Review: In vitro generation of red blood cells for transfusion medicine: Progress,prospects and challenges

In vitro generation of red blood cells (RBCs) has the potential to circumvent the shortfalls in global demand for blood for transfusion applications. The conventional approach for RBC generation has been from differentiation of hematopoietic stem cells (HSCs) derived from cord blood, adult bone marrow or peripheral blood. More recently, RBCs have been generated from human induced pluripotent stem cells (hiPSCs) as well as from immortalized adult erythroid progenitors. In this review, we highlight the recent advances to RBC generation from these different approaches and discuss the challenges and new strategies that can potentially make large-scale in vitro generation of RBCs a feasible approach.     

Kinetics of Choline Uptake into Isolated Rat Forebrain Microvessels: Evidence of Endocrine Modulation

The active uptake of [methyl-3H]choline into isolated rat brain microvessel suspension was studied as a likely guide to the transport of choline across the blood-brain barrier. The method consisted primarily of incubation of the suspension with a fixed concentration of labeled choline in the presence of increasing concentrations of unlabeled choline or any other inhibitor (I) of active uptake, defined as the difference in uptake at 37 degrees and 0 degrees C. From the linear regression of (1/V) against [I], the following values of Vmax (nmol g-1 min-1) and Km (microM) were obtained for choline: 2-month-old males, 10.6 +/- 3.8 and 6.1 +/- 0.9; 3-month old random females, 28.4 +/- 5.9 and 12.6 +/- 4.0; females at metaestrus, 17.8 +/- 10.3 and 8.3 +/- 5.0; at diestrus, 31.1 +/- 9.3 and 13.0 +/- 2.6; at proestrus, 54.9 +/- 2.2 and 14.0 +/- 1.5; at estrus, 19.2 +/- 2.2 and 2.6 +/- 1.7. The differences between males and random females (p less than 0.018) and between females at proestrus and estrus (p less than 0.005) are significant. It is suggested that these inter- and intrasex variations in choline uptake reflect a dynamic adjustment of supply in accordance with brain demand for choline at the time of assay. Hemicholinium-3 was an effective inhibitor of choline uptake, Ki = 14.0 +/- 8.5 microM; dimethylaminoethanol was much less effective; and imipramine had no measurable effect.     

A single element within the hepatitis B virus enhancer binds multiple proteins and responds to multiple stimuli.

The hepatitis B virus enhancer can be dissected into multiple functional elements, one of which is the E element. We show here that the E element binds multiple nuclear proteins that are essential for its enhancer activity. These findings, together with the ability of this element to respond to at least two different viral transactivators, suggest that the E element is an enhancer modulator capable of binding different factors and responding to multiple stimuli.     

Adaptation to Short-Term Cataclysmic Events: Flooding in Premodern Riverine Societies

This article examines flooding and resilience in two riverine systems in the premodern Eastern Mediterranean. Flooding represents a distinct type of short-term cataclysmic events (SCEs) because of its frequency and long-term predictability which facilitates societal adaptation. We discuss the sources for premodern floods and their limitations before surveying Mesopotamia and Ancient Egypt as case studies. Both societies are compared with regard to their environment and how it shaped local flood management practices. We argue that although floods caused short-term societal disruption in these societies, they also stimulated the reorganization and regeneration of economic resources. Both Mesopotamian and Egyptian societies systematically managed and mitigated their risks and were, in general, resilient to flooding events.     

Nitroxide Radicals Prevent Metal-aggravated Reperfusion Injury in Isolated Rat Heart

The effects of Cu(II) and the stable nitroxide radical 4-OH-2, 2, 6, 6-tetramethyl-piperidine-1-oxyl (TPL) on reperfusion injury following global myocardial ischemia have been studied using the isolated rat heart model in the Langendorff configuration. Hearts were equilibrated with Krebs-Henseleit buffer (KH-buffer) for 10 min and subjected to 18 min of normothermic global ischemia. After 20 min reperfusion, hemodynamic parameters recovered as follows: ventricular developed pressure (77%), dP/dt (71%) and -dP/dt (80%), heart rate (91%), and work index (70%). End-diastolic pressure was 16 mm Hg. When 10μM Cu-nitrilotriacetate or Cu-(histidine)₂ was included in the perfusate before, during, and following ischemia, the heart injury was more extensive and the work index only recovered to 17% of the preischemic value. The inclusion of 100μM TPL during reperfusion abolished the copper-induced sensi-tization. In the absence of copper, TPL did not provide any protection against ischemia-reperfusion damage to the heart. The inclusion of 100μM 1, 4-dihydroxy-2, 2, 6, 6-tetramethylpiperidine (TPL-H) during reperfusion, partially abolished the copper-induced sensitization. Since conversion between TPL and TPL-H takes place, the fact that both forms provide protection can increase their protective efficacy.     

Phosphatidylcholine Biosynthesis in the Neuroblastoma-Glioma Hybrid Cell Line NG 108-15: Stimulation by Phorbol Esters

Studies were conducted on curaremimetic neurotoxin binding to the nicotinic acetylcholine receptor present on membrane fractions derived from the human medulloblastoma clonal line, TE671. High-affinity binding sites (KD = 2 nM for 1-h incubation at 20 degrees C) and low-affinity binding sites (KD = 40 nM) for 125I-labeled alpha-bungarotoxin are present in equal quantities (60 fmol/mg membrane protein). The kinetically determined dissociation constant for high-affinity binding of toxin is 0.56 nM (k1 = 6.3 X 10(-3) min-1 nM-1; k-1 = 3.5 X 10(-3) min-1) at 20 degrees C. Nicotine, d-tubocurarine, and acetylcholine are among the most effective inhibitors of high-affinity toxin binding. The quantity of toxin binding sites and their affinity for cholinergic agonists is sensitive to reduction, alkylation, and/or oxidation of membrane sulfhydryl residues. High-affinity toxin binding sites that have been subjected to reaction with the sulfhydryl reagent dithiothreitol are irreversibly blocked by the nicotinic receptor affinity reagent bromoacetylcholine. High-affinity toxin binding is inhibited in the presence of either of two polyclonal antisera or a monoclonal antibody raised against nicotinic acetylcholine receptors from fish electric tissue. Taken together, these results indicate that curaremimetic neurotoxin binding sites on membrane fractions of the TE671 cell line share some properties with nicotinic acetylcholine receptors of peripheral origin and with toxin binding sites on other neuronal tissues.     

The effects of temperature and drought on autumnal senescence and leaf shed in apple under warm,east mediterranean climate

Key message

Autumnal senescence of apple in a warm climate corresponds to accumulated degree-days beneath 22 °C. Summer drought delays senescence and enables replenishment of carbohydrate reserves. Recovery of the root system plays a key role.

Abstract

Autumnal senescence of apple (Malus domestica Borkh.), a deciduous, temperate climate species, is triggered by a rather abrupt temperature drop, down to the lower teens. Under the warmer, east Mediterranean climate of northern Israel, the temperature drop is gradual and much more moderate. Another characteristic of this climate is the complete lack of precipitation during summer. The aim of the present study was to elucidate the effects of summer drought on seasonal leaf senescence in a warm autumn. We hypothesized that summer drought delays senescence due to an increased demand for carbohydrates during autumn. The advent of autumnal senescence was followed for 3 years (2009–2011) on trees exposed to various levels of drought. Total canopy green area (effective leaf area, ELA) and hue angle were estimated periodically by means of image analysis, as a measure of leaf drop and autumnal color change. Photosynthesis, midday stem water potential, and roots’ non-structural carbohydrate contents were measured on several occasions. The time course of leaf drop followed the decline in air and soil temperatures. The rate of decline in ELA closely corresponded to accumulated degree-days beneath 22 °C in the soil, a much higher temperature threshold than previously reported for apple. Drought stress during the summer delayed leaf senescence even further, when compared with well-irrigated trees. Leaves maintained their photosynthetic functionality throughout autumn, until late December. The delayed senescence enabled replenishment of root carbohydrate reserves, which is critical for next year’s growth and fruiting. The eco-physiological significance of the findings is discussed.     

Structural changes in bacteriorhodopsin following retinal photoisomerization from the 13-cis form

Bacteriorhodopsin (BR), a light-driven proton pump in Halobacterium salinarum, accommodates two resting forms of the retinylidene chromophore, the all-trans form (AT-BR) and the 13-cis,15-syn form (13C-BR). Both isomers are present in thermal equilibrium in the dark, but only the all-trans form has proton-pump activity. In this study, we applied low-temperature Fourier-transform infrared (FTIR) spectroscopy to 13C-BR at 77 K and compared the local structure around the chromophore before and after photoisomerization with that in AT-BR. Strong hydrogen-out-of-plane (HOOP) vibrations were observed at 964 and 958 cm(-)(1) for the K state of 13C-BR (13C-BR(K)) versus a vibration at 957 cm(-)(1) for the K state of AT-BR (AT-BR(K)). In AT-BR(K), but not in 13C-BR(K), the HOOP modes exhibit isotope shifts upon deuteration of the retinylidene at C15 and at the Schiff base nitrogen. Whereas the HOOP modes of AT-BR(K) were significantly affected by the mutation of Thr89, this was not the case for the HOOP modes of 13C-BR(K). These observations imply that, while the chromophore distortion is localized near the Schiff base in AT-BR(K), it is located elsewhere in 13C-BR(K). By use of [zeta-(15)N]lysine-labeled BR, we identified the N-D stretching vibrations of the 13C-BR Schiff base (in D(2)O) at 2173 and 2056 cm(-)(1), close in frequency to those of AT-BR. These frequencies indicate strong hydrogen bonding of the Schiff base in 13C-BR, presumably with a water molecule as in AT-BR. In contrast, the N-D stretching vibration appears at 2332 and 2276 cm(-)(1) in 13C-BR(K) versus values of 2495 and 2468 cm(-)(1) for AT-BR(K), suggesting that the rupture of the Schiff base hydrogen bond that occurs in AT-BR(K) does not occur in 13C-BR(K). Rotational motion of the Schiff base upon retinal isomerization is probably smaller in magnitude for 13C-BR than for AT-BR. These differences in the primary step are possibly related to the absence of light-driven proton pumping by 13C-BR.