Vertebrate Red Blood Cells Research Articles

Comparison of the human’s and camel’s red blood cell deformability by optical tweezers and Raman spectroscopy

Red blood cells of vertebrates have undergone evolutionary changes over time, leading to the diversification of morphological and mechanical properties of red blood cells (RBCs). Among the vertebrates, camelids have the most different RBC characteristics. As a result of adaptation to the desert environment, camelid RBCs can expand twice as much of their total volume in the case of rapid hydration yet are almost undeformable under mechanical stress. In this work, the mechanical and chemical differences in the RBC properties of the human and camelid species were examined using optical tweezers and Raman spectroscopy. We measured the deformability of camel and human RBCs at the single-cell level using optical tweezers. We found that the deformability index (DI) of the camel and the human RBCs were 0.024 ± 0.019 and 0.215 ± 0.061, respectively. To investigate the chemical properties of these cells, we measured the Raman spectra of the whole blood samples. The result of our study indicated that some of the Raman peaks observed on the camel’s blood spectrum were absent in the human blood’s spectrum, which further points to the difference in chemical contents of these two species’ RBCs.

Purification and partial characterization of carbohydrate-recognition protein C-type lectin from Hemifusus pugilinus

A mannose binding lectin (C-type lectin) was detected in a molluscan snail Hemifusus pugilinus, this lectin molecule was isolated and purified from the plasma using mannose-fixed sepharose CL-4B column affinity chromatography. The purified protein corresponds to the molecular weight of 118 kDa on an SDS-PAGE gel. The divalent cation-dependent nature of the H. pugilinus lectin (Hp-Lec) evidenced through pH and thermal stability analysis using Circular Dichroism (CD) and Surface Plasmon Resonance (SPR) respectively. Functional investigations of the Hp-Lec reveal a broad spectrum of bacterial agglutination activity against wide range of Gram-positive and Gram-negative bacterial strains. Furthermore, Hp-Lec displayed the haemo agglutination activity against vertebrate red blood cells (RBCs) and its titers were recorded. Excitingly, microbial virulent pathogens such as fungal strains tested against the purified Hp-Lec (25 and 50 μg/ml), which exhibits the effective antifungal activity against tested fungal pathogens such as Aspergillus niger and A. flavus.

Wdr26 regulates nuclear condensation in developing erythroblasts

AbstractMammalian red blood cells lack nuclei. The molecular mechanisms underlying erythroblast nuclear condensation and enucleation, however, remain poorly understood. Here we show that Wdr26, a gene upregulated during terminal erythropoiesis, plays an essential role in regulating nuclear condensation in differentiating erythroblasts. Loss of Wdr26 induces anemia in zebrafish and enucleation defects in mouse erythroblasts because of impaired erythroblast nuclear condensation. As part of the glucose-induced degradation-deficient ubiquitin ligase complex, Wdr26 regulates the ubiquitination and degradation of nuclear proteins, including lamin B. Failure of lamin B degradation blocks nuclear opening formation leading to impaired clearance of nuclear proteins and delayed nuclear condensation. Collectively, our study reveals an unprecedented role of an E3 ubiquitin ligase in regulating nuclear condensation and enucleation during terminal erythropoiesis. Our results provide mechanistic insights into nuclear protein homeostasis and vertebrate red blood cell development.

7SL RNA in vertebrate red blood cells.

We report that 7SL, the RNA component of the signal recognition particle (SRP), is an abundant noncoding RNA (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 coimmunoprecipitates 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 nonfunctional remnant of the protein synthetic machinery. Alternatively, it could play a new, as yet undefined role in RBC metabolism.

Hypoxia inhibits the regulatory volume decrease in red blood cells of common frog (Rana temporaria)

Red blood cells of vertebrates can restore their cellular volume after hyposmotic swelling. The process strictly depends on oxygen availability in the environment. However, the role of hemoglobin in regulation of cell volume recovery is not clear yet. Little is known about the osmotic reactions and regulatory volume decrease of amphibian red blood cells. We investigated volume recovery process in oxygenated (oxyhemoglobin concentration 97 ± 3% of total hemoglobin) deoxygenated (96 ± 2% of deoxyhemolobin) and oxidized (47 ± 2% of methemoglobin, 41 ± 3% of deoxyhemoglobin) red blood cells of common frog (Rana temporaria) after hyposmotic swelling. Using the low-angle light scattering method we demonstrated the regulatory volume decrease in oxygenated cells and showed that the process was eliminated in hypoxic conditions. Reoxygenation of hypoxic cells restored the regulatory volume decrease. Oxidation of cellular hemoglobin to methemoglobin inhibited the volume recovery response in hyposmotically swollen oxygenated and reoxygenated hypoxic cells.

Intracellular iron and heme trafficking and metabolism in developing erythroblasts.

Vertebrate red blood cells (RBCs) arise from erythroblasts in the human bone marrow through a process known as erythropoiesis. Iron uptake is a crucial hallmark, essential for heme biosynthesis in the differentiating erythroblasts, which are dedicated to producing hemoglobin. Erythropoiesis is facilitated by a network of intracellular transport proteins, chaperones, and circulating hormones. Intracellular iron is targeted to the mitochondria for incorporation into a porphyrin ring to form heme and cytosolic iron-sulfur proteins, including Iron Regulatory Protein 1 (IRP1). These processes are tightly regulated to prevent both excess and insufficient levels of iron and heme precursors. Crosstalk between the heme and iron-sulfur synthesizing pathways has been demonstrated to serve as a regulatory feedback mechanism. The activity of δ-aminolevulinic acid synthase (ALAS), the first and rate-limiting enzyme of heme biosynthesis, is a fundamental node of this regulation. Recently, the mitochondrial unfoldase, ClpX, has received attention as a novel key player that modulates this step in heme biogenesis, implicating a role in the pathophysiology of anemic diseases. This chapter reviews the canonical pathways in intracellular iron and heme trafficking and recent findings of iron and heme metabolism in vertebrate red cells. A discussion of the molecular approaches to studying iron and heme transport is provided to highlight opportunities for revealing therapeutic targets.

Bone Microvasculature Tracks Red Blood Cell Size Diminution in Triassic Mammal and Dinosaur Forerunners

Vertebrate red blood cells (RBCs) display a range ofsizes, spanning orders of magnitude in volume in different clades [1]. The importance of this size variation to diffusion during exercise is reinforced by functional links between RBC and capillary diameters [2, 3]. Small RBCs, such as those of mammals (which lack nuclei) and birds, contribute to shorter diffusion distances and permit relatively fast O2 uptake kinetics [4]. Although constraints on RBC size have been tied to the cell's need to attend capillary sizes for effective gas diffusion [3], as well as to genome size evolution [5, 6], major questions persist concerning patterns of RBC size evolution and its paleobiological significance. Here, we evaluate the relationship between RBC sizes and bone histometry and use microstructural evidence to trace their evolution in a phylogeny of extinct tetrapods. We find that several fossilizable aspects of bone microstructure, including the sizes of vascular and lacunar (cellular) spaces, provide useful indicators of RBC size in tetrapods. We also show that Triassic non-mammalian cynodonts had reduced and densely packed vascular canals identical to those of some mammals and likely accommodated smaller, more mammal-like RBCs. Reduced channel diameters accommodating smaller RBCs predated the origin of crown mammals by as much as 70 million years. This discovery offers a new proxy for the physiologic status of the mammal and avian stem groups and contextualizes the independent origins of their increased activity metabolism.

Phytoglobin: a novel nomenclature for plant globins accepted by the globin community at the 2014 XVIII conference on Oxygen-Binding and Sensing Proteins

Hemoglobin (Hb) is a heme-containing protein found in the red blood cells of vertebrates. For many years, the only known Hb-like molecule in plants was leghemoglobin (Lb). The discovery that other Hb-like proteins existed in plants led to the term “nonsymbiotic Hbs (nsHbs)” to differentiate them from the Lbs. While this terminology was adequate in the early stages of research on the protein, the complexity of the research in this area necessitates a change in the definition of these proteins to delineate them from red blood cell Hb. At the 2014 XVIII Conference on Oxygen-Binding and Sensing Proteins, the group devoted to the study of heme-containing proteins, this issue was discussed and a consensus was reached on a proposed name change. We propose Phytoglobin (Phytogb) as a logical, descriptive name to describe a heme-containing (Hb-like) protein found in plants. It will be readily recognized by the research community without a prolonged explanation of the origin of the term. The classification system that has been established can essentially remain unchanged substituting Phytogb in place of nsHb. Here, we present a guide to the new nomenclature, with reference to the existing terminology and a phylogenetic scheme, placing the known Phytogbs in the new nomenclature.

Phytoglobin: a novel nomenclature for plant globins accepted by the globin community at the 2014 XVIII conference on Oxygen-Binding and Sensing Proteins.

Hemoglobin (Hb) is a heme-containing protein found in the red blood cells of vertebrates. For many years, the only known Hb-like molecule in plants was leghemoglobin (Lb). The discovery that other Hb-like proteins existed in plants led to the term "nonsymbiotic Hbs (nsHbs)" to differentiate them from the Lbs. While this terminology was adequate in the early stages of research on the protein, the complexity of the research in this area necessitates a change in the definition of these proteins to delineate them from red blood cell Hb. At the 2014 XVIII Conference on Oxygen-Binding and Sensing Proteins, the group devoted to the study of heme-containing proteins, this issue was discussed and a consensus was reached on a proposed name change. We propose Phytoglobin (Phytogb) as a logical, descriptive name to describe a heme-containing (Hb-like) protein found in plants. It will be readily recognized by the research community without a prolonged explanation of the origin of the term. The classification system that has been established can essentially remain unchanged substituting Phytogb in place of nsHb. Here, we present a guide to the new nomenclature, with reference to the existing terminology and a phylogenetic scheme, placing the known Phytogbs in the new nomenclature.

More than hemoglobin - the unexpected diversity of globins in vertebrate red blood cells.

In many multicellular organisms, oxygen is transported by respiratory proteins, which are globins in vertebrates, between respiratory organs and tissues. In jawed vertebrates, eight globins are known which are expressed in a highly tissue-specific manner. Until now, hemoglobin (Hb) had been agreed to be the only globin expressed in vertebrate erythrocytes. Here, we investigate for the first time the mRNA expression of globin genes in nucleated and anucleated erythrocytes of model vertebrate species by quantitative real-time reverse transcription PCR (qRT-PCR). Surprisingly, we found transcripts of the whole gnathostome globin superfamily in RBCs. The mRNA expression levels varied among species, with Hb being by far the dominant globin. Only in stickleback, a globin previously thought to be neuron-specific, neuroglobin, had higher mRNA expression. We furthermore show that in birds transcripts of globin E, which was earlier reported to be transcribed only in the eye, are also present in RBCs. Even in anucleated RBCs of mammals, we found transcripts of myoglobin, neuroglobin, and cytoglobin. Our findings add new aspects to the current knowledge on the expression of globins in vertebrate tissues. However, whether or not the mRNA expression of these globin genes has any functional significance in RBCs has to be investigated in future studies.

PbGEST mediates malaria transmission to both mosquito and vertebrate host

The malaria life cycle relies on the successful transfer of the parasite between its human and mosquito hosts. We identified a Plasmodium berghei secreted protein (PBANKA_131270) that plays distinct roles in both the mammal-to-mosquito and the mosquito-to-mammal transitions. This protein, here named gamete egress and sporozoite traversal (GEST), plays an important role in the egress of male and female gametes from the vertebrate red blood cell. Interestingly, GEST is also required following the bite of the infected mosquito, for sporozoite progression through the skin. We found PbGEST to be secreted shortly after activation of the intraerythrocytic gametocyte, and during sporozoite migration. These findings indicate that a single malaria protein may have pleiotropic roles in different parasites stages mediating transmission between its insect and mammalian hosts.

Misdiagnosis of Spider Bites: Bacterial Associates, Mechanical Pathogen Transfer, and Hemolytic Potential of Venom From the Hobo Spider, Tegenaria agrestis (Araneae: Agelenidae)

The European spider Tegenaria agrestis (Walckenaer) (hobo spider) has been implicated as a spider of medical importance in the Pacific Northwest since its introduction in the late 1980s. Studies have indicated that the hobo spider causes necrotic tissue lesions through hemolytic venom or through the transfer of pathogenic bacteria introduced by its bite. Bacterial infections are often diagnosed as spider bites, in particular the pathogenic bacteria methicillin-resistant Staphylococcus aureus (MRSA). This study examines three aspects of the potential medical importance of hobo spiders in part of its introduced range, Washington State. First, the bacterial diversity of the spider was surveyed using a polymerase chain reaction-based assay to determine whether the spider carries any pathogenic bacteria. Second, an experiment was conducted to determine the ability of the spiders to transfer MRSA. Third, the venom was evaluated to assess the hemolytic activity. We found 10 genera of ubiquitous bacteria on the exterior surface of the spiders. In addition, none of the spiders exposed to MRSA transferred this pathogen. Finally, the hemolytic venom assay corroborates previous studies that found hobo spider venom was not deleterious to vertebrate red blood cells.

Allosteric modulation of haemoglobin function in response to changes in oxygen supply and demand: insights from aquatic ectotherms

Phosphate compounds in the red blood cells of vertebrates play an important role in modulating haemoglobin function whereby tissue oxygenation is maintained despite changes in both environmental oxygen supply and metabolic demand. Three examples from Gordon Grigg's work on aquatic ectotherms illustrate how our thinking has developed in terms of adaptive responses to seasonal temperature changes, environmental hypoxia, and ontogeny.

Specificity and Developmental Changes in Hemagglutinating Activity of Serum of the Desert LocustSchistocerca gregaria(Orthoptera: Acrididae)

Hemagglutinating activity (HA) of the serum of the 5th-instar desert locust Schistocerca gregaria was detected against a range of vertebrate red blood cells (RBCs). Serum strongly agglutinates RBCs of rabbit. In contrast, no agglutination was observed for RBCs from guinea pig or horse and a very low level was observed for human, sheep, and rat RBCs. HA is Ca2 dependent, heat-labile, and was strongly inhibited by α-linked-D-galactosides and rhamnose. Developmentally, a relatively low level of HA (≤32) is present in the extracted fluids from centrifuged homogenate of 1 to 12 day-old eggs. Also, a limited level of HA was observed in the 2nd and 3rd instars. However, prominent and cyclical patterns were observed in the 4th and 5th instars. In each of these cycles, HA starts low (64), maximizes (128, 512, in the two instars respectively) at about midstadium, then declines again prior to ecdysis to the next stage. On the other hand, adult stage HA starts at a low value (512), then maximizes and is sustained at a fairly constant value (1024), without any difference in the sexes throughout the period of measurements (up to day 73 of the life cycle). HA is independent of sex and season but conspicuously and reproducibly varies with stadium, stage and age.

Changes in Dry State Hemoglobin over Time Do Not Increase the Potential for Oxidative DNA Damage in Dried Blood

BackgroundHemoglobin (Hb) is the iron-containing oxygen transport protein present in the red blood cells of vertebrates. Ancient DNA and forensic scientists are particularly interested in Hb reactions in the dry state because both regularly encounter aged, dried bloodstains. The DNA in such stains may be oxidatively damaged and, in theory, may be deteriorated by the presence of Hb. To understand the nature of the oxidative systems potentially available to degrade DNA in the presence of dried Hb, we need to determine what molecular species Hb forms over time. These species will determine what type of iron (i.e. Fe2+/Fe3+/Fe4+) is available to participate in further chemical reactions. The availability of “free” iron will affect the ability of the system to undergo Fenton-type reactions which generate the highly reactive hydroxyl radical (OH•). The OH• can directly damage DNA.Methodology/Principal FindingsOxygenated Hb (oxyHb) converts over time to oxidized Hb (metHb), but this happens more quickly in the dry state than in the hydrated state, as shown by monitoring stabilized oxyHb. In addition, dry state oxyHb converts into at least one other unknown species other than metHb. Although “free” iron was detectable as both Fe2+ and Fe3+ in dry and hydrated oxyHb and metHb, the amount of ions detected did not increase over time. There was no evidence that Hb becomes more prone to generating OH• as it ages in either the hydrated or dry states.ConclusionsThe Hb molecule in the dried state undergoes oxidative changes and releases reactive Fe(II) cations. These changes, however, do not appear to increase the ability of Hb to act as a more aggressive Fenton reagent over time. Nevertheless, the presence of Hb in the vicinity of DNA in dried bloodstains creates the opportunity for OH•-induced oxidative damage to the deoxyribose sugar and the DNA nucleobases.

Quaternary Transition Pathway in Sol-Gel Encapsulated Haemoglobin Tracked by NIR and UV Spectral Relaxations

→T structural transition of haemoglobin (hb), the protein responsible for oxygen (o) transport in the red blood cells of vertebrates, is the hall mark example. This transition, which regu lates o2 uptake in the lungs and o2 release in the tissues, is a switch in the quaternary structure of the protein from a low-affinity state (T) to a high-affinity state (R), two well-characterised structures. The struc tural pathway connecting the end states of this transition remains unclear, however, although recently several experimental 1 or

In vivored blood cell sickling and mechanism of recovery in whiting,Merlangius merlangus

Haemoglobin concentrations in vertebrate red blood cells are so high that in human sickle cell disease a single surface amino acid mutation can result in formation of large insoluble haemoglobin aggregates at low oxygen levels, causing peculiar cell deformations or 'sickling'. This may cause vascular occlusion and thereby severe pain, organ failure and death. Here, using light and transmission electron microscopy, we demonstrate extensive in vivo sickling of whiting red blood cells after capture stress without any apparent haemolysis and show its subsequent recovery. We show exceptionally high cooperative proton binding during the sickling process in vitro and identify the reduction of extracellular pH below resting values as the primary cause for in vivo sickling, although the response is modulated to a lesser extent also by oxygen tension. Using isotope tracer fluxes, we further show that beta-adrenergic hormones, which are released under capture stress, activate a powerful endogenous Na/H exchanger in these fish red blood cells, which is known to elevate intracellular pH. beta-adrenergic treatment further leads to a marked reduction of acid-induced in vitro sickling, which is impaired when Na/H exchange is inhibited by amiloride. We propose that this mechanism protects red blood cells of some fishes against the problem of haemoglobin aggregation and red blood cell sickling, except under most severe acidosis. This system offers a unique example of how, over evolutionary time, nature may have overcome what is still a deadly disease in humans.

Comparative physiology of O2sensitive ion transport in vertebrate red blood cells

Comparative physiology of O2sensitive ion transport in vertebrate red blood cells

Soft tissue and cellular preservation in vertebrate skeletal elements from the Cretaceous to the present.

Soft tissues and cell-like microstructures derived from skeletal elements of a well-preserved Tyrannosaurus rex (MOR 1125) were represented by four components in fragments of demineralized cortical and/or medullary bone: flexible and fibrous bone matrix; transparent, hollow and pliable blood vessels; intravascular material, including in some cases, structures morphologically reminiscent of vertebrate red blood cells; and osteocytes with intracellular contents and flexible filipodia. The present study attempts to trace the occurrence of these four components in bone from specimens spanning multiple geological time periods and varied depositional environments. At least three of the four components persist in some skeletal elements of specimens dating to the Campanian. Fibrous bone matrix is more altered over time in morphology and less likely to persist than vessels and/or osteocytes. Vessels vary greatly in preservation, even within the same specimen, with some regions retaining pliability and other regions almost crystalline. Osteocytes also vary, with some retaining long filipodia and transparency, while others present with short and stubby filipodia and deeply pigmented nuclei, or are pigmented throughout with no nucleus visible. Alternative hypotheses are considered to explain the origin/source of observed materials. Finally, a two-part mechanism, involving first cross-linking of molecular components and subsequent mineralization, is proposed to explain the surprising presence of still-soft elements in fossil bone. These results suggest that present models of fossilization processes may be incomplete and that soft tissue elements may be more commonly preserved, even in older specimens, than previously thought. Additionally, in many cases, osteocytes with defined nuclei are preserved, and may represent an important source for informative molecular data.

Red blood cell size and the Fåhraeus–Lindqvist effect

The reason for the large interspecies variation in vertebrate red blood cell (RBC) size is poorly understood. To test the effect of RBC size on blood viscosity, blood samples from two vertebrates, the dog (Canis familiaris L., 1758) and the greater siren (Siren lacertina L., 1766), with extremely different RBC sizes were examined. In this study, we examined whether RBC size altered the relationship of viscosity and viscometer tube diameter, the well-known Fåhraeus–Lindqvist effect (FLE). We used a glass capillary viscometer that incorporates tubes with a diameter narrow enough to evoke the FLE. At similar RBC concentrations, viscosity of suspensions with the larger siren RBCs were greater than the viscosity of suspensions with the smaller dog RBCs. However, the relationship between viscosity and tube diameter is independent of RBC size. The results of this study allow us to conclude that the FLE is not related to RBC size. While packaging hemoglobin in RBCs appears adaptive by reducing blood viscosity through the FLE, RBC size does not contribute to the reductions in tube-relative viscosity.