Single point mutation detection in living cancer cells by far-red emitting PNA-FIT probes.

Peptide nucleic acid bis-quinoline conjugates are reported as attractive far-red emitting probes that detect mutated mRNA in living cells at SNP resolution.

Base stimulation of phospholipid metabolism in neuroblastoma cells. I. Kinetics of incorporation of N-methylated ethanolamine bases.

The effects of nitrogen bases on the regulation of phospholipid metabolism in neuroblastoma cell cultures were investigated. An increase in the total cellular phospholipids was observed up to 24 h following plating. Addition of monomethyl- and dimethylethanolamine bases resulted in a stimulation of the synthesis of their corresponding phospholipids. The average rates of synthesis of phosphatidylmonomethyl- and phosphatidyldimethylethanolamine were 0.09 and 0.12 nmol/microgram DNA per h, respectively. The labeling patterns of the various phospholipid species from ortho[32P]phosphate have been determined. They suggest that the synthesis of the analogs proceeded entirely via a phosphate mediated pathway rather than through a base exchange mechanism. A number of distinct patterns for the incorporation of bases into acyl-, alkyl- and alkenyl-containing phosphoglyceride species were indicated. The polar head group composition appeared to be intimately related to the type of bond of the hydrocarbon residue.

Translocation and turnover of phospholipid analogs in plasma membrane-derived vesicles from cell cultures.

The time-dependent accumulation of phosphatidyldimethylethanolamine in formaldehyde-induced vesicles obtained from a somatic cell hybrid line was investigated. From a number of considerations including a two-fold enrichment of cholesterol and sphingomyelin it was concluded that these vesicles were derived from the cell plasma membrane. A progressive depletion of phosphatidylcholine, the major vesicle phospholipid, was observed in cells supplemented for various time periods with dimethylethanolamine. This depletion was accompanied by a concomitant increase in the amount of lipid analog. The time-dependent alteration of the phospholipid polar head group in intact cells was almost identical to that observed in isolated plasma membrane vesicles, suggesting a rapid equilibration of the de novo synthesized phospholipid with the cell surface compartment. From the initial velocity rate, the time required for the phosphatidylcholine pool to double was about 12 h. Agarose-linked phospholipase A2 was used to measure the relative composition of choline- and dimethylethanolamine-phosphoglycerides in the outer surface of vesicles prepared from cells with different degrees of polar head group substitution. The gradual appearance of lysodimethylethanolamine lipid analog in vesicles treated with phospholipase A2 suggested an asymmetric distribution of the phospholipid between the interior and the exterior part of the vesicle. This asymmetry was maximal up to about 4 h following the addition of dimethylethanolamine to the culture medium and was of a transient nature as the lipid analog accumulated on both sides of the plasma membrane. Based on these measurements a fast followed by a slow translocation component could be distinguished with apparent doubling times of 7 and 43 h for the lipid analog, respectively. As the analog becomes the predominant cellular phospholipid a significant increase in the vesicle lipid fluidity was measured.

Tetanus toxin interaction with human erythrocytes. I. Properties of polysialoganglioside association with the cell surface.

Human erythrocytes in suspension acquire gangliosides containing di- and trisialosyl residues added to the maintenance medium. This is reflected in the increased cell-associated sialic acid content and ability to bind 125I-labeled tetanus toxin. A salt-sensitive and a salt-insensitive ganglioside-mediated toxin-cell surface association is detected which is reduced after sialidase treatment of ganglioside-supplemented cells. The salt-insensitive ganglioside-cell association is saturable after 2 h incubation in 0.3 M mannitol buffer and has an optimum at pH 5. The association process is higher at 37 degrees C than at 4 degrees C, depends on cell density, and is considerably higher in metabolically active cells compared to lysed cells. Pretreatment of cells with trypsin decreases the salt-resistant toxin association with ganglioside-supplemented cells. In contrast, glutaraldehyde-fixed cells treated with trypsin and supplemented with gangliosides bind more toxin which is insensitive to salt. Ganglioside-mediated tetanus toxin binding to the intact erythrocyte membrane can be utilized as a model system for studying the role of glycolipids in membrane function.

Tetanus toxin interaction with human erythrocytes. II. Kinetic properties of toxin association and evidence for a ganglioside-toxin macromolecular complex formation.

The properties of tetanus toxin interaction with human erythrocytes supplemented with disialo- and trisialo-gangliosides have been investigated. Binding of toxin is linear with time for 1 h and is 3-4-fold higher at 37 degrees C than at 4 degrees C during incubation of long duration. It exhibits saturation at toxin concentrations between 0.1 and 1 microgram/ml; however, it is nonsaturable between 1 and up to 50 micrograms/ml. It is effectively prevented by free gangliosides and antibodies or by pretreatment with sialidase but is unaffected by a number of closely related ligands including toxoid and toxin fragments. NaCl (1 M) removes a great portion (86%) of cell-associated toxin while Triton X-100 extracts an additional fraction (30%) of the salt-resistant cell-bound toxin. The residual sequestred toxin after detergent extraction is sensitive to proteolytic degradation. The trypsin-stable fraction (1.5%) is biotoxic and may be indicative of internalization of toxin. A macromolecular complex of about 700 kDa containing toxin and gangliosides has been isolated and characterized by Sephacryl S-300 gel permeation chromatography, SDS-gel electrophoresis, immunoprecipitability and biotoxicity. This complex is obtained only in ganglioside-supplemented cells and not when free 3H-labeled GD1b is reacted with 125I-labeled toxin in solution in the absence of cells. The hydrophobicity properties acquired as a result of ganglioside-toxin interaction, presumably at the cell surface, suggest a conformational change of the toxin which may enable its penetration into the bilayer.

Ethyl docosahexaenoate-associated decrease in fetal brain lipid peroxide production is mediated by activation of prostanoid and nitric oxide pathways.

Previously we have shown that intraamniotic administration of ethyl docosahexaenoate (Et-DHA) to pregnant rats resulted in decreased lipid peroxidation in the fetal brain, under a variety of conditions (S. Glozman, P. Green, E. Yavin, J. Neurochem. 70 (1998) 2482-2491). In the present study we examine the potential mechanisms to explain this effect. This was done by a pharmacological approach, utilizing brain slice preparations from Et-DHA treated or control rats in the presence of various agents and examining the formation of products in the tissue slices or incubation medium. Et-DHA treated brains produced 2-3-fold more prostanoids (PN) than control brains, indicating cyclooxygenase (COX) activation. Indomethacin at 50 microM inhibited PN formation and also abolished Et-DHA induced decrease in lipid peroxides, as evident by the levels of thiobarbituric acid reactive substances (TBARS) released in the medium. The phospholipase A2 inhibitors quinacrine and p-bromophenacyl bromide added at 0.1 mM concentration each to either slices from controls or Et-DHA treated fetal brains, decreased TBARS production. Et-DHA treated brains released 2.2-fold more nitric oxide (NO) than control brains and NO synthase (NOS) inhibitors abolished this effect. Increasing the concentration of NO by the addition of an NO donor greatly decreased the concentration of the TBARS in the medium. These results suggest that at least some of the effect of Et-DHA on decreased lipid peroxidation may be explained by a shift of oxygen species utilization via enzymatically regulated, therefore metabolically controlled, COX and NOS activities.

Enhanced free radical scavenging and decreased lipid peroxidation in the rat fetal brain after treatment with ethyl docosahexaenoate.

In order to explore possible mechanisms to explain previously observed decreases in fetal brain lipid peroxidation (LPO) following intraamniotic administration of ethyl docosahexaenoate (Et-DHA) to near term fetuses, the hydroxyl radical trapping capacity of Et-DHA treated fetal brain preparations was compared to control ethyl oleate injected fetuses by electron spin resonance using 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) probe. Lipid extracts from control brains showed little hydroxyl radical scavenging activity, whereas those from the Et-DHA injected animals exhibited an almost 70% decrease in the amount of DMPO-OH adducts. A marked decrease (58%) in LPO formation was noticed in the Et-DHA treated animals compared to controls. The Et-DHA treatment related trapping capacity resided in the phospholipid fraction of the lipid extract, which was enriched in both docosahexaenoic acid and aminophospholipid contents. The decreased LPO production, as well as increased production of prostaglandin E(2) and nitric oxide by the fetal brain following Et-DHA administration, could be mimicked by a synthetic quinone possessing both hydroxyl radical producing and LPO propagation inhibiting properties. The data are consistent with the possibility that the neuroprotective effect of Et-DHA might be due to possible free radical scavenging ability of the brain tissue and interference with LPO propagation.

Docosahexaenoic acid-deficient phosphatidyl serine and high alpha-tocopherol in a fetal mouse brain over-expressing Cu/Zn-superoxide dismutase.

The over-expressed Cu/Zn-superoxide dismutase (Cu/Zn-SOD) gene has been found in some circumstances phenotypically deleterious and associated with oxidative injury-mediated aberrations while in other studies it was considered neuroprotective. In this work we examine a number of biochemical markers in fetal and adult brain from transgenic (tg) mice expressing the human Cu/Zn-SOD gene, which may determine this dual characteristic. These markers include the polyunsaturated fatty acid (PUFA) profile in discrete phospholipid species, the alpha-tocopherol levels, a marker for lipid anti-oxidant status, and thiobarbituric acid reactive substance (TBARS), a marker for the tissue oxidative status. The PUFA profile in choline- and ethanolamine-phosphoglycerides was similar in tg and nontransgenic (ntg) animals of either fetal or adult brain. Serine-phosphoglycerides, however, showed a marked decrease from 20. 07+/-0.53 to 14.92+/-0.87 wt% and 14.52+/-1.15 wt% in docosahexaenoic acid (DHA; 22:6 n3), in the tg 51 and tg 69 fetal brains, respectively, but not in the comparable adult tissues. The alpha-tocopherol levels were significantly higher in the fetal compared to the adult brain. There were no differences in the anti-oxidant levels between the ntg and tg fetal brains, but there were differences in the adult animals; the tg mice were higher by at least two-fold than the control animals. The basal TBARS in the tg 51 fetal brain was 35% lower than that of ntg mouse and in the presence of Fe(2+), brain slices from the former released less TBARS (57% reduction) into the medium than the latter. These results suggest that higher dosages of Cu/Zn-SOD gene are compatible with increased alpha-tocopherol levels, reduced basal TBARS levels and a DHA deficiency in the fetal, but not the adult, tg brain.

S-Allylmercaptoglutathione: the reaction product of allicin with glutathione possesses SH-modifying and antioxidant properties.

The reaction between allicin (diallylthiosulfinate), the active component of garlic and reduced glutathione was investigated. The product of this reaction, mixed disulfide S-allylmercaptoglutathione (GSSA) was separated by high performance liquid chromatography and identified by 1H and (13)C nuclear magnetic resonance and mass spectroscopy. The reaction is fast (with an apparent bimolecular reaction rate constant of 3.0 M(-1) s(-1)). It is pH-dependent, which reveals a direct correlation to the actual concentration of mercaptide ion (GS(-)). Both GSSA and S-allylmercaptocysteine (prepared from allicin and cysteine) reacted with SH-containing enzymes, papain and alcohol dehydrogenase from Thermoanaerobium brockii yielding the corresponding S-allylmercapto proteins, and caused inactivation of the enzymes. The activity was restored with dithiothreitol or 2-mercaptoethanol. In addition, GSSA also exhibited high antioxidant properties. It showed significant inhibition of the reaction between OH radicals and the spin trap 5,5'-dimethyl-1-pyroline N-oxide in the Fenton system as well as in the UV photolysis of H2O2. In ex vivo experiments done with fetal brain slices under iron-induced oxidative stress, GSSA significantly lowered the production levels of lipid peroxides. The similar activity of GSSA and allicin as SH-modifiers and antioxidants suggests that the thioallyl moiety has a key role in the biological activity of allicin and its derivatives.

Improved representation of gene markers on microarray by PCR-Select subtracted cDNA targets.

Despite the success and popularity of microarrays as a high-throughput technology for gene-expression studies, its sensitivity is as yet fairly limited. We have successfully combined the use of PCR-Select cDNA subtraction and Affymetrix GeneChips (AGC) to identify differentially expressed gene markers. Total RNA (totRNA) from combined hippocampus and cerebellum tissues of 2-week-old rat pups maintained for 5 weeks on an n-3 fatty acid (FA) deficient diet supplied to dams was isolated, SMART-amplified, and used for PCR-Select subtraction versus an adequately fed control litter preparation. Subtracted and amplified ds-cDNA end products were fragmented, terminally labeled with biotin-ddUTP and hybridized with RN-U34A AGC. At least 10-fold more potential gene markers with log2(T/D) > or = 1.4 were found versus the traditional AGC technology when the same chip was tested using nonsubtracted targets. Of this set of markers, 30% were robustly validated by real-time relative RT-PCR (rtrRT-PCR) and grouped as "confirmed" markers while the remaining were ascribed as "latent" markers. An improved and universal protocol to provide a rapid assessment for gene profiling in biological specimens is indicated.

Gonadotropin-releasing hormone stimulates phospholipid labeling in cultured granulosa cells.

Cultured ovarian granulosa cells from preantral and preovulatory follicles were incubated with [32P]Pi to label endogenous phospholipids. Labeled cells were then incubated with FSH, GnRH, or a GnRH agonist analog [D-Ala6]GnRH (GnRHa), cellular phospholipids were separated by two-dimensional thin layer chromatography, and the radioactivity was determined. Phosphatidylcholine was the major labeled phospholipid accounting for 64% of the total radioactivity. The remaining labeling was distributed among choline plasmalogen (8.4%), phosphatidylinositol (6.3%), lyso phosphatidylcholine (3.7%), phosphatidylethanolamine (3.4%), phosphatidic acid (1.75%), phosphatidylserine (1.65%), and cardiolipin (1.3%). GnRH and its agonist analog GnRHa, but not FSH, increased 32P incorporation into phospholipids by 2-fold. Analysis of the several phospholipids revealed that GnRHa (10(-7) M) increased 32P labeling of phosphatidylcholine and lyso phosphatidylcholine by 1.5- and 2.5-fold respectively, and that of phosphatidic acid and phosphatidylinositol by 5- and 7-fold, respectively, during 60 min of incubation. The natural decapeptide GnRH was 30 times less potent than its agonist analog. Labeling of other phospholipids was not affected by GnRHa treatment, and FSH had no effect on 32P incorporation under similar conditions. The stimulatory effect of GnRHa was blocked by the potent GnRH antagonist [D-pGlu1,pClPhe2, D-Trp3,6]GnRH. The minimal stimulating dose of GnRHa was 10(-12) M, and increased phospholipid labeling could be detected after 10 min of incubation with the analog. These results indicate that phospholipids, in particular phosphatidylinositol and phosphatidic acid, might be involved in the mechanism by which GnRH exerts its gonadal effects.

Tetanus toxin-induced protein kinase C activation and elevated serotonin levels in the perinatal rat brain.

A single intracerebral injection of tetanus toxin (TeTox) is able to produce a time-dependent translocation of Ca2(+)-phosphatidylserine-dependent protein kinase C (PKC) in close-to-term rat brain. TeTox-triggered translocation of PKC is dose- and time-dependent, can be prevented by tetanus antitoxin, and does not occur upon administration of toxin fragments B and C. TeTox-triggered PKC translocation is accompanied by a time-dependent increase in brain serotonin (5-HT). Increase of brain 5-HT is independent of monoamine oxidase inhibition by pargyline. Phorbol ester and TeTox cause a significant increase in serotonin while H-7, a kinase inhibitor, does not affect serotonin levels but abolishes the effect of TeTox. Gangliosides prevent TeTox-triggered 5-HT increase. The data are consistent with the possibility that TeTox acts effectively on the serotonergic innervation, presumably in conjunction with PKC to cause accumulation of serotonin.

Serum amyloid A-derived peptides, present in human rheumatic synovial fluids, induce the secretion of interferon-gamma by human CD(4)(+) T-lymphocytes.

Serum amyloid A (SAA) is a major acute-phase protein whose biochemical functions remain largely obscure. Human rheumatic synovial fluids were screened by high performance liquid chromatography mass spectrometry for SAA-derived peptides, specifically the sequence AGLPEKY (SAA(98-104)) which was previously shown to modulate various leukocyte functions. Two such fluids were found to contain a truncated version of SAA(98-104). Synthetic SAA(98-104) and several of its analogs were shown capable of binding isolated human CD(4)(+) T-lymphocytes and stimulating them to produce interferon-gamma. Given the high acute-phase serum level of SAA and its massive proteolysis by inflammatory related enzymes, SAA-derived peptides may be involved in host defense mechanisms.

Isolation and characterization by cell density adjustment of a PC12 pheochromocytoma variant with altered Ca2+ homeostasis.

Rat PC12 pheochromocytoma cells loaded with the fluorescent Ca2+ dye fluo-3 or indo-1 and scanned fluorimetrically on a cell sorter apparatus showed a rapid cell density-dependent increase in free cytosolic calcium concentration [Ca2+]i when maintained in suspension cultures. Cell adhesion, measured under a defined set of conditions, was low when cells were seeded at 1.5 x 10(4) cells/ml but reached maximal levels after addition of A23187 calcium ionophore. A six to sevenfold increase in cell density mimicked the effect of the ionophore. Densities above 2 x 10(6) cells/ml caused a decrease in cell adhesion, which was further reduced by the addition of A23187. BAPTA, AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and nifedipine (10 microM each), partially inhibited cell attachment (34% and 44% reduction), but at 0.25 microM and 1 microM, respectively, they enhanced attachment (46% and 67% increase). The data suggest that a certain permissive level of [Ca2+]i, attained by either increasing cell density or by the presence of a calcium ionophore, is sufficient for maximal cell adhesion. Above the permissive level, manipulation of [Ca2+]i either by altering cell density or by the addition of calcium blocking agents in high concentrations results in a significant reduction in cell adhesion. Based on these observations, we were able to isolate a biochemically and morphologically distinct cell population. The variant, designated PC12ds (density selected), differed substantially from the original cells. Most notable was a relatively lower content of free [Ca2+]i in the PC12ds cells, as independently assayed by using fluo-3 and indo-1 dyes. In addition, the variant cells exhibited a significantly diminished rate of 45Ca2+ uptake, most likely due to less efficient L-type voltage-dependent calcium (VDC) channels. Addition of several calcium channels agonists and antagonists suggested that PC12ds cells contained relatively more N-type VDC channels, possibly indicating a shift to a neuronal phenotype.

Docosahexaenoic acid accumulation in the prenatal brain: prooxidant and antioxidant features.

Docosahexaenoic acid (DHA; 22:6n-3) is the major polyunsaturated fatty acid (FA) in the adult rat brain and it accumulates significantly more than any other FA prior to birth. Under normal nutritional conditions, fetal-brain DHA accumulation is substantial, with a "DHA accretion spurt" being demonstrated in the last period of gestation. Under stress conditions, this spurt may be harmful owing to an increase in multiple double-bond targets for lipid peroxidation. The "DHA accretion spurt" is supported by the maternal supply of DHA or its precursor. Under maternal dietary n-3 FA deficiency, DHA content in the fetal brain can be restored by direct intraamniotic injection of mM concentrations of ethyl-DHA (Et-DHA). This approach may hold a potential advantage in the event of maternal-fetal insufficiency, a stress that may cause intrauterine growth retardation. It also revealed a potential beneficial effect after in utero ischemic stress; brain slices from Et-DHA-treated fetuses formed less oxidation products, as detected by thiobarbituric acid (TBA), compared to controls. Furthermore, brain-lipid extracts from Et-DHA but not ethyl-oleate treated fetuses, exhibited hydroxyl radical scavenging activity, as demonstrated by electron spin-resonance technique. Part of the beneficial effect of Et-DHA administration on the fetal brain may be attributed to enhanced free-radical scavenging capability, a phenomenon not directly related to vitamin E or lipid-soluble antioxidant levels.

Cyclic GMP alterations in fetal rat cerebrum after global intrauterine ischemia: role of guanylate cyclase phosphorylation.

Changes in the levels of cyclic AMP (cAMP) and cyclic GMP (cGMP) have been measured in brains of 20-day-old rat fetuses exposed to global intrauterine ischemia. Ischemia of different duration (0.5-30 minutes) did not alter the level of cAMP. In contrast, cGMP levels increased as a result of ischemia. This increase was seen even after a short period of ischemia (less than 5 minutes) and was maximal after 5 minutes, where a threefold increase could be observed. This stimulation was transient: after 30 min of ischemia, cGMP returned to the control level. Accumulation of cGMP can be related to the activation of guanylate cyclase, the activity of which is doubled after 15 minutes of ischemia. Immunoprecipitation of guanylate cyclase after in vivo labeling of the fetal brain with 32Pi revealed a threefold increase in the phosphorylation of the enzyme after 15 minutes of ischemia. The possible role of these modifications in cGMP metabolism during the course of ischemia is discussed.

Overexpression of genes in the CA1 hippocampus region of adult rat following episodes of global ischemia.

Ischemic stress is associated with marked changes in gene expression in the hippocampus--albeit little information exists on the activation of nonabundant genes. We have examined the expression of several known genes and identified novel ones in the adult rat hippocampus after a mild, transient, hypovolemic and hypotensive, global ischemic stress. An initial differential screening using a prototype array to assess gene expression after stress followed by a suppression subtractive hybridization protocol and cDNA microarray revealed 124 nonoverlapped transcripts predominantly expressed in the CA1 rat hippocampus region in response to ischemic stress. About 78% of these genes were not detected with nonsubtracted probes. Reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization on these 124 transcripts confirmed the differential expression of at least 83. Most robustly expressed were gene sequences NFI-B, ATP1B1, RHOGAP, PLA2G4A, BAX, CASP3, P53, MAO-A, FRA1, HSP70.2, and NR4A1 (NUR77), as well as sequence tags of unknown function. New stress-related genes of similar functional motifs were identified, reemphasizing the importance of functional grouping in the analysis of multiple gene expression profiles. These data indicate that ischemia elicits expression of an array of functional gene clusters that may be used as an index for stress severity and a template for target therapy design.

Hydrogen peroxide induces nuclear translocation of p53 and apoptosis in cells of oligodendroglia origin.

The observation that apoptosis is an inherent pathway in oligodendrocytes development coupled with the notion that wild-type p53 is expressed in these cells, prompted us to investigate the interrelationship between the two phenomena. Using a permanent oligodendroglia-like cell line (OLN 93), we examined the role of p53 protein in apoptosis following a DNA insult induced by a brief exposure to H2O2. A marked translocation of p53 from the cytosolic to the nuclear compartment was notable by 20 min, following a 5 min treatment with 1 mM H2O2 as identified by cell immunostaining. By 48 h following H2O2 addition, nearly 60% of the cells exhibited p53 in the nuclei. At this time, a large proportion of the cells underwent apoptosis as identified by DAPI nuclear staining. The genotoxic-induced p53 relocalization appeared to be cell cycle phase specific; thus OLN 93 cultures enriched for cells in the G0/G1 stage by serum starvation, and abundant in nuclear-associated p53, were more susceptible to H2O2-induced apoptosis than their untreated counterparts and than double thymidine block, G1/S enriched, cultures. Analysis of the expression of p53 downstream genes indicated that p21 and mdm2 were upregulated following p53 nuclear translocation. From the kinetics of protein accumulation, it appears that mdm2 enhancement accelerated the exit of p53 from the nucleus to the cytosol. Our results suggest that following stress, oligodendroglia-like cells are induced to undergo p53-dependent apoptosis, an event that coincides with p53 nuclear translocation and is cell-cycle related.

Ischemia stress and arachidonic acid metabolites in the fetal brain.

Hypoxic-ischemic insults caused by placental insufficiency in perinatal life are today considered a major cause for neuronal injury and impaired postnatal development. A major consequence of placental insufficiency and ischemia is the change in metabolism of arachidonic acid and its oxidation products. A burst of postischemic production of prostaglandins, unequivocally shown in many systems, is documented in the fetal rabbit brain as well as in placenta tissue soon after vascular restriction. PGE2, a most abundant prostaglandin of the fetal brain, is particularly elevated. Similarly, thromboxane B2 and 6-keto PGF1 alpha, the stable metabolites of thromboxane A2 and prostacyclin, are both increased over the control values. However, after 48 h of restriction, the levels of these eicosanoids are restored to near-normal values. The metabolic machinery responsible for the conversion of arachidonic acid into eicosanoids in brain and placenta tissues appears to be impaired following a period of placental insufficiency. This inhibition can be accounted for by excessive production of eicosanoids and also by formation of an endogenous inhibitor or free radicals. Studies are in progress to test these possibilities.