Calcium Ionophore-Induced Degradation of Neurofilament and Cell Death in MSN Neuroblastoma Cells

Extensive necrotic death of MSN neuroblastoma cells could be induced after incubation with the calcium ionophore, A23187. The reaction was concentration-dependent and time course-dependent. Levels of the 66 kd/-internexin neurofilament protein (NF-66) and the cognate heat shock protein 70 (Hsc 70) decreased during the Ca²⁺-activated cell death. Addition of the calcium chelator, ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid (EGTA) restored the normal level of NF-66 and partially that of the Hsc 70. Use of either calpain I or calpain II inhibitor could alleviate the reduction of 66 kd protein during the ionophore treatment whereas only calpain I inhibitor treatment was effective in restoring the normal level of the Hsc 70. Neither of these calpain inhibitors could block the ionophore triggered cell death. EQTA was toxic to cells in a wide range of concentration suggesting a calcium-independent activation of cell death mechanism.     

Heterogeneity of calcium compartmentation: Electron probe analysis of renal tubules

Summary The objective of this study has been to determine the intracellular localization of calcium in cryofixed, cryosectioned suspensions of kidney proximal tubules using quantitative electron probe X-ray microanalysis. Two populations of cells have been identified: 1) Viable cells, representing the majority of cells probed, are defined by their relatively normal K/Na concentration ratio of 41. Their measured Ca content is 4.1±1.4 (sem) mmol/kg dry wt in the cytoplasm and 3.1 ± 1.1 mmol/kg dry wt in the mitochondria, or an average cell calcium content of 3.8 mmol/kg dry wt. 2) Nonviable cells, defined by the presence of dense inclusions in their mitochondria and a K/Na concentration ratio of 1. The Ca content is 15±2 mmol/kg dry wt in the cytoplasm and 685±139 mmol/kg dry wt in the mitochondria of such cells. Assuming 25 to 30% of the cell volume is mitochondrial, the overall calcium content of such nonviable cells is 210 mmol/kg dry wt. The presence of these inclusions in 4 to 5% of the cells would account for the average total Ca content measured in perchloric acid extracts of isolated proximal tubule suspensions ( 18 nmol/mg protein or 12.6 mmol/kg dry wt). Whole kidney tissues display a large variability in toal Ca content (4.5 to 18 nmol/mg protein, or 3.4 to 13.5 mmol/kg dry wt), which could be accounted for by inclusion in 0 to 4% of the cells. The electron probe X-ray microanalysis (EPXMA) data conclusively demonstrate that thein situ mitochondrial Ca content of viable cells from the kidney, proximal tubule is low and support the idea that mitochondrial Ca may regulate dehydrogenase activity but probably does not normally control cytosolic free Ca.     

Studies on the Cytosolic Phospholipase A2 in Immortalized Astrocytes (DITNC) Revealed New Properties of the Calcium Ionophore,A23187

Besides playing an important role in the maintenance of cell membrane phospholipids, phospholipases A₂ (PLA₂) are responsible for the release of arachidonic acid (AA) which is a precursor for prostaglandin biosynthesis. The cytosolic PLA₂ has been the focus of recent studies, probably due to its ability to respond to protein kinases and changes in intracellular calcium levels. In this study, we examined agents for stimulation of the cytosolic phospholipase A₂ in immortalized astrocytes (DITNC). Incubation of DITNC cells with [¹⁴C]arachidonic acid (AA) resulted in a time-dependent uptake of the label into phospholipids (PL) and neutral glycerides. In prelabeled cells, release of labeled AA could be stimulated by calcium mobilizing agents such as calcium ionophore A23187 (4–20 M) and thimerosal (100 M), and by phorbol myristate acetate (PMA, 100 nM), an agent for activation of protein kinase C. The release of AA could also be stimulated by ATP (200 M), probably through activation of the purinergic receptor but not by glutamate (1 mM). The stimulated release of AA was dependent on extracellular Ca²⁺ and was inhibited by mepacrine (50 M), a non-specific PLA₂ inhibitor. Western blot analysis further confirmed the presence of an 85 kDa cPLA₂ in both membrane and cytosol fractions of these cells and stimulation by A23187 resulted in translocation of this protein to the membrane fraction. Besides labeled fatty acids, A23187 also stimulated the concomitant release of labeled PL into the culture medium and this event was accompanied by the increased release in lactate dehydrogenase (LDH). Results thus revealed that besides activation of cPLA₂, the calcium ionophore A23187 is capable of perturbating cell membrane integrity.     

Induction,polarity and spatial control of cytokinesis in some abnormal subsidiary cell mother cells ofZea mays

Summary Cytokinesis in the subsidiary cell mother cells (SMCs) ofZea mays leaves grown in the presence of 5 mM of caffeine solution is usually partially inhibited. A continuous wall strip, resembling a portion of the subsidiary cell (SC) wall, is laid down in the preprophase microtubule band (PMB) cortical zone. Sometimes, the incomplete SC (SC) wall grows centripetally in the absence of a phragmoplast and the gap becomes smaller or closes. The SC nucleus escapes through the SC wall gap into the larger SMC compartment and may fuse with the other nucleus.The aberrant SMCs (a-SMCs) pass through another division cycle, reattempting to produce a SC. A typical PMB is found in the SC space, in the site of the previous PMB. Moreover, in some preprophase SMCs, the cytoplasm adjacent to the SC wall is traversed by a small number of microtubules. The preprophase nuclei are partly or totally separated from the PMB by the perforated SC wall and may lie far from the latter.Usually, one mitotic spindle is assembled. The cycling paired polarized nuclei appear to synchronize and their chromosomes line up together on a single metaphase plate. Although the mitotic spindle axis is diversely oriented, one of its poles tends to be stabilized in the proximity of the SC wall gap. These divisions separate abnormal cells. Most or all the cell plate edges fuse with wall regions far from the PMB cortical zone. However, when some of them approach the SC wall strips, they are attracted and intersect their rims. In rare occasions the cell plate, invading the SC space is guided by the PMB cortical zone to create a SC-like curved wall portion, in absence of a daughter nucleus.Observations show that the cell plate arrangement in redividing aberrant SMCs is not subjected to a strict spatial control. The disorder of polarization sequence generated by the SC wall ring and especially the perturbation of the spatial (and functional?) relationship between PMB-PMB cortical zone and the nucleus—mitotic spindle is a causal factor of the variable cell plate arrangements.     

Allospecific T cell recognition of HLA-A2 antigens: Evidence for group-specific and subgroup-specific epitopes

Interleukin 2-dependent alloreactive cytotoxic T cell lines, with activity predominantly directed against the HLA-A2 antigen, have been generated in vitro by stimulating blood mononuclear cells from donors nonimmune to the Epstein-Barr (EB) virus with appropriate numbers of EB virus-transformed B cells from A2-homozygous individuals. Such effector cells were tested against a panel of EB virus-transformed target cell lines all expressing the serologically defined A2 antigen but typed into common A2 and variant A2 subgroups on the basis of their recognition by A2-restricted EB virus-specific cytotoxic T cells. Variant A2 responder cells cocultivated with common A2-bearing stimulators gave rise to effector T cell lines which recognized only the common A2-bearing subgroup of targets. By contrast, responder cells from A2-negative donors stimulated with common A2-bearing cells produced effector T cell lines in which the strong lysis of common A2-bearing targets was accompanied by a lower, but still significant, lysis directed against all targets within the variant A2 subgroup. In both cases, lysis of the target cells was blocked equally well by the anti-A2-specific monoclonal antibody MA2.1 as by the monoclonal antibody W6/32 specific for HLA-A, -B, and -C determinants. This suggests that HLA-A2 molecules possess at least two distinct sets of epitopes capable of inducing alloreactive T cell cytotoxicity: first, epitopes probably associated with T cell-restricting sites, which generate subgroup-specific responses, and second, epitopes shared by all A2 molecules, and perhaps associated with serologically defined sites, which generate pan A2 group-specific responses.Abbreviations used in this paper EB Epstein-Barr - IL-2 Interleukin 2 - UM unfractionated mononuclear - AET aminoethylisothiouroniumbromide hydrobromide     

Phenotypic analyses of lymphokine-activated killer cells that release interferon γ and tumor necrosis factor α

Summary We recently reported that interleukin-2(IL-2)-activated peripheral blood lymphocytes and CD3⁺, lymphokine-activated killer (LAK) cell clones release tumor necrosis factor (TNF) and interferon (IFN) when stimulated with K562 erythroleukemia cells. We examined the phenotype of IL-2-activated peripheral blood leukocytes that secrete TNF and IFN when stimulated with K562 cells and demonstrated that TNF secretion is not due to the presence of contaminating mononuclear phagocytes. Further, we demonstrate that IL-2-activated natural killer (NK) cells release only IFN when stimulated with K562 cells while T lymphocytes exposed to monoclonal anti-CD3 and K562 cells secrete both TNF and IFN. However, T cells stimulated only with K562 cells did not release IFN or TNF while the admixture of these T cells with NK cells, when stimulated with K562 cells, released levels of TNF comparable to those produced by the unseparated cells. At present it is unclear whether only one or both effector cell types respond to K562 by releasing TNF or why the presence both cell types is needed.This work was supported by grants from the national Institutes of Health (CA 23074 and CA 17094) and the Arizona Disease Commission (8277-000000-1-0-YR-9301)     

Purification and characterization of cyclic 3′5′-nucleotide phosphodiesterase D3 from Sinorhizobium fredii MAR-1

The cyclic 35-nucleotide phosphodiesterase D3 was purified from Sinorhizobium fredii MAR-1. The native enzyme had a molecular weight of approximately 44.5kDa and a subunit molecular weight of approximately 21kDa as judged by SDS-gel electrophoresis. The pH optimum of the enzyme for the hydrolysis of cyclic AMP was approximately 6.0 with both acetate and Tris-maleate buffers. The optimum temperature for hydrolysing cyclic AMP was approximately 50C. No metal ion was required for activity and EDTA up to 2.5mM did not markedly affect the enzyme. However, methylxanthines, adenine and adenosine as well as 5-AMP, ATP, ADP and metal ions like Zn²⁺, Fe²⁺, Pb²⁺, Al³⁺ and Fe³⁺, were strongly inhibitory at 2.5mM.The D3 enzyme could hydrolyse both cyclic AMP and cyclic GMP with the apparent K _m for cyclic AMP of approximately 0.23M.     

Enhanced acetyl esterase production by Fusarium oxysporum

Production of acetyl esterase (EC 3.1.1.6) by Fusarium oxysporum strain F3 was enhanced by optimization of growth conditions. Under optimal conditions, activities as high as 0.89U/ml of culture medium were obtained. The culture filtrate was equally active on p-nitrophenyl acetate and acetylxylan. The enzyme produced 71% deacetylation of acetylxylan in 2h at 40C. Activity was optimized at pH6.5 and at 55^C. The respective K_m values for p-nitrophenyl acetate and acetylxylan were 0.25mM and 1.05% (w/v) and the V_m values were 0.65 and 0.43mol acetate/min/mg protein.     

Effects of D‐mannoheptulose upon D‐glucose metabolism in tumoral pancreatic islet cells

D[³H]mannoheptulose was recently reported to be poorly taken up by tumoral pancreatic islet cells of the RINm5F and INS1 lines. We have now investigated the effects of Dmannoheptulose upon Dglucose metabolism in these two cell lines. Dmannoheptulose (1.0–10.0 mM) only caused a minor decrease of Dglucose metabolism in RINm5F cells, whether at low (1.1 mM) or higher (8.3 mM) Dglucose concentration. A comparable situation was found in INS1 cells examined after more than 20 passages. In both cases, however, the hexaacetate ester of Dmannoheptulose (5.0 mM) efficiently inhibited Dglucose metabolism. In the INS1 cells, the relative extent of the inhibitory action of Dmannoheptulose upon Dglucose metabolism increased from 12.4 ± 2.6 to 38.3 ± 3.8% as the number of passages was decreased from more than 20 to 13–15 passages, the latter percentage remaining lower, however, than that recorded in INS1 cells also examined after 13–15 passages but exposed to Dmannoheptulose hexaacetate (66.9 ± 2.2%). These findings when compared to our recent measurements of D[³H]mannoheptulose uptake, reinforce the view that the entry of the heptose into cells and, hence, its inhibitory action on Dglucose metabolism are dictated by expression of the GLUT2 gene.     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact     

Comparison of free and immobilizedCephalosporium acremonium for β-lactam antibiotic production

Summary Cephalosporium acremonium cells were immobilized in calcium alginate beads. Immobilized cells were used to produce -lactam antibiotics in rest medium under various oxygen concentrations, and the results were compared with free cell performance. Cell growth rate of immobilized cells was 35% of the growth rate of free cells. -Lactam antibiotic production rate of immobilized cells was also limited by mass transfer of oxygen. -Lactam antibiotic production rate of immobilized cells was 70% of that of free cells at oxygen saturation condition (i.e., 0.27 mM O₂). Specific antibiotic production of immobilized cells was about 200% of that of free cells at 0.27 mM O₂.     

Photoreversion und Photoprotektion bei UV-induzierten Mutationen des nichtphotoreaktivierbaren Bacteriums E.coli phr?

Summary Stationary phase cells of strain phr^–/MC 2 ofE. coli are not photoreactivable but the frequency of UV-induced mutations to low Streptomycine-resistance (S 3, 3/ml) is decreased strongly by illumination with light of fluorescence tubes (310 to 500 nm) after UV-irradiation. Also dark-reversion (DRM) of these mutations due to keeping UV-irradiated cells in saline is observed. Illumination before UV-irradiation decreases the frequency of the mutations (photoprotection against mutation=PPM) to the same extent as the combined action of photoreversion (PRM) and DRM. The lag-phase of cell division is prolonged strongly by illumination from 80 min without light to 150 min by the light-dose of highest activity. The additional lag is nearly the same if the illumination is done before, after or without UV-irradiation; this lag is about additive to the small lag caused by UV. Pre-illumination of the stationary-phase cells does not cause photoprotection against killing (PP), it even decreases the survival after high UV-doses. The observations support the hypothesis that PRM in this strain may be indirect, i.e. caused by the light-induced additional division lag which enhances the dark repair of UV-premutations. Also spontaneous premutations which are apparently present in the stationary-phase cells seem to be influenced by the light in this way.     

Colorectal cancer diagnosis by a direct leukocyte migration test using a panel of tumour extracts

Summary In a direct leukocyte migration test, peripheral blood leukocytes were pulsed with a high dose (2.5 and 0.5 mg/ml) of 3 M KCl extracts from 5 different colorectal tumours as well as with one 3 M KCl extract of normal colonic mucosa. Patients showing a pathological migration index (0.80 and 1.17), with 3 or more out of 5 tumour extracts, were considered as positives.With this test mode 93% (55/59) of patients with colorectal carcinomas were reactive, irrespective of the tumour stage, while only 7% (2/27) of patients with non-malignant colorectal diseases showed a positive reaction. Patients with malignant and non-malignant diseases of other organs were reactive in 2–3% of cases. No positive reactivity was observed with leukocytes from 37 healthy volunteers. Pulsing leukocytes with the normal colonic mucosal extract, a pathological migration index was found in about 20% of colorectal cancer patients, but not in healthy volunteers.Evaluating 10 single tumour extracts individually, reactivity of cancer patients' leukocytes ranged from 65–89% of tests, the difference being not statistically significant. Leukocytes from healthy volunteers showed a pathological migration index with the different extracts in 0–6% of tests.With the leukocyte migration test we could not differentiate between tumours of the colon, sigma or rectum. Patients bearing tumours in any part of the large bowel showed pathological leukocyte migration with extracts of colon-, sigma- and rectum tumours. When the cross-reactivity study was extended to tumours of the gastrointestinal tract, it was found that patients with colorectal tumours were reactive, in a high percentage of tests, with extracts of gastric tumours, but gastric as well as oesophageal and pancreatic cancer patients' leukocytes only reacted occasionally with colorectal tumour extracts.In the follow-up study, a positive reactivity was still found 10–14 days after surgery in 27/31 patients. After more than 2 months, the frequency of positive reactivity decreased to 10/70 cases. Patients with local recurrence or metastases exhibited positive reactivity in 6/7 cases.Abbreviations LMT leukocyte migration test - LM leukocyte migration - LMI leukocyte migration inhibition - LME leukocyte migration enhancement - MI migration index     

Effects of inducer levels on a recombinant bacterial biofilm formation and gene expression

Summary A segregationally stable host-plasmid system, E. coli DH5 (pTKW106), was used to study the effect of induction on the accumulation rate of cells and gene expression in biofilm cultures. Isopropyl -D-thiogalactoside (IPTG) was used to induce the expression of -galactosidase from the plasmid. The biofilm cell net accumulation rates decreased with increasing induction levels. At 0.17 and 0.34 mM of IPTG, the biofilm cell net accumulation rates ranged between 17 and 30% when compared to the uninduced case. At 0.51 mM of IPTG, the biofilm cell density never increased. At 0.17 and 0.34 mM of IPTG, -galactosidase contents reached maxima 36 hours after induction with both amounts representing about 7.5% of total protein. At 0.51 mM of IPTG, -galactosidase production reached its maximum, about 16% of total protein, 48 hours after induction. The -galactosidase mRNA synthesis rates increased with increasing inducer levels. Maximum -galactosidase mRNA synthesis rates were reached 36 hours after induction for each IPTG concentration.     

Structure of the parathyroid glands,as revealed by different methods of fixation

Summary Stereology and semi-automatic image analysis were used with the aim of comparing the structure of parathyroid glands from untreated adult Mongolian gerbils fixed by immersion with those fixed by perfusion. Subclassification of the chief cells based upon the staining affinity or electron density of the cytoplasm was readily performed only in glands fixed by immersion, and so-called atrophic cells were observed only in these glands. The atrophic cells were often surrounded by light chief cells. In glands fixed by perfusion, light chief cells were only rarely encountered. A significant difference between glands fixed by immersion and those fixed by perfusion was found only with regard to the form of cells and nuclei, those fixed by perfusion being more spherical. When comparing individual cells within glands fixed by immersion, light chief cells were more spherical and had a significantly larger nuclear and cellular size, and a lower mitochondrial volume density than the intermediate/dark chief cells. Otherwise there were no significant differences in any of the parameters investigated. These data indicate that occurrence of socalled light chief cells and atrophic cells is a result of improper fixation. The results of this study do not favour the concept of a functional cycle with a simultaneous occurrence of active and inactive cells within parathyroid glands.     

T-cell receptor gene rearrangements and expression in normal human large granular lymphocytes (LGL) and their pathological expansions

The lineage to which normal large granular lymphocytes/natural killer (LGL/NK) cells belong is controversial; in fact they share some surface markers and functional activities with monocytes, but also with T lymphocytes. The relationship of LGL to the T cell lineage by analysis with the T cell receptor (T-rec) gene has been investigated. Pure preparations of human LGL and their CD11⁺ CD8^- and CD11^- CD8⁺ subsets had the T gene in its unrearranged germline configuration. Expression of T and T genes was not detectable. The organization of T gene, which is of particular importance because it occurs early in T cell ontogeny, was also found in its germline configuration.A rare type of lymphoproliferative disorder, termed T-LPD, is characterized by expansion of cells very similar to LGL for morphology, phenotype, and functional activity. Of 17 patients with T-LPD studied for T-rec rearrangement, 15 displayed rearrangement of T and T loci and were CD3+ (14/15 had monoclonal rearrangement), while 2 cases were in germline configuration and were CD3–. Similarly to very small subsets of CD3+ LGL recently described, most T-LPD cases are CD3+ and have T-rec genes rearranged. These data suggest that either a subset of LGL or a particular step of differentiation may be related to the T cell lineage; they also demonstrate that, in contrast to previous views, most TLPD are monoclonal, presumably neoplastic, lymphoproliferative disorders.Abbreviations LGL large granular lymphocytes - NK natural killer - T-rec T-cell receptor - TLPD T lymphoproliferative disease     

Production,purification, and properties of β-glucosidase from Candida wickerhamii

Summary Candida wickerhamii growing on cellobiose produced -glucosidase with high activity against -nitrophenyl glucoside (PNPG) but low activity against cellobiose. -glucosidase production was constitutive, and was repressed by -glucosides and glucose. -glucosides containing an aromatic moiety in the aglycon were the best substrates for -glucosidase indicating that the enzyme is an aryl--glucosidase. A -glucosidase from C. wickerhamii cells was purified by (NH₄)₂SO₄ precipitation, dialysis, ion-exchange chromatography and gel filtration. The purified enzyme was homogeneous as shown by sodium-dodecyl-sulphate polyacrylamide gel electrophoresis and discontinuous gel electrophoresis. The purified enzyme hydrolysed PNPG but not cellobiose. The K_m of the enzyme was 0.185 mM. Glucose inhibited the enzyme competitively and the K_i was 7.5 mM. The apparent molecular mass was 97,000. The optimum pH and temperature for enzyme activity were between pH 7 and 7.4 and 40°C respectively. At temperatures of 45°C and greater the enzyme was inactivated. The activation energy of the enzyme was 29.4 kJ · mol^-1.     

Regulation ofN-acetylglucosaminyltransferase V by protein kinases

When 7721 human hepatocarcinoma cells were treated with 100nm phorbol-12-myristate-13-acetate (PMA), the activity ofN-acetylglucosaminyltransferase V(GnT-V) in the cells varied in accordance with the activity of membranous protein kinase C (PKC), but not with that of cytosolic PKC. Quercetin, a non-specific inhibitor of Ser/Thr protein kinase, andd-sphingosine and staurosporine, two specific inhibitors of PKC, blocked the activation of membranous PKC and GnT-V by PMA. Among the three inhibitors, quercetin was least effective. The inhibitory rates of quercetin and staurosporine toward membranous PKC and GnT V were proportional to the concentrations of the two inhibitors. The activities of GnT V and membranous protein kinase A (PKA) were also induced in parallel by dibutyryl cAMP (db-cAMP) and this induction was blocked by a specific PKA inhibitor. When cell free preparations of 7721 cells and human kidney were treated with alkaline phosphatase (ALP) to remove the phosphate groups, the GnT V activities were decreased. These results suggest that GnT V may be activated by membranous PKC or PKA, indirectly or directly, via phosphorylation of Ser/Thr residues.Abbreviations UDP uridine diphospho- - GnT N-acetylglucosaminyltransferase - GlcNAc Gn N-acetylglucosamine - M mannose - PMA phorbol-12-myristate-13-acetate - PKC protein kinase C - PKA protein kinase A - cAMP adenosine 3, 5-cyclic monophosphate - db-cAMP dibutyryl cAMP - TPK tyrosine protein kinase - MES 2-[N-morpholino]ethanesulfonic acid - DTT dithiothreitol - PMSF phenylmethylsulfonyl fluoride - EDTA ethylene diamine tetraacetic acid - EGTA glycol-bis-(-aminoethyl) etherN,N,N,N-tetraacetic acid - PA 2-aminopyridine - ALP alkaline phosphatase - C₂C₂ GlcNAc1-2 Man1-6(GlcNAc1-2Man1-3)ManR - C_2,4C₂ GlcNAc1-2Man1-6(GlcNAc1-4[GlcNAc1-2]Man1-3)ManR - C₂C_2,6 GlcNAc1-6[GlcNAc1-2]Man1-6(GlcNAc1-2Man1-3)ManR - C_2,4C_2,6 GlcNAc1-6[GlcNAc1-2]Man1-6(GlcNAc1-4[GlcNAc1-2]Man1-3)ManR where R=1-4GlcNAc1-4GlcNAcAsnX - Gn₂M₃Gn₂-PA C₂C₂ where R=1-4GlcNAc1-4GlcNAc-PA - Gn₃M₃Gn₂-PA C₂C_2,6 where R=1-4GlcNAc1-4GlcNAc-PA     

Transfer of chloramphenicol‐resistant mitochondrial DNA into the chimeric mouse

The mitochondrial DNA (mtDNA) chloramphenicol (CAP)resistance (CAP^R) mutation has been introduced into the tissues of adult mice via female embryonic stem (ES) cells. The endogenous CAPsensitive (CAP^S) mtDNAs were eliminated by treatment of the ES cells with the lipophilic dye Rhodamine6G (R6G). The ES cells were then fused to enucleated cell cytoplasts prepared from the CAP^R mouse cell line 5011. This procedure converted the ES cell mtDNA from 100% wildtype to 100% mutant. The CAP^R ES cells were then injected into blastocysts and viable chimeric mice were isolated. Molecular testing for the CAP^R mutant mtDNAs revealed that the percentage of mutant mtDNAs varied from zero to approximately 50% in the tissues analyzed. The highest percentage of mutant mtDNA was found in the kidney in three of the chimeric animals tested. These data suggest that, with improved efficiency, it may be possible to transmit exogenous mtDNA mutants through the mouse germline.     

Decrease of P-Glycoprotein Activity in K562/ADR Cells by MβCD and Filipin and Lack of Effect Induced by Cholesterol Oxidase Indicate That This Transporter Is Not Located in Rafts

The effect of low-density membrane domains on function of the plasma membrane transporter P-glycoprotéine (P-gp), involved in multidrug resistance (MDR) phenotype, has been investigated in K562/ADR cells. To this end we reversibly altered the cholesterol content of K562/ADR cells by using methyl--cyclodextrin as a cholesterol chelator and conversely we repleted them through incubation with cholesterol in culture medium. We also used the cholesterol-binding fluorochrome filipin and cholesterol oxidase. Our data show that either cholesterol depletion or complex formation with filipin resulted in a strong decrease of P-gp activity. However, when cells were incubated with cholesterol oxidase that are known to disrupt rafts, no modification of the P-gp activity was observed. In addition, using a free-detergent methodology to separate by ultracentrifugation, light, heavy, and extra heavy fractions we show that no P-gp is found in the light fraction where rafts are usually detected. Altogether, our data strongly suggest that, in this cell line, P-gp is not localized in rafts.