Enhanced suicidal death of erythrocytes from gene-targeted mice lacking the Cl-/HCO(3)(-) exchanger AE1

Genetic defects of anion exchanger 1 (AE1) may lead to spherocytic erythrocyte morphology, severe hemolytic anemia, and/or cation leak. In normal erythrocytes, osmotic shock, Cl^– removal, and energy depletion activate Ca²⁺-permeable cation channels with Ca²⁺-induced suicidal erythrocyte death, i.e., surface exposure of phosphatidylserine, cell shrinkage, and membrane blebbing, all features typical for apoptosis of nucleated cells. The present experiments explored whether AE1 deficiency favors suicidal erythrocyte death. Peripheral blood erythrocyte numbers were significantly smaller in gene-targeted mice lacking AE1 (AE1^–/– mice) than in their wild-type littermates (AE1^+/+ mice) despite increased percentages of reticulocytes (AE1^–/–: 49%, AE1^+/+: 2%), an indicator of enhanced erythropoiesis. Annexin binding, reflecting phosphatidylserine exposure, was significantly larger in AE1^–/–erythrocytes/reticulocytes (10%) than in AE1^+/+ erythrocytes (1%). Osmotic shock (addition of 400 mM sucrose), Cl^– removal (replacement with gluconate), or energy depletion (removal of glucose) led to significantly stronger annexin binding in AE1^–/– erythrocytes/reticulocytes than in AE1^+/+ erythrocytes. The increase of annexin binding following exposure to the Ca²⁺ ionophore ionomycin (1 µM) was, however, similar in AE1^–/– and in AE1^+/+ erythrocytes. Fluo3 fluorescence revealed markedly increased cytosolic Ca²⁺ permeability in AE1^–/– erythrocytes/reticulocytes. Clearance of carboxyfluorescein diacetate succinimidyl ester-labeled erythrocytes/reticulocytes from circulating blood was more rapid in AE1^–/– mice than in AE1^+/+ mice and was accelerated by ionomycin treatment in both genotypes. In conclusion, lack of AE1 is associated with enhanced Ca²⁺ entry and subsequent scrambling of cell membrane phospholipids. annexin; cell volume; osmolarity; phosphatidylserine; energy depletion     

Identification of a novel galactosyl transferase involved in biosynthesis of the mycobacterial cell wall

The possibility of the Rv3782 protein of Mycobacterium tuberculosis being a putative galactosyl transferase (GalTr) implicated in galactan synthesis arose from its similarity to the known GalTr Rv3808c, its classification as a nucleotide sugar-requiring inverting glycosyltransferase (GT-2 family), and its location within the "possible arabinogalactan biosynthetic gene cluster" of M. tuberculosis. In order to study the function of the enzyme, active membrane and cell wall fractions from Mycobacterium smegmatis containing the overexpressed Rv3782 protein were incubated with endogenous decaprenyldiphosphoryl-N-acetylglucosaminyl-rhamnose (C(50)-P-P-GlcNAc-Rha) as the primary substrate for galactan synthesis and UDP-[(14)C]galactopyranose as the immediate precursor of UDP-[(14)C]galactofuranose, the ultimate source of all of the galactofuranose (Galf) units of galactan. Obvious increased and selective synthesis of C(50)-P-P-GlcNAc-Rha-Galf-Galf, the earliest product in the pathway leading to the fully polymerized galactan, was observed, suggesting that Rv3782 encodes a GalTr involved in the first stages of galactan synthesis. Time course experiments pointed to a possible bifunctional enzyme responsible for the initial synthesis of C(50)-P-P-GlcNAc-Rha-Galf, followed by immediate conversion to C(50)-P-P-GlcNAc-Rha-Galf-Galf. Thus, Rv3782 appears to be the initiator of galactan synthesis, while Rv3808c continues with the subsequent polymerization events.     

Neural stem cells transplanted into intact brains as neurospheres form solid grafts composed of neurons, astrocytes and oligodendrocyte precursors

Neural stem cells (NSCs) are tissue-specific stem cells with self-renewal potential that can give rise to neurons and glia in vivo and in vitro. The aim of this study was to transplant NSCs as whole neurospheres into intact brain and assess the fate and phenotype of their progeny generated in vivo. We isolated NSCs from E14 foetal rat forebrains and cultured them in basic fibroblast and epidermal growth factor-supplemented serum-free medium in the form of neurospheres in vitro. Neurospheres were transplanted into the intact brains of 2 Wistar rats and after a period of 3 weeks, grafted brains were examined immunohistochemically. Neurospheres formed solid grafts that were found in the lateral ventricle and in the velum interpositum under the hippocampus. The majority of cells in the transplanted tissue were identified as beta-III-tubulin(+), NeuN(+), PanNF(+) and synaptophysin(+) neurons and were accumulated throughout the graft centre. GFAP(+) astrocytes were scattered throughout the entire graft and astrocyte processes delimited the outer and perivascular surfaces. A great number of NG2(+) oligodendrocyte precursors was detected. Nestin(+) endothelial cells were found to line capillaries growing in the transplant. These data indicate that nestin(+) NSCs prevailing in neurospheres differentiate following transplantation into nestin(-) neuronal and glial cells which confirms the multipotency of NSCs. Three weeks posttransplantation neuronal and astrocyte cells reached terminal differentiation (formation of synaptic vesicles and superficial and perivascular limiting membranes) while elements of oligodendroglial cell lineage remained immature. Grafting stem cells as non-dissociated neurospheres provide cells with favourable conditions which facilitate cell survival, proliferation and differentiation. However, in the intact brain, grafted neurosphere cells were not found to integrate with the brain parenchyma and formed a compact structure demarcated from its surroundings.     

Somatic alterations in mitochondrial DNA produce changes in cell growth and metabolism supporting a tumorigenic phenotype

There have been many reports of mitochondrial DNA (mtDNA) mutations associated with human malignancies. We have observed allelic instability in UV-induced cutaneous tumors at the mt-Tr locus encoding the mitochondrial tRNA for arginine. We examined the effects of somatic alterations at this locus by modeling the change in a uniform nuclear background by generating cybrids harboring allelic variation at mt-Tr. We utilized the naturally occurring mtDNA variation at mt-Tr within the BALB/cJ (BALB) and C57BL/6J (B6) strains of Mus musculus to transfer their mitochondria into a mouse ρ(0) cell line that lacked its own mtDNA. The BALB haplotype containing the mt-Tr 9821insA allele produced significant changes in cellular respiration (resulting in lowered ATP production), but increased rates of cellular proliferation in cybrid cells. Furthermore, the mtDNA genotype associated with UV-induced tumors endowed the cybrid cells with a phenotype of resistance to UV-induced apoptosis and enhanced migration and invasion capabilities. These studies support a role for mtDNA changes in cancer.     

Red antenna states of photosystem I from Synechocystis PCC 6803

Single-molecule spectroscopy at low temperatures was used to elucidate spectral properties, heterogeneities, and dynamics of the red-shifted chlorophyll a (Chl a) molecules responsible for the fluorescence from photosystem I (PSI). Emission spectra of single PSI complexes from the cyanobacterium Synechocystis PCC 6803 show zero-phonon lines (ZPLs) as well as broad intensity distributions without ZPLs. ZPLs are found most frequently on the blue side of the broad intensity distributions. The abundance of ZPLs decreases almost linearly at longer wavelengths. The distribution of ZPLs indicates the existence of at least two pools with maxima at 699 and 710 nm. The pool with the maximum at 710 nm is assigned to chlorophylls absorbing around 706 nm (C706), whereas the pool with the maximum at 699 nm (F699) can be assigned to chlorophylls absorbing at 692, 695, or 699 nm. The broad distributions dominating the red side of the spectra are made up of a low number of emitters assigned to the red-most pool C714. The properties of F699 show close relation to those of F698 in Synechococcus PCC 7002 and C708 in Thermosynechococcus elongatus. Furthermore, a high similarity is found between the C714 pool in Synechocystis PCC 6803 and C708 in Synechococcus PCC 7002 as well as C719 in T. elongatus.     

Diurnal changes in photosystem II photochemistry, photoprotective compounds and stress-related phytohormones in the CAM plant, Aptenia cordifolia

Acclimation of photosynthetic light reactions to daily changes in solar radiation requires adjustments in photosystem II photochemistry and may be affected by environmental stresses, such as drought. In this study, we examined the effects of a short-term, severe water deficit on diurnal variations in photosystem II photochemistry, photoprotective compounds (tocopherols and carotenoids, including the xanthophyll cycle) and stress-related phytohormones (abscisic acid and salicylic acid) in the CAM plant, Aptenia cordifolia L. f. Schwantes. Violaxanthin was rapidly converted to zeaxanthin under high light, the de-epoxidation state of the xanthophyll cycle reaching maximum levels of 0.95 at midday in irrigated plants. Under a higher photoprotective demand caused by water deficit, plants showed significant increases in abscisic acid and γ-tocopherol levels, which were followed by decreases in β-carotene and the F_v/F_m ratio at later stages of stress. Decreases in this ratio below 0.70 correlated with sustained increases in the de-epoxidation state of the xanthophyll cycle, which kept above 0.90 at night after 15 days of water deficit. In contrast to abscisic acid, salicylic acid levels kept constant under water deficit and showed a sharp decrease during the day both under irrigated and water stress conditions. We conclude that the CAM plant, A. cordifolia showed several strategies of acclimation to short-term water deficit, including abscisic acid and γ-tocopherol accumulation, as well as sustained increases in the de-epoxidation state of the xanthophyll cycle, which was tightly coupled to daily variations in photosystem II photochemistry. The differential accumulation of tocopherol homologues under water deficit and the diurnal fluctuations of salicylic acid levels in this CAM plant will also be discussed.     

Glucans from fruit bodies of cultivated mushrooms Pleurotus ostreatus and Pleurotus eryngii: Structure and potential prebiotic activity

Cultivated oyster mushrooms (genus Pleurotus) are interesting as a source of biologically active glucans. Partially, β-glucan from Pleurotus sp. (pleuran) has been used as food supplements due to its immunosuppressive activity. Like other dietary fibre components, oyster mushroom polysaccharides can stimulate the growth of colon microorganisms (probiotics), i.e. act as prebiotics. Specific glucans were isolated from stems of Pleurotus ostreatus and Pleurotus eryngii by subsequent boiling water and alkali extraction. Obtained water soluble (L1), alkali soluble (L2) and insoluble (S) fractions were characterised by various analytical methods. Spectroscopic analysis detected glucans in all the fractions: branched 1,3-1,6-β-d-glucan predominated in L1 and S, while linear 1,3-α-d-glucan in L2. Fractions L1 also contained marked amount of proteins partially in complex with glucans; protein content in L2 was insignificant. Effective deproteinisation of L1 and separation of α- and β-glucans in L2 was achieved by the treatment with phenolic reagent. Small amount of chitin was found in S as a component of cell wall chitin–glucan complex. Potential prebiotic activity of extracts L1 and L2 was testing using nine probiotic strains of Lactobacillus, Bifidobacterium and Enterococcus. These probiotics showed different growth characteristics dependently on used extract and strain specificity due to the presence of structurally diverse compounds. The extracts L1 and L2 can be applied to synbiotic construction only for carefully selected probiotic strains. This exploitation of fruit body extracts extends the use of mushrooms P. ostreatus and P. eryngii for human health.     

Continuous fluorescence microphotolysis and correlation spectroscopy using 4Pi microscopy

Continuous fluorescence microphotolysis (CFM) and fluorescence correlation spectroscopy (FCS) permit measurement of molecular mobility and association reactions in single living cells. CFM and FCS complement each other ideally and can be realized using identical equipment. So far, the spatial resolution of CFM and FCS was restricted by the resolution of the light microscope to the micrometer scale. However, cellular functions generally occur on the nanometer scale. Here, we develop the theoretical and computational framework for CFM and FCS experiments using 4Pi microscopy, which features an axial resolution of ∼100 nm. The framework, taking the actual 4Pi point spread function of the instrument into account, was validated by measurements on model systems, employing 4Pi conditions or normal confocal conditions together with either single- or two-photon excitation. In all cases experimental data could be well fitted by computed curves for expected diffusion coefficients, even when the signal/noise ratio was small due to the small number of fluorophores involved.