Sickle Cell Hemoglobin with Mutation at αHis-50 Has Improved Solubility

The unliganded tetrameric Hb S has axial and lateral contacts with neighbors and can polymerize in solution. Novel recombinants of Hb S with single amino acid substitutions at the putative axial (recombinant Hb (rHb) (βE6V/αH20R) and rHb (βE6V/αH20Q)) or lateral (rHb (βE6V/αH50Q)) or double amino acid substitutions at both the putative axial and lateral (rHb (βE6V/αH20R/αH50Q) and rHb (βE6V/αH20Q/αH50Q)) contact sites were expressed in Escherichia coli and purified for structural and functional studies. The ¹H NMR spectra of the CO and deoxy forms of these mutants indicate that substitutions at either αHis-20 or αHis-50 do not change the subunit interfaces or the heme pockets of the proteins. The double mutants show only slight structural alteration in the β-heme pockets. All mutants have similar cooperativity (n₅₀), alkaline Bohr effect, and autoxidation rate as Hb S. The oxygen binding affinity (P₅₀) of the single mutants is comparable with that of Hb S. The double mutants bind oxygen with slightly higher affinity than Hb S under the acidic conditions. In high salt, rHb (βE6V/αH20R) is the only mutant that has a shorter delay time of polymerization and forms polymers more readily than Hb S with a dextran-Csat value of 1.86 ± 0.20 g/dl. Hb S, rHb (βE6V/αH20Q), rHb (βE6V/αH50Q), rHb (βE6V/αH20R/αH50Q), and rHb (βE6V/αH20Q/αH50Q) have dextran-Csat values of 2.95 ± 0.10, 3.04 ± 0.17, 11.78 ± 0.59, 7.11 ± 0.66, and 10.89 ± 0.83 g/dl, respectively. rHb (βE6V/αH20Q/αH50Q) is even more stable than Hb S under elevated temperature (60 °C).     

Sickle Cell Hemoglobin in the Ferryl State Promotes βCys-93 Oxidation and Mitochondrial Dysfunction in Epithelial Lung Cells (E10)

Polymerization of intraerythrocytic deoxyhemoglobin S (HbS) is the primary molecular event that leads to hemolytic anemia in sickle cell disease (SCD). We reasoned that HbS may contribute to the complex pathophysiology of SCD in part due to its pseudoperoxidase activity. We compared oxidation reactions and the turnover of oxidation intermediates of purified human HbS and HbA. Hydrogen peroxide (H₂O₂) drives a catalytic cycle that includes the following three distinct steps: 1) initial oxidation of ferrous (oxy) to ferryl Hb; 2) autoreduction of the ferryl intermediate to ferric (metHb); and 3) reaction of metHb with an additional H₂O₂ molecule to regenerate the ferryl intermediate. Ferrous and ferric forms of both proteins underwent initial oxidation to the ferryl heme in the presence of H₂O₂ at equal rates. However, the rate of autoreduction of ferryl to the ferric form was slower in the HbS solutions. Using quantitative mass spectrometry and the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide, we found more irreversibly oxidized βCys-93in HbS than in HbA. Incubation of the ferric or ferryl HbS with cultured lung epithelial cells (E10) induced a drop in mitochondrial oxygen consumption rate and impairment of cellular bioenergetics that was related to the redox state of the iron. Ferryl HbS induced a substantial drop in the mitochondrial transmembrane potential and increases in cytosolic heme oxygenase (HO-1) expression and mitochondrial colocalization in E10 cells. Thus, highly oxidizing ferryl Hb and heme, the product of oxidation, may be central to the evolution of vasculopathy in SCD and may suggest therapeutic modalities that interrupt heme-mediated inflammation.     

The Role of Altered Cell–Cell Communication in Melanoma Progression

Under normal homeostasis, melanocyte growth and behaviour is tightly controlled by the surrounding keratinocytes. Keratinocytes regulate melanocyte behaviour through a complex system of paracrine growth factors and cell-cell adhesion molecules. Pathological changes, leading to development of malignant melanoma, upset this delicate homeostatic balance and can lead to altered expression of cell-cell adhesion and cell-cell communication molecules. In particular, there is a switch from the E-cadherin-mediated keratinocyte-melanocyte partnership to the N-cadherin-mediated melanoma-melanoma and melanoma-fibroblast interaction. Other changes include the alteration in the gap junctions formed between the melanocyte and keratinocyte. Changes in the connexin expression, in particular the loss of connexin 43, may result in a reduction or a loss of gap junctional activity, which is thought to contribute towards tumour progression. In the current review we describe the alterations in cell-cell adhesion and communication associated with melanoma development and progression, and discuss how a greater understanding of these processes may aid the future therapy of this disease.     

Effect of X-Irradiation at Different Stages in the Cell Cycle on Individual Cell–Based Kinetics in an Asynchronous Cell Population

Using an asynchronously growing cell population, we investigated how X-irradiation at different stages of the cell cycle influences individual cell–based kinetics. To visualize the cell-cycle phase, we employed the fluorescent ubiquitination-based cell cycle indicator (Fucci). After 5 Gy irradiation, HeLa cells no longer entered M phase in an order determined by their previous stage of the cell cycle, primarily because green phase (S and G2) was less prolonged in cells irradiated during the red phase (G1) than in those irradiated during the green phase. Furthermore, prolongation of the green phase in cells irradiated during the red phase gradually increased as the irradiation timing approached late G1 phase. The results revealed that endoreduplication rarely occurs in this cell line under the conditions we studied. We next established a method for classifying the green phase into early S, mid S, late S, and G2 phases at the time of irradiation, and then attempted to estimate the duration of G2 arrest based on certain assumptions. The value was the largest when cells were irradiated in mid or late S phase and the smallest when they were irradiated in G1 phase. In this study, by closely following individual cells irradiated at different cell-cycle phases, we revealed for the first time the unique cell-cycle kinetics in HeLa cells that follow irradiation.     

Single Nucleotide Polymorphisms in the Wilms’ Tumour Gene 1 in Clear Cell Renal Cell Carcinoma

The Wilms’ tumour gene 1 (WT1) single nucleotide polymorphism (SNP) rs16754 has recently been described as an independent prognostic factor in acute myeloid leukaemia (AML) patients. It is of great interest to test whether WT1 SNPs can be used as a molecular marker in other cancer types in order to improve risk and treatment stratification. We performed sequencing analysis on all 10 exons of the WT1 gene in a total of 182 patients with clear cell renal cell carcinoma (ccRCC). Six different SNPs were identified, in descending order for minor allele frequency: rs2234582, rs16754, rs1799925, rs5030315, rs2234583, and rs2234581. At least one minor allele for WT1 SNP was identified in 61% of ccRCC patients. In the entire study population, only 6% carried two copies of the minor allele. The genotypes of WT1 SNPs in 78 tumour-free kidney tissue specimens were found to be in 95% concordance with corresponding tumour samples. No correlation was observed between WT1 SNP genotypes and RNA expression level. WT1 SNP genotypes did not associate with clinical and pathological characteristics. We found favourable outcomes associated with the homozygous minor allele for WT1 SNP. However, SNP genotypes did not show to be of prognostic significance when comparing wild-type versus homozygous or heterozygous for the minor allele in the entire cohort. None of the previously reported WT1 mutations in AML was found in the present study. A novel WT1 missense mutation was identified in only one patient. Our data suggest that common WT1 mutations are not involved in ccRCC. Due to too few cases harbouring the homozygous minor allele, the prognostic impact needs to be verified in larger study populations.     

Global Burden of Sickle Cell Anaemia in Children under Five, 2010–2050: Modelling Based on Demographics,Excess Mortality,and Interventions

Background

The global burden of sickle cell anaemia (SCA) is set to rise as a consequence of improved survival in high-prevalence low- and middle-income countries and population migration to higher-income countries. The host of quantitative evidence documenting these changes has not been assembled at the global level. The purpose of this study is to estimate trends in the future number of newborns with SCA and the number of lives that could be saved in under-five children with SCA by the implementation of different levels of health interventions.

Methods and Findings

First, we calculated projected numbers of newborns with SCA for each 5-y interval between 2010 and 2050 by combining estimates of national SCA frequencies with projected demographic data. We then accounted for under-five mortality (U5m) projections and tested different levels of excess mortality for children with SCA, reflecting the benefits of implementing specific health interventions for under-five patients in 2015, to assess the number of lives that could be saved with appropriate health care services. The estimated number of newborns with SCA globally will increase from 305,800 (confidence interval [CI]: 238,400–398,800) in 2010 to 404,200 (CI: 242,500–657,600) in 2050. It is likely that Nigeria (2010: 91,000 newborns with SCA [CI: 77,900–106,100]; 2050: 140,800 [CI: 95,500–200,600]) and the Democratic Republic of the Congo (2010: 39,700 [CI: 32,600–48,800]; 2050: 44,700 [CI: 27,100–70,500]) will remain the countries most in need of policies for the prevention and management of SCA. We predict a decrease in the annual number of newborns with SCA in India (2010: 44,400 [CI: 33,700–59,100]; 2050: 33,900 [CI: 15,900–64,700]). The implementation of basic health interventions (e.g., prenatal diagnosis, penicillin prophylaxis, and vaccination) for SCA in 2015, leading to significant reductions in excess mortality among under-five children with SCA, could, by 2050, prolong the lives of 5,302,900 [CI: 3,174,800–6,699,100] newborns with SCA. Similarly, large-scale universal screening could save the lives of up to 9,806,000 (CI: 6,745,800–14,232,700) newborns with SCA globally, 85% (CI: 81%–88%) of whom will be born in sub-Saharan Africa. The study findings are limited by the uncertainty in the estimates and the assumptions around mortality reductions associated with interventions.

Conclusions

Our quantitative approach confirms that the global burden of SCA is increasing, and highlights the need to develop specific national policies for appropriate public health planning, particularly in low- and middle-income countries. Further empirical collaborative epidemiological studies are vital to assess current and future health care needs, especially in Nigeria, the Democratic Republic of the Congo, and India. Please see later in the article for the Editors'' Summary     

Cell Bound α-Amylase in Aspergillus oryzae

It was previously reported that α-amylase accumulation is caused within the mycelium grown in a phosphate deficient medium and the concentration of anions or pH in a surrounding medium is responsible for its liberation. As it was subsequently found that α-amylase liberation from the mycelium of Aspergillus oryzae is stimulated by peptone, an attempt was made on purification of effective substances from it. The present paper describes on purification and properties of phosphopeptides found as an effective substance for α-amylase liberation, and discusses on the stimulation effect, comparing with the effects on pH and concentration of anions which were previously observed.     

Influenza Hemifusion Phenotype Depends on Membrane Context: Differences in Cell–Cell and Virus–Cell Fusion

Influenza viral entry into the host cell cytoplasm is accomplished by a process of membrane fusion mediated by the viral hemagglutinin protein. Hemagglutinin acts in a pH-triggered fashion, inserting a short fusion peptide into the host membrane followed by refolding of a coiled-coil structure to draw the viral envelope and host membranes together. Mutations to this fusion peptide provide an important window into viral fusion mechanisms and protein–membrane interactions. Here, we show that a well-described fusion peptide mutant, G1S, has a phenotype that depends strongly on the viral membrane context. The G1S mutant is well known to cause a “hemifusion” phenotype based on experiments in transfected cells, where cells expressing G1S hemagglutinin can undergo lipid mixing in a pH-triggered fashion similar to virus but will not support fusion pores. We compare fusion by the G1S hemagglutinin mutant expressed either in cells or in influenza virions and show that this hemifusion phenotype occurs in transfected cells but that native virions are able to support full fusion, albeit at a slower rate and 10–100 × reduced infectious titer. We explain this with a quantitative model where the G1S mutant, instead of causing an absolute block of fusion, alters the protein stoichiometry required for fusion. This change slightly slows fusion at high hemagglutinin density, as on the viral surface, but at lower hemagglutinin density produces a hemifusion phenotype. The quantitative model thus reproduces the observed virus–cell and cell–cell fusion phenotypes, yielding a unified explanation where membrane context can control the observed viral fusion phenotype.     

Cell—cell interactions in bacterial populations

In developing bacterial populations many essential processes, such as division, genetic transformation, sporulation, and synthesis of antibiotics and secondary metabolites, are regulated by intercellular communication mediated by secretion of signaling molecules, such as homoserine lactones and peptides. Another intercellular communication type, namely a physical contact between cells (cell aggregation), plays a key role in formation of biofilms or cellular consortia and in cell proliferation under unfavorable conditions. The mechanisms involved in these two types of bacterial communication are discussed in this review.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1555–1564.Original Russian Text Copyright © 2004 by Voloshin, Kaprelyants     

Quantification of Biomass and Cell Motion in Human Pluripotent Stem?Cell?Colonies

Somatic cell reprogramming to pluripotency requires an immediate increase in cell proliferation and reduction in cell size. It is unknown whether proliferation and biomass controls are similarly coordinated with early events during the differentiation of pluripotent stem cells (PSCs). This impasse exists because PSCs grow in tight clusters or colonies, precluding most quantifying approaches. Here, we investigate live cell interferometry as an approach to quantify the biomass and growth of HSF1 human PSC colonies before and during retinoic acid-induced differentiation. We also provide an approach for measuring the rate and coordination of intracolony mass redistribution in HSF1 clusters using live cell interferometry images. We show that HSF1 cells grow at a consistent, exponential rate regardless of colony size and display coordinated intracolony movement that ceases with the onset of differentiation. By contrast, growth and proliferation rates show a decrease of only ∼15% decrease during early differentiation despite global changes in gene expression and previously reported changes in energy metabolism. Overall, these results suggest that cell biomass and proliferation are regulated independent of pluripotency during early differentiation, which is distinct from what occurs with successful reprogramming.     

Developmental and Stress-Induced Remodeling of Cell–Cell Communication in the Adrenal Medullary Tissue

The adrenal medullary tissue contributes to maintain body homeostasis in reaction to stressful environmental changes via the release of catecholamines into the blood circulation in response to splanchnic nerve activation. Accordingly, chromaffin cell stimulus-secretion coupling undergoes temporally restricted periods of anatomo-functional remodeling in response to prevailing hormonal requirements of the organism. The postnatal development of the adrenal medulla and response to stress are remarkable physiological situations in which the stimulus-secretion coupling is critically affected. Catecholamine secretion from rat chromaffin cells is under a dual control involving an incoming initial command arising from the sympathetic nervous system that releases acetylcholine at the splanchnic nerve terminal-chromaffin cell synapses and a local gap junction-mediated intercellular communication. Interestingly, these two communication pathways are functionally interconnected within the gland and exhibit coordinated plasticity mechanisms. This article reviews the physiological and molecular evidence that the adrenal medullary tissue displays anatomical and functional adaptative remodeling of cell–cell communications upon physiological (postnatal development) and/or physiopathological (stress) situations associated with specific needs in circulating catecholamine levels.     

Cell–cell communication in Arabidopsis early embryogenesis

The basic body plan of the adult plant is established during embryogenesis, resulting in the juvenile form of the seedling. Arabidopsis embryogenesis is distinguished by a highly regular pattern of cell divisions. Some of these divisions are asymmetric, generating daughter cells with different fates. However, their subsequent differentiation might still depend on cell–cell communication to be fully accomplished or maintained. In some cases, cell fate specification solely depends on cell–cell communication that in general plays an important role in the generation of positional information within the embryo. Although auxin-dependent signalling has received much attention, other ways of cell–cell communication have also been demonstrated or suggested. This review focuses on aspects of pattern formation and cell–cell communication during Arabidopsis embryogenesis up to the mid-globular stage of development.     

Cell–cell Signaling in the Neurovascular Unit

Historically, the neuron has been the conceptual focus for almost all of neuroscience research. In recent years, however, the concept of the neurovascular unit has emerged as a new paradigm for investigating both physiology and pathology in the CNS. This concept proposes that a purely neurocentric focus is not sufficient, and emphasizes that all cell types in the brain including neuronal, glial and vascular components, must be examined in an integrated context. Cell–cell signaling and coupling between these different compartments form the basis for normal function. Disordered signaling and perturbed coupling form the basis for dysfunction and disease. In this mini-review, we will survey four examples of this phenomenon: hemodynamic neurovascular coupling linking blood flow to brain activity; cellular communications that evoke the blood–brain barrier phenotype; parallel systems that underlie both neurogenesis and angiogenesis in the CNS; and finally, the potential exchange of trophic factors that may link neuronal, glial and vascular homeostasis. Special issue in honor of Naren Banik.     

Cell specificity in auxin- and ethylene-induced ‘supergrowth’ in Riella helicophylla

Summary In gemmalings of Riella helicophylla, auxin and ethylene stimulate elongation growth, especially of pillar cells. When the two hormones are supplied simultaneously, the effects are additive, i.e. the result is supergrowth. In the cells of the meristem, elongation is enhanced by auxin, but not by ethylene when given alone. However, these cells also respond with supergrowth to a combined treatment with auxin and ethylene. The antiauxin p-chlorophenoxyisobutyric acid suppresses both the ethylene stimulation of cell growth and the additive supergrowth. The results support the concept that auxin pre-conditions the cells to the ethylene-dependent growth event. We suggest that the response elicited by the specific cell types could be related to differences in their level of endogenous auxin.Abbreviations IAA indole-3-acetic acid - PCIB p-chlorophenoxyisobutyric acid