species and genera. Phylogenetic placement of the genera was determined from analysis of nuclear gene sequences for D1/D2 large subunit rRNA, small subunit rRNA, translation elongation...

Scintigraphic detection of occult hemorrhage using RBCs labeled in vitro with technetium Tc 99m sodium pertechnetate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bunker, S.R.; Kolina, J.S.; Kaplan, K.A.

1983-05-01

Scintigraphy with RBCs labeled with technetium Tc 99m sodium pertechnetate effectively located the source of hemorrhage in a patient receiving long-term anticoagulant therapy. (The patient was initially seen with a large hematoma on the flank.) More important, the procedure was used to monitor activity in this otherwise-occult bleeding site. Scintigraphic studies may be useful in the management of these difficult clinical problems.

Raman spectroscopy of stored red blood cells: evaluating clinically-relevant biochemical markers in donated blood

NASA Astrophysics Data System (ADS)

Atkins, Chad G.; Buckley, Kevin; Chen, Deborah; Schulze, H. G.; Devine, Dana V.; Blades, Michael W.; Turner, Robin F. B.

2015-07-01

Modern transfusion medicine relies on the safe, secure, and cost-effective delivery of donated red blood cells (RBCs). Once isolated, RBCs are suspended in a defined additive solution and stored in plastic blood bags in which, over time, they undergo chemical, physiological, and morphological changes that may have a deleterious impact on some patients. Regulations limit the storage period to 42 days and the cells do not routinely undergo analytical testing before use. In this study, we use Raman spectroscopy to interrogate stored RBCs and we identify metabolic and cell-breakdown products, such as haemoglobin and membrane fragments, that build-up in the blood bags as the cells age. Our work points the way to the development of an instrument which could quickly and easily assess the biochemical nature of stored RBC units before they are transfused.

Stationary scalar clouds around a BTZ black hole

NASA Astrophysics Data System (ADS)

Ferreira, Hugo R. C.; Herdeiro, Carlos A. R.

2017-10-01

We establish the existence of stationary clouds of massive test scalar fields around BTZ black holes. These clouds are zero-modes of the superradiant instability and are possible when Robin boundary conditions (RBCs) are considered at the AdS boundary. These boundary conditions are the most general ones that ensure the AdS space is an isolated system, and include, as a particular case, the commonly considered Dirichlet or Neumann-type boundary conditions (DBCs or NBCs). We obtain an explicit, closed form, resonance condition, relating the RBCs that allow the existence of normalizable (and regular on and outside the horizon) clouds to the system's parameters. Such RBCs never include pure DBCs or NBCs. We illustrate the spatial distribution of these clouds, their energy and angular momentum density for some cases. Our results show that BTZ black holes with scalar hair can be constructed, as the non-linear realization of these clouds.

Volumetric measurement of human red blood cells by MOSFET-based microfluidic gate.

PubMed

Guo, Jinhong; Ai, Ye; Cheng, Yuanbing; Li, Chang Ming; Kang, Yuejun; Wang, Zhiming

2015-08-01

In this paper, we present a MOSFET-based (metal oxide semiconductor field-effect transistor) microfluidic gate to characterize the translocation of red blood cells (RBCs) through a gate. In the microfluidic system, the bias voltage modulated by the particles or biological cells is connected to the gate of MOSFET. The particles or cells can be detected by monitoring the MOSFET drain current instead of DC/AC-gating method across the electronic gate. Polystyrene particles with various standard sizes are utilized to calibrate the proposed device. Furthermore, RBCs from both adults and newborn blood sample are used to characterize the performance of the device in distinguishing the two types of RBCs. As compared to conventional DC/AC current modulation method, the proposed device demonstrates a higher sensitivity and is capable of being a promising platform for bioassay analysis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Measurement of red blood cell mechanics during morphological changes

PubMed Central

Park, YongKeun; Best, Catherine A.; Badizadegan, Kamran; Dasari, Ramachandra R.; Feld, Michael S.; Kuriabova, Tatiana; Henle, Mark L.; Levine, Alex J.; Popescu, Gabriel

2010-01-01

The human red blood cell (RBC) membrane, a fluid lipid bilayer tethered to an elastic 2D spectrin network, provides the principal control of the cell’s morphology and mechanics. These properties, in turn, influence the ability of RBCs to transport oxygen in circulation. Current mechanical measurements of RBCs rely on external loads. Here we apply a noncontact optical interferometric technique to quantify the thermal fluctuations of RBC membranes with 3 nm accuracy over a broad range of spatial and temporal frequencies. Combining this technique with a new mathematical model describing RBC membrane undulations, we measure the mechanical changes of RBCs as they undergo a transition from the normal discoid shape to the abnormal echinocyte and spherical shapes. These measurements indicate that, coincident with this morphological transition, there is a significant increase in the membrane’s shear, area, and bending moduli. This mechanical transition can alter cell circulation and impede oxygen delivery. PMID:20351261

Relative deformability of red blood cells in sickle cell trait and sickle cell anemia by trapping and dragging

NASA Astrophysics Data System (ADS)

Solomon, Rance; Cooper, James; Welker, Gabriel; Aguilar, Elaura; Flanagan, Brooke; Pennycuff, Chelsey; Scott, David; Farone, Anthony; Farone, Mary; Erenso, Daniel; Mushi, Robert; del Pilar Aguinaga, Maria

2013-06-01

Genetic mutation of the β-globin gene or inheritance of this mutated gene changes the chemical composition of the oxygen-carrying hemoglobin molecule that could lead to either the heterozygote genotype, resulting in sickle cell trait (SCT), or the homozygote genotype, resulting in sickle cell anemia (SCA). These mutations could affect the reversible elastic deformations of the red blood cells (RBCs) which are vital for biological functions. We have investigated this effect by studying the differences in the deformability of RBCs from blood samples of an individual with SCT and an untreated patient with SCA along with hemoglobin quantitation of each blood sample. Infrared 1064 nm laser trap force along with drag shear force are used to induce deformation in the RBCs. Ultra2-High Performance Liquid Chromatography (UHPLC) is used for the hemoglobin quantitation.

Thiolation mediated pegylation platform to generate functional universal red blood cells.

PubMed

Nacharaju, Parimala; Manjula, Belur N; Acharya, Seetharama A

2007-01-01

The PEGylation that adds an extension arm on protein amino groups with the conservation of their positive charge masks the A and D antigens of erythrocytes efficiently. In the present study, the efficiency of masking the antigens of RBC by PEGylation protocols that do not conserve the charge with and without adding extension arms is compared. The conjugation of PEG-5000 to RBCs through the addition of extension arms masked the D antigen more efficiently than the other protocol. A combination of PEG-5 K and PEG-20 K is needed to mask the A antigen, irrespective of the PEGylation approach. The oxygen affinity of the PEGylated RBCs increased by the extension arm facilitated PEGylation. The protocol involving the conjugation of PEG-chains without adding extension arm did not alter the oxygen affinity of RBCs. A combination of PEGylation protocols is an alternate strategy to generate universal red blood cells with good levels of oxygen affinity.

Novel inhibitors of the Gardos channel for the treatment of sickle cell disease.

PubMed

McNaughton-Smith, Grant A; Burns, J Ford; Stocker, Jonathan W; Rigdon, Gregory C; Creech, Christopher; Arrington, Susan; Shelton, Tara; de Franceschi, Lucia

2008-02-28

Sickle cell disease (SCD) is a hereditary condition characterized by deformation of red blood cells (RBCs). This phenomenon is due to the presence of abnormal hemoglobin that polymerizes upon deoxygenation. This effect is exacerbated when dehydrated RBCs experience a loss of both water and potassium salts. One critical pathway for the regulation of potassium efflux from RBCs is the Gardos channel, a calcium-activated potassium channel. This paper describes the synthesis and biological evaluation of a series of potent inhibitors of the Gardos channel. The goal was to identify compounds that were potent and selective inhibitors of the channel but had improved pharmacokinetic properties compared to 1, Clotrimazole. Several triarylamides such as 10 and 21 were potent inhibitors of the Gardos channel (IC50 of <10 nM) and active in a mouse model of SCD. Compound 21 (ICA-17043) was advanced into phase 3 clinical trials for SCD.

Molecular cloning and characterization of ADP-glucose pyrophosphorylase cDNA clones isolated from pea cotyledons.

PubMed

Burgess, D; Penton, A; Dunsmuir, P; Dooner, H

1997-02-01

Three ADP-glucose pyrophosphorylase (ADPG-PPase) cDNA clones have been isolated and characterized from a pea cotyledon cDNA library. Two of these clones (Psagps1 and Psagps2) encode the small subunit of ADPG-PPase. The deduced amino acid sequences for these two clones are 95% identical. Expression of these two genes differs in that the Psagps2 gene shows comparatively higher expression in seeds relative to its expression in other tissues. Psagps2 expression also peaks midway through seed development at a time in which Psagps1 transcripts are still accumulating. The third cDNA isolated (Psagp11) encodes the large subunit of ADPG-PPase. It shows greater selectivity in expression than either of the small subunit clones. It is highly expressed in sink organs (seed, pod, and seed coat) and undetectable in leaves.

GABAA receptors: Various stoichiometrics of subunit arrangement in α1β3 and α1β3ε receptors.

PubMed

Has, Ahmad Tarmizi Che; Chebib, Mary

2018-05-15

GABAA receptors (GABAARs) are members of the Cys-loop ligand-gated ion channel (LGIC) superfamily, which includes nicotinic acetylcholine, glycine, and serotonin (5HT3) receptors [1,2,3,4]. LGICs typically mediate fast synaptic transmission via the movement of ions through channels gated by neurotransmitters, such as acetylcholine for nicotinic receptors and GABA for GABAARs [5]. The term Cys-loop receptors originates from the presence of a conserved disulphide bond (or bridge) which holds together two cysteine amino acids of the loop that forms from the structure of polypeptides in the extracellular domain of the receptor's subunit [6]. GABAARs are pentameric transmembrane protein complexes consisting of five subunits from a variety of polypeptide subunits [7,8]. All of these subunits are pseudo-symmetrically organized in the plane of the membrane, with a Cl--selective channel in the middle of the complex. To date, nineteen GABAAR subunits have been identified and categorized into eight classes, α1-6, β1-3, γ1-3, δ, ε, θ, π and ρ1-3, but their variety is further broadened by the existence of several splice forms for certain subunits (e.g., α6, β2 and γ2) [9,10,11,12]. The subunits within each class have an amino acid sequence homology of 70% or more, whereas those with a sequence homology of 30% or less are grouped into different classes [13,14]. A subunit consists of four transmembrane domains (TM1-TM4), each forming an α-helix; a large extracellular N-terminal domain that incorporates part of the orthosteric agonist/antagonist binding site; a large intracellular loop between the TM3 and TM4; a small intracellular loop between TM1 and TM2; a small extracellular loop between TM2 and TM3; and a C-terminal extracellular domain [15,16]. Each subunit is arranged in such a way as to create principal and complementary interfaces with the other subunits, and in a position such that the TM2 of each subunit forms the wall of the channel pore [17,18,19]. The major subunit combination found in the brain comprises α1, β2 and γ2 subunits with the stoichiometry 2α1:2β2:1γ2 [18,20]. For the GABAA α1β2γ2 receptors, the subunits form a specific arrangement in which α1 and β2 subunits alternate with each other and are connected by a γ2 subunit (Figure A) [16,20,21]. For minor subtypes, different α and β subunits have been detected to co-exist as proven by the existence of GABAARs containing α1α2, α1α3, α1α5, α2α3, α3α5, α4α1, α4α2 and α4α3 in the central nervous system [22,23]. Meanwhile, the same pattern has been detected with β and γ subunits, at least the co-occurrence of β in the same GABAAR as well as γ2 with γ3, indicating that these subunits have the capacity to co-exist with each other [24,25,26]. Since different subunits can actually occur in one receptor, GABAARs are considered to exist in a multi-subunit arrangement, leading to ambiguity in the determination of a receptor's stoichiometry. In this review, we first briefly discuss the different subunit arrangements of α1 and β3 subunits in the binary α1β3 receptors. Then we review the GABAA ε-containing receptors predominantly in terms of the ability of ε subunit to present in different position in the ternary α1β3ε receptors, which introduce distinct populations of receptor. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase

PubMed Central

Bowman, Lisa; Flanagan, Lindsey; Fyfe, Paul K.; Parkin, Alison; Hunter, William N.; Sargent, Frank

2014-01-01

Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydro-genase was observed to comprise associated large and small subunits. The structure indicated that His229 from the large subunit was close to the proximal [4Fe–3S] cluster in the small subunit. In addition, His229 was observed to lie close to a buried glutamic acid (Glu73), which is conserved in oxygen-tolerant hydrogenases. His229 and Glu73 of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His229 or Glu73 with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that His229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73could play a supporting role in fine-tuning the chemistry of His229 to enable this function. PMID:24428762

Novel Insights in the Regulation of Phosphatidylserine Exposure in Human Red Blood Cells.

PubMed

Wesseling, Mauro C; Wagner-Britz, Lisa; Nguyen, Duc Bach; Asanidze, Salome; Mutua, Judy; Mohamed, Nagla; Hanf, Benjamin; Ghashghaeinia, Mehrdad; Kaestner, Lars; Bernhardt, Ingolf

2016-01-01

In previous publications we were able to demonstrate the exposure of phosphatidylserine (PS) in the outer membrane leaflet after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA), phorbol-12 myristate-13acetate (PMA), or 4-bromo-A23187 (A23187). It has been concluded that three different mechanisms are responsible for the PS exposure in human RBCs: (i) Ca2+-stimulated scramblase activation (and flippase inhibition) by A23187, LPA, and PMA; (ii) PKCα activation by LPA and PMA; and (iii) enhanced lipid flip flop caused by LPA. Further studies aimed to elucidate interconnections between the increased Ca2+ content, scramblase- and PKCα-activation. In addition, the role of the Ca2+-activated K+ channel (Gardos channel) activity in the process of PS exposure needs to be investigated. The intracellular Ca2+ content and the PS exposure of RBCs have been investigated after treatment with LPA (2.5 µM), PMA (6 µM), or A23187 (2 µM). Fluo-4 and annexin V-FITC has been used to detect intracellular Ca2+ content and PS exposure, respectively. Both parameters (Ca2+ content, PS exposure) were studied using flow cytometry. Inhibitors of the scramblase, the PKCα, and the Gardos channel have been applied. The percentage of RBCs showing PS exposure after activation with LPA, PMA, or A23187 is significantly reduced after inhibition of the scramblase using the specific inhibitor R5421 as well as after the inhibition of the PKCα using chelerythrine chloride or calphostin C. The inhibitory effect is more pronounced when the scramblase and the PKCα are inhibited simultaneously. Additionally, the inhibition of the Gardos channel using charybdotoxin resulted in a significant reduction of the percentage of RBCs showing PS exposure under all conditions measured. Similar results were obtained when the Gardos channel activity was suppressed by increased extracellular K+ content. PS exposure is mediated by the Ca2+-dependent scramblase but also by PKCα activated by LPA and PMA in a Ca2+-dependent and a Ca2+-independent manner. Furthermore, we hypothesize that a hyperpolarisation of RBCs caused by the opening of the Gardos channel is essential for the scramblase activity as well as for a fraction of the LPA-induced Ca2+ entry. © 2016 The Author(s) Published by S. Karger AG, Basel.