Transport and distribution of α-tocopherol in lymph,serum and liver cells in rats

Rats were cannulated in the major mesenteric lymph duct and given an intraduodenal bolus of unlabeled and α-[³H]tocopherol, and [¹⁴C]oleic acid in soybean oil. The appearance of α-tocopherol in lymph was negligible during the first 2 h and peaked 4–15 h after feeding, whereas no detectable amount was recovered in the portal vein. Intestinal absorption via the lymphatic pathway was 15.4 ± 8.9% (n = 10) and 45.9 ± 10.8% (n = 4) for α-tocopherol and [¹⁴C]oleic acid, respectively. About 99% of α-tocopherol in lymph was associated with the chylomicron fraction (d < 1.006 g/ml). In non-fasting rats, 51% of serum α-tocopherol was associated with chylomicrons/VLDL (very-low-density lipoprotein, d < 1.006 g/ml) and 47% with HDL (high-density lipoprotein, 1.05 < d < 1.21 g/ml). Our study revealed that the liver, skeletal muscle and adipose tissue contain approx. 92% of the total mass of α-tocopherol measured in ten different organs. Parenchymal and nonparenchymal liver cells contributed to 75% and 25% of the total mass of α-tocopherol in the liver, respectively.     

Anaplerosis from glucose, α-ketoisocaproate, and pyruvate in pancreatic islets, INS-1 cells and liver mitochondria

Methyl succinate (MS) and alpha-ketoisocaproate (KIC) when applied alone to cultured pancreatic islets or INS-1 832/13 cells do not stimulate insulin release. However, when the two metabolites are combined together they strongly stimulate insulin release. Studying the possible explanations for this complementarity has provided clues to the pathways involved in insulin secretion. MS increased carbon incorporation of KIC into acid-precipitable material and lipid in INS-1 cells. In isolated mitochondria, MS alone increased malate, but MS plus KIC increased citrate, alpha-ketoglutarate, and isocitrate. These data and the known pathways of their metabolism suggest that MS supplies the oxaloacetate component of citrate and KIC supplies the acetate component of citrate. Other citric acid cycle intermediates can be formed from citrate enabling anaplerosis to supply precursors for extramitochondrial pathways. In addition, KIC, glucose and pyruvate can be metabolized to acetoacetate. In an INS-1 cell line deficient in ATP citrate lyase, incorporation of carbon from pyruvate into acid-precipitable material and lipid was not lowered. This negative result is in agreement with our recent discovery that citrate is not the only carrier of acyl groups from the mitochondria to the cytosol in the beta cell and that acetoacetate can also transfer acyl carbon to the cytosol.     

Expression of α2, α5 and α6 subunits of integrin in de-differentiated NIH3T3 cells by cell-free extract of embryonic stem cells

Generation of patient specific stem cells is among the ultimate goals in regenerative medicine. Such a cell needs to be functional when it transplants. Interaction between the matrix proteins and integrin adjust many cells' function such as adhesion, migration, cell cycle and self renewal in stem cells. In this study, NIH3T3 cells were dedifferentiated by mouse Embryonic Stem Cell (mESC) extract. The expression of pluripotency markers as well as a2, a5 and a6 integrin subunits were determined. NIH3T3 cells treated with mESC extract showed noticeable changes in cell morphology as early as day 2 post-treatment forming colonies similar to typical mESC morphology by day 8, after three passages. Alkaline phosphatase (ALP) assay and immunocytochemistry staining were performed for the induced reprogrammed cells. The results indicated that these colonies showed the ALP activity and they express Sox2 and Nanog. RT-PCR revealed that the colonies also express Oct3/4. NIH3T3 cells, ESC and reprogrammed cells expressed a2 integrin. a5 integrin expression was greatest in reprogrammed cells followed by the expression of this integrin in NIH3T3 which in turn was more than in ESC. a6A integrin was expressed in NIH3T3 cells while a6B integrin was expressed in ESC and in very low quantity was expressed in reprogrammed cells. These data provide evidence for both the generation of ES like cells from differentiated somatic cells and the expression profile of integrins after de-differentiation by mESC extract.     

Cell kinetic status of mouse lens epithelial cells lacking αA- and αB-crystallin

alphaA- and alphaB-crystallins are small heat shock proteins and molecular chaperones that are known to prevent non-specific aggregation of denaturing proteins. Recent work indicates that alphaA-/- lens epithelial cells grow at a slower rate than wild-type cells, and cultured alphaB-/- cells demonstrate increased hyperproliferation and genomic instability, suggesting that these proteins may exert a direct effect on the cell cycle kinetics, and influence cell proliferation. However, the cell cycle parameters of alphaA/alphaBKO (double knockout) cells have not been analyzed. Here we investigate the cell cycle kinetics of synchronized mouse lens epithelial cultures derived from wild-type and alphaA/alphaB double knockout (alphaA/alphaBKO) mice using BrdU labeling of proliferating cells, and flow cytometric analysis. We also provide data on the changing pattern of expression of HSP25, a small heat shock protein in alphaA/alphaBKO and wild-type cells during the cell cycle. Using serum starvation to synchronize cells in the quiescent G0 phase, and restimulation with serum followed by BrdU labeling and flow cytometry, the data indicated that as compared to wild-type cells, a <50% smaller fraction of the alphaA/alphaBKO cells entered the DNA synthetic S phase of the cell cycle. Furthermore, there was a delay in cell cycle transit through S phase in alphaA/alphaBKO cells, suggesting that although capable of entering S phase, the alphaA/alphaBKO cells are blocked in G1 phase, and are delayed in their cell cycle progression. Immunoblot analysis with antibodies to the small heat shock protein HSP25 indicated that although HSP25 increased in G1 phase of wild-type cells, and remained elevated on further progression through the cell cycle, HSP25 accumulation was delayed to S phase in alphaA/alphaBKO cells. These data can be interpreted to indicate that mouse lens epithelial cell progression through the cell cycle is significantly affected by expression of alphaA and alphaB-crystallin.     

Of fat, β cells, and diabetes

The molecular mechanisms linking diet, obesity, and type 2 diabetes are still poorly understood. In a recent paper, Ohtsubo et?al. (2011) show that high lipid levels induce nuclear exclusion of Foxa2 and HNF1α in β cells, leading to impaired expression and glycosylation of proteins controlling glucose-stimulated insulin secretion.     

Multiple aggregates and aggresomes of C-terminal truncated human αA-crystallins in mammalian cells and protection by αB-crystallin

Background

Cleavage of 11 (αA162), 5 (αA168) and 1 (αA172) residues from the C-terminus of αA-crystallin creates structurally and functionally different proteins. The formation of these post-translationally modified αA-crystallins is enhanced in diabetes. In the present study, the fate of the truncated αA-crystallins expressed in living mammalian cells in the presence and absence of native αA- or αB-crystallin has been studied by laser scanning confocal microscopy (LSM).

Methodology/Principal Findings

YFP tagged αAwt, αA162, αA168 and αA172, were individually transfected or co-transfected with CFP tagged αAwt or αBwt, expressed in HeLa cells and studied by LSM. Difference in protein aggregation was not caused by different level of α-crystallin expression because Western blotting results showed nearly same level of expression of the various α-crystallins. The FRET-acceptor photo-bleaching protocol was followed to study in situ protein-protein interaction. αA172 interacted with αAwt and αBwt better than αA168 and αA162, interaction of αBwt being two-fold stronger than that of αAwt. Furthermore, aggresomes were detected in cells individually expressing αA162 and αA168 constructs and co-expression with αBwt significantly sequestered the aggresomes. There was no sequestration of aggresomes with αAwt co-expression with the truncated constructs, αA162 and αA168. Double immunocytochemistry technique was used for co-localization of γ-tubulin with αA-crystallin to demonstrate the perinuclear aggregates were aggresomes.

Conclusions/Significance

αA172 showed the strongest interaction with both αAwt and αBwt. Native αB-crystallin provided protection to partially unfolded truncated αA-crystallins whereas native αA-crystallin did not. Aggresomes were detected in cells expressing αA162 and αA168 and αBwt co-expression with these constructs diminished the aggresome formation. Co-localization of γ-tubulin in perinuclear aggregates validates for aggresomes.     

α-Actinin 1 and α-actinin 4: Contrasting roles in the survival, motility, and RhoA signaling of astrocytoma cells

α-Actinin is a prominent actin filament associated protein for which different isoforms exist. Here, we have examined whether the two highly homologous non-muscle α-actinin isoforms 1 and 4 exhibit functional differences in astrocytoma cells. The protein levels of these isoforms were differentially regulated during the development and progression of astrocytomas, as α-actinin 1 was higher in astrocytomas compared to normal brains whereas α-actinin 4 was elevated in high-grade astrocytomas compared to normal brains and low grade astrocytomas. RNAi demonstrated contrasted contributions of α-actinin 1 and 4 to the malignant behavior of U-373, U-87 and A172 astrocytoma cells. While α-actinin 1 appeared to favor the expansion of U-373, U-87 and A172 astrocytoma cell populations, α-actinin 4 played this role only for U-373 cells. On the other hand, downregulation of α-actinin 4, but not 1, reduced cell motility, adhesion, cortical actin, and RhoA levels. Finally, in the three astrocytoma cell lines examined, α-actinin 1 and 4 had contrasted biochemical properties as α-actinin 4 was significantly more abundant in the actin cytoskeleton than α-actinin 1. Collectively, these findings suggest that α-actinin 1 and 4 are differentially regulated during the development and progression of astrocytomas because each of these isoforms uniquely contributes to distinct malignant properties of astrocytoma cells.     

New syntheses of α-methyl- and α,α′-dimethylspermine

-Methylspermine and ,-dimethylspermine were synthesized in high overall yields starting from N-(benzyloxycarbonyl)-3-aminobutanol in order to study polyamine biochemistry in vitro and in vivo.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 200–205.Original Russian Text Copyright © 2005 by Grigorenko, Vepsalainen, Jarvinen, Keinanen, Alhonen, Janne, Khomutov.     

Caldesmon,calponin and α-smooth muscle actin expression in subcultured smooth muscle cells from human airways

Calponin and caldesmon are two proteins considered to play a regulatory role in smooth muscle contraction, which have never previously been found to be expressed in subcultured cells. In the present study, immunocytochemistry and immunoblotting were performed to identify these proteins in smooth muscle cells (SMC) from human bronchi. It was found that human airway SMC, kept in a non-proliferative state, continued to express caldesmon and calponin at least until the 8th passage. The expression of -smooth muscle actin studied under the same conditions was also shown to be preserved in subcultured bronchial SMC.     

γ-Synuclein: seeding of α-synuclein aggregation and transmission between cells

Protein misfolding and aggregation is a ubiquitous phenomenon associated with a wide range of diseases. The synuclein family comprises three small naturally unfolded proteins implicated in neurodegenerative diseases and some forms of cancer. α-Synuclein is a soluble protein that forms toxic inclusions associated with Parkinson's disease and several other synucleinopathies. However, the triggers inducing its conversion into noxious species are elusive. Here we show that another member of the family, γ-synuclein, can be easily oxidized and form annular oligomers that accumulate in cells in the form of deposits. Importantly, oxidized γ-synuclein can initiate α-synuclein aggregation. Two amino acid residues in γ-synuclein, methionine and tyrosine located in neighboring positions (Met(38) and Tyr(39)), are most easily oxidized. Their oxidation plays a key role in the ability of γ-synuclein to aggregate and seed the aggregation of α-synuclein. γ-Synuclein secreted from neuronal cells into conditioned medium in the form of exosomes can be transmitted to glial cells and cause the aggregation of intracellular proteins. Our data suggest that post-translationally modified γ-synuclein possesses prion-like properties and may induce a cascade of events leading to synucleinopathies.     

Subcellular distribution of tissue kallikrein and Na,K-ATPase α-subunit in rat parotid striated duct cells

Intracellular protein distribution and sorting were examined in rat parotid striated duct cells, in which tissue kallikrein is apical, and Na,K-ATPase is basolateral. Electron-microscopic immunogold cytochemistry, with both polyclonal and monoclonal antibodies, demonstrated these enzymes at opposite poles of the cells and in distinct intracellular sites. Kallikrein was found within apical secretory granules, whereas Na,K-ATPase was present on basolateral cell membranes. In addition, kallikrein was localized throughout cisternae of all Golgi profiles, whereas Na,K-ATPase (-subunit) was found only in small peripheral vesicles and/or lateral cisternal extensions of a basal subset of Golgi profiles. These differences in the subcellular distribution of the two marker antigens were most clearly seen with double immunogold labelling. Our results suggest that kallikrein, an apical, regulated secretory protein, and Na,K-ATPase, a basolateral, constitutively transported membrane protein, are segregated at (or prior to) the level of the Golgi apparatus rather than in the trans-Golgi network (TGN), as was expected.Abbreviations ATP adenosine tri-phosphate - HBSS Hanks' balanced salt solution - GaM goat anti-mouse - GaR goat anti-rabbit - PBS phosphate-buffered saline - RaM rabbit anti-mouse - RER rough endoplasmic reticulum - TGN trans-Golgi network     

N-terminal deletion does not affect α-synuclein membrane binding, self-association and toxicity in human neuroblastoma cells, unlike yeast

α-Synuclein causes Parkinson's disease if mutated or aberrantly produced in neurons. α-Synuclein-lipid interactions are important for the normal function of the protein, but can also contribute to pathogenesis. We previously reported that deletion of the first 10 N-terminal amino acids dramatically reduced lipid binding in vitro, as well as membrane binding and toxicity in yeast. Here we extend this study to human neuroblastoma SHSY-5Y cells, and find that in these cells the first 10 N-terminal residues do not affect α-synuclein membrane binding, self-association and cell viability, contrary to yeast. Differences in lipid composition, membrane fluidity and cytosolic factors between yeast and neuronal cells may account for the distinct binding behavior of the truncated variant in these two systems. Retinoic acid promotes differentiation and α-synuclein oligomer formation in neuroblastoma cells, while addition of a proteasomal inhibitor induces neurite outgrowth and toxicity to certain wild-type and truncated α-synuclein clones. Yeast recapitulate several features of α-synuclein (patho)biology, but its simplicity sets limitations; verification of yeast results in more relevant model systems is, therefore, essential.     

Studies on steroid hormone refeptors (5 α=dihidrotestosterone,estradiol, and dexamethasone) in cultured human fibroblasts and amniotic fluid cells

Summary Cultured human fibroblasts and amniotic fluid cells (AF cells) were examined for the presence of steroid hormone receptors. In both cell types, the androgen (DHT) or glucocorticoid (dexamethasone) receptor was detected, but not the estrogen receptor. Binding parameters in fibroblasts were: for androgen: K_D=3.7×10^-9M, B_max=13 fmol/mg; for dexamethasone: K_D=4.5×10^-9M, B_max=120 fmol/mg. Binding parameters in AF cells were: for androgen: K_D=4×10^-9M, B_max=8 fmol/mg; for dexamethasone: K_D=1.9×10^-8M, B_max=375 fmol/mg. Cultured cells derived from the gonads (of a patient with 17-ketosteroid reductase deficiency) seem to have more receptors than cells from extragenital body parts (B_max=21 fmol). With the aid of gel chromatography, the molecular weight of the androgen receptor was estimated to be 30–40 000D.     

Surface α2-3- and α2-6-sialylation of human monocytes and derived dendritic cells and its influence on endocytosis

Several glycoconjugates are involved in the immune response. Sialic acid is frequently the glycan terminal sugar and it may modulate immune interactions. Dendritic cells (DCs) are antigen-presenting cells with high endocytic capacity and a central role in immune regulation. On this basis, DCs derived from monocytes (mo-DC) are utilised in immunotherapy, though many features are ignored and their use is still limited. We analyzed the surface sialylated glycans expressed during human mo-DC generation. This was monitored by lectin binding and analysis of sialyltransferases (ST) at the mRNA level and by specific enzymatic assays. We showed that α2-3-sialylated O-glycans and α2-6- and α2-3-sialylated N-glycans are present in monocytes and their expression increases during mo-DC differentiation. Three main ST genes are committed with this rearrangement: ST6Gal1 is specifically involved in the augmented α2-6-sialylated N-glycans; ST3Gal1 contributes for the α2-3-sialylation of O-glycans, particularly T antigens; and ST3Gal4 may contribute for the increased α2-3-sialylated N-glycans. Upon mo-DC maturation, ST6Gal1 and ST3Gal4 are downregulated and ST3Gal1 is altered in a stimulus-dependent manner. We also observed that removing surface sialic acid of immature mo-DC by neuraminidase significantly decreased its endocytic capacity, while it increased in monocytes. Our results indicate the STs expression modulates the increased expression of surface sialylated structures during mo-DC generation, which is probably related with changes in cell mechanisms. The ST downregulation after mo-DC maturation probably results in a decreased sialylation or sialylated glycoconjugates involved in the endocytosis, contributing to the downregulation of one or more antigen-uptake mechanisms specific of mo-DC.     

A Specific and Essential Role for Na,K-ATPase α3 in Neurons Co-expressing α1 and α3

Most neurons co-express two catalytic isoforms of Na,K-ATPase, the ubiquitous α1, and the more selectively expressed α3. Although neurological syndromes are associated with α3 mutations, the specific role of this isoform is not completely understood. Here, we used electrophysiological and Na⁺ imaging techniques to study the role of α3 in central nervous system neurons expressing both isoforms. Under basal conditions, selective inhibition of α3 using a low concentration of the cardiac glycoside, ouabain, resulted in a modest increase in intracellular Na⁺ concentration ([Na⁺]_i) accompanied by membrane potential depolarization. When neurons were challenged with a large rapid increase in [Na⁺]_i, similar to what could be expected following suprathreshold neuronal activity, selective inhibition of α3 almost completely abolished the capacity to restore [Na⁺]_i in soma and dendrite. Recordings of Na,K-ATPase specific current supported the notion that when [Na⁺]_i is elevated in the neuron, α3 is the predominant isoform responsible for rapid extrusion of Na⁺. Low concentrations of ouabain were also found to disrupt cortical network oscillations, providing further support for the importance of α3 function in the central nervous system. The α isoforms express a well conserved protein kinase A consensus site, which is structurally associated with an Na⁺ binding site. Following activation of protein kinase A, both the α3-dependent current and restoration of dendritic [Na⁺]_i were significantly attenuated, indicating that α3 is a target for phosphorylation and may participate in short term regulation of neuronal function.     

Developmental potency of mouse primitive ectoderm cells,embryonic ectoderm cells,embryonic ectoderm cells and primordial germ cells after blastocyst injection

Developmental potency of primitive and embryonic ectoderm cells from 4.50-day to 6.25-day post-coitum (p.c.) mouse embryos and primordial germ cells from 12.50-day p.c.male genital ridges of fetal mice were studied by direct introducing them into 3.50-day p.c.blastocysts.Sixteen (61.5) overt chimaeras out of 26(50%) offsprings were obtained after transfer of 52 blastocysts injected with 4.50-day primitive ectoderm cells;four (16.0%) overt chimaeras were obtained out of 25 (51.0%) offsprings with 4.75-day primitive ectoderm cells from 49 transferred blastocysts.However,no overt chimaera was obtained with either 5.25-day or 6.25-day embryonic ectoderm cells or 12.50-day male primordial germ cells.GPI analysis of mid-gestation conceptuses developed from injected blastocysts showedthat 5.25-day embryonic ectoderm cells could only contributed to yolk sac of conceptus.Results suggested that implantation acts as a trigger for the determination of primitive ectoderm cells,and their developmental potency becomes limited within a short period of time in normal development.     

Inhibitors of α-glucosidase, α-amylase and lipase from Chrysanthemum morifolium

A new endoperoxysesquiterpene lactone, 10α-hydroxy-1α,4α-endoperoxy-guaia-2-en-12,6α-olide (1), together with a flavanone, eriodictyol (2), and two flavone glycosides, acacetin-7-O-β-d-glucopyranoside (3) and acacetin-7-O-α-l-rhamopyranoside (4), were isolated from the methanol extract of Chrysanthemum morifolium flowers by a bioassay-guided fractionation. Compound 1 showed strong inhibitory effects against α-glucosidase and lipase activities, with IC₅₀ values of 229.3 and 161.0 μM, respectively. The flavone glycosides 3 and 4 inhibited both α-glucosidase and α-amylase, while flavanone 2 was only effective against α-amylase.