Effect of Inhibitors of GABA Transaminase on the Synthesis, Binding, Uptake and Metabolism of GABA

Abstract: Four catalytic inhibitors of GABA aminotransferase (gabaculine, γ-acetylenic GABA, γ-vinyl GABA, ethanolamine O -sulphate) as well as aminooxyacetic acid and valproate were studied for effects on neurochemical assays for GABA synthesis, receptor binding, uptake and metabolism in mouse and rat brain preparations. Gabaculine did not interfere with GABA synthesis as reflected by the activity of glutamate decarboxylase (GAD), it was only a weak inhibitor (IC₅₀= 0.94 mM) of GABA receptor binding sites but was a moderately potent inhibitor of GABA uptake (IC₅₀= 81 μM) and very potent (IC₅₀= 1.8 μM) with respect to inhibition of the GABA-metabolizing enzyme GABA aminotransferase (GABA-T). γ-Acetylenic GABA was a weak inhibitor of GAD and GABA binding (IC₅₀ > 1 mM), but virtually equipotent to inhibit uptake and metabolism of GABA (IC₅₀ 560 and 150 μM, respectively). This was very similar to γ-vinyl GABA, except that this drug did not decrease GAD activity. Ethanolamine O -sulphate was found to show virtually no inhibition of GAD and GABA uptake, but was a fairly potent inhibitor of GABA binding (IC₅₀= 67 μM) and in this respect, 500 times more potent than as an inhibitor of GABA-T. Aminooxyacetic acid was a powerful inhibitor of both GAD and GABA-T (IC₅₀ 14 and 2.7 μM, respectively), but had very little affinity to receptor and uptake sites for GABA. Valproate showed no effects on GABA neurochemical assays which could be related to anticonvulsant action. The present results suggest that the anticonvulsant properties of the four catalytic inhibitors of GABA-T tested are at least in part mediated through a direct influence on GABA receptors and uptake sites.     

Effects of di-n-butyl phthalate on growth and photosynthesis in algae and on isolated organelles from higher plants

Di- n -butyl phthalate (DBF) is widely used as a plasticizer and has been found in all types of ecosystems. It inhibits growth and photosynthesis of green algae ( Chlorella emersonii CCAP strain 211/8 h and Selenastrum capricornutum CCAP strain 278/4) at concentrations higher than 10-⁵ M . The IC₅₀ value for CO₂-dependent oxygen evolution in algae was 3 × 10^-4M. The CO₂-reduction in isolated protoplasts prepared from barley ( Hordeum vulgare L. cv. Simba) was also inhibited by phthalate. The IC₅₀ value was 2 × 10^-4 M . The electron transport in isolated thylakoids prepared from spinach was inhibited with an IC₅₀ value of 3 × 10^-4 M . The IC₅₀ value for uncoupled electron transport extrapolated to zero chlorophyll concentration was 2.5 × 10^-5 M . The effect of di-n-butyl phthalate was localized to reactions in photosystem II. Di-n-butyl phthalate could thus be a pollutant which affects growth and photosynthesis of plants. The reported IC₅₀ values may be underestimated since di- n -butyl phthalate can attach to surfaces. The results are discussed in relation to observed effects of di- n -butyl phthalate on other organisms.     

Effects of Polyamines on the Uptake of Neurotransmitters by Rat Brain Synaptosomes

Abstract: The influence of putrescine, spermidine, spermine, and some aliphatic α,ω-diamines on the uptake of neurotransmitters by rat forebrain synaptosomes was investigated. Choline uptake was most effectively inhibited by spermine (IC₅₀= 0.22 m M ), less so by spermidine (IC₅₀= 4.0 m M ), but not by putrescine (IC₅₀ > 100 m M ). At 10 m M, 1,3-diaminopropane, cadaverine, and 1,8-diaminooctane all inhibited choline uptake by 50% or more. Spermine and spermidine inhibited the uptake of dopamine with IC₅₀ values of 2.7 and 2.2 m M , respectively. Putrescine was only slightly inhibitory (IC₅₀= 17.3 m M ) and the other diamines were inactive. The uptake of γ-aminobutyrate (GABA) was only slightly inhibited (15–40%) by the polyamines at 10 m M . With the exception of inhibition of glycine uptake by 1,8-diaminooctane (60%) and of glutamate uptake by cadaverine (35%) none of the polyamines, tested at 10 m M , affected the uptake of adenosine, glutamate, and glycine significantly. A possible modulatory role for polyamines in synaptic transmission through interaction by negatively charged groups of the synaptic membrane with the polycationic compounds is discussed.     

Proadrenomedullin N-Terminal 20 Peptide (PAMP), an Endogenous Anticholinergic Peptide: Its Exocytotic Secretion and Inhibition of Catecholamine Secretion in Adrenal Medulla

Abstract: In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca²⁺-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC₅₀ = 50.1 µ M ) and catecholamines (EC₅₀ = 63.0 µ M ), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1–20]NH₂ inhibited carbachol-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀ = 2.5 µ M ) in a noncompetitive manner and thereby reduced carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels (IC₅₀ = 1.0 µ M ) and catecholamine secretion (IC₅₀ = 1.6 µ M ). It did not alter high K⁺-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels or veratridine-induced ²²Na⁺ influx via voltage-dependent Na⁺ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca²⁺-dependent exocytosis in response to nicotinic receptor stimulation.     

Coupling of the Human Y2 Receptor for Neuropeptide Y and Peptide YY to Guanine Nucleotide Inhibitory Proteins in Permeabilized SMS-KAN Cells

Abstract: Using guanine nucleotides, pertussis toxin, and specific antisera against the COOH-terminals of the α-subunits of G_i1/2, G_i3, and G_o, the binding and biological response of the Y2 receptor (Y2R) for peptide YY (PYY) was probed in SMS-KAN neuroblastoma cells. The specific binding of radiolabeled PYY exhibited a single apparent dissociation constant, K _D = 76 p M for intact cells and K _D = 906 p M for permeabilized cells. However, other data suggested existence of multiple receptor affinity states. A shift in K _D and a decrease in apparent number of binding sites ( B _max) was observed in permeabilized cells when incubated with guanine nucleotides. By contrast, in membrane preparations guanine nucleotides induced only a decrease in B _max. In intact cells, agonist exposure inhibited the intracellular accumulation of forskolin-stimulated cyclic AMP by 80% (IC₅₀ = 420 n M ) compared with 94% inhibition (IC₅₀ = 380 n M ) in permeabilized cells. In permeabilized cells, preincubation with antisera against α_i1/2 and α_i3 blocked the functional response of PYY, with anti-α_i3 being the most potent; whereas anti-α_o failed to affect the cyclic AMP levels. These results suggest that permeabilized SMS-KAN cells serve as a good model system for analysis of Y2R binding kinetics and functional response and that the Y2R interacts directly with several different G_is (but not G_o).     

Induction of differentiation and apoptosis in three human leukemia cell lines by a new compound from Dendrostellera lessertii

It has previously been shown that Dendrostellera lessertii(Thymelaeaceae)has stronganticancer activity.In this study,the antileukemic activity of another new compound from the same plantextract is reported.Promyelocytic(NB4 and HL-60)and erythroleukemia(K562)cells were cultured in thepresence of various concentrations of the new compound(0.5-3.0 μtg/ml)for 3d.The cell numbers werethen determined by trypan blue exclusion test.The new compound inhibited growth and proliferation ofNB4,HL-60 and K562 with IC_(50) values of 1.5,2.0 and 2.5μg/ml,respectively.We also found that the newcompound inhibited cell proliferation in a dose-and time-dependent manner.At low concentrations and after48h of treatment,approximately 50%-70% of NB4 and HL-60 cells were differentiated to monocyte/macrophage lineage and approximately 30%-40% of the treated K562 cells were differentiated in the mega-karyocytic lineage,as evidenced by morphological changes and nitro blue tetrazolium reduction assays.Results of Hoechst 33258 staining also indicated that the new compound induced NB4 and HL-60 cellapoptosis at their respective IC_(50) values after 72h of treatment.Based on the present data,the new com-pound seems a good candidate for further evaluation as an effective chemotherapeutic agent acting throughinduction of differentiation and apoptosis.     

Inhibition of Neuroblastoma Cell Phosphatidylinositol 3-Kinase by CDP-Diacylglycerol and Phosphatidate

Abstract: Phosphatidylinositol (PI) 3-kinase is activated by a variety of agents, including various growth factors, and has been proposed to play a role in initiation of cell growth, proliferation, and differentiation. We here investigate the effect of various membrane lipids on PI 3-kinase immunopurified from human SH-SY5Y neuroblastoma cells. CDP-diacylglycerol (CDP-DAG) inhibited PI 3-kinase activity with an IC₅₀ of 6 µ M . Phosphatidate (PA) was also inhibitory (IC₅₀ = 38 µ M ) as was lysophosphatidate. Neither DAG nor any of the other phospholipids examined affected PI 3-kinase activity. The results offer the possibility that CDP-DAG or PA at critical membrane sites may exert functionally significant metabolic regulation at the point of convergence of the PI 3-kinase-directed and the PI 4-kinase-directed phosphoinositide signal transduction pathways.     

A Comparison of 2-Hydroxyestradiol and U-0521 (3'4'-Dihydroxy-2-methylpropiophenone, Upjohn) as In Situ and In Vitro Inhibitors of Tyro sine Hydroxylase

Abstract: Feedback inhibition of tyrosine hydroxylase by catechols was evaluated using in situ and in vitro enzyme assays. The three catechol compounds used were norepinephrine, 2-hydroxyestradiol, and 3'4'-dihydroxy-2-methylpropiophenone (U-0521, Upjohn); representing endogenous catechol-amines, catechol estrogens, and a synthetic catechol, respectively. The in situ experiments were performed with dissociated retinal cells from rats and with stationary phase adrenergic-like neuroblastoma cells (N1E-115). The catechol estrogen, 2-hydroxyestradiol, resembled the endogenous catecholamines in its potency to inhibit in vitro and in situ tyrosine hydroxylations with IC₅₀ values of 10 μM in vitro and 100 μM in situ. The drug U-0521, which has been used as an inhibitor of catechol- O -methyltransferase (COMT), was also found to be an inhibitor of tyrosine hydroxylase. Further, it was shown to be more potent than the natural catechols, both in vitro and in situ , with IC₅₀ values of 30–600 nM.     

Selective Inhibition of Cholesterol Biosynthesis in Brain Cells by Squalestatin 1

Abstract: The effect of squalestatin 1 (SQ) on squalene synthase and other enzymes utilizing farnesyl pyrophosphate (F-P-P) as substrate was evaluated by in vitro enzymological and in vivo metabolic labeling experiments to determine if the drug selectively inhibited cholesterol biosynthesis in brain cells. Direct in vitro enzyme studies with membrane fractions from primary cultures of embryonic rat brain (IC₅₀ = 37 n M ), pig brain (IC₅₀ = 21 n M ), and C6 glioma cells (IC₅₀ = 35 n M ) demonstrated that SQ potently inhibited squalene synthase activity but had no effect on the long-chain cis -isoprenyltransferase catalyzing the conversion of F-P-P to polyprenyl pyrophosphate (Poly-P-P), the precursor of dolichyl phosphate (Dol-P). SQ also had no effect on F-P-P synthase; the conversion of [³H]F-P-P to geranylgeranyl pyrophosphate (GG-P-P) catalyzed by partially purified GG-P-P synthase from bovine brain; the enzymatic farnesylation of recombinant H-p21 ^ras by rat brain farnesyltransferase; or the enzymatic geranylgeranylation of recombinant Rab1A, catalyzed by rat brain geranylgeranyltransferase. Consistent with SQ selectively blocking the synthesis of squalene, when C6 glial cells were metabolically labeled with [³H]mevalonolactone, the drug inhibited the incorporation of the labeled precursor into squalene and cholesterol (IC₅₀ = 3–5 µ M ) but either had no effect or slightly stimulated the labeling of Dol-P, ubiquinone (CoQ), and isoprenylated proteins. These results indicate that SQ blocks cholesterol biosynthesis in brain cells by selectively inhibiting squalene synthase. Thus, SQ provides a useful tool for evaluating the obligatory requirement for de novo cholesterol biosynthesis in neurobiological processes without interfering with other critical reactions involving F-P-P.     

Laboratory appraisal of optimal gaseous conditions for growth of zoonotic Helicobacter felis ATCC 49179

An attempt was made to assess the hitherto undescribed optimal gaseous conditions for growth of zoonotic Helicobacter felis, focusing on the ratio of spiral-forms amongst the whole cells examined. The largest mean colony diameter was obtained under the gaseous condition of O₂ 12% and CO₂ 10%. In analyzing the five day old colonies, the highest percentage of spiral forms (85.5%) was observed under the condition of O₂ 18% and CO₂ 5%. In contrast, the lowest percentage of spiral forms (2.3%) was demonstrated under the condition of O₂ 1% and CO₂ 10%. The condition of O₂ 12% and CO₂ 10% was concluded to be optimal for obtaining cells with the largest colony sizes, although colonies proliferated under such conditions definitely contain many more coccoid cells than spiral forms. In culturing H. felis strains, optimal gaseous conditions should be employed according to the purposes or preferences of study designs.     

Structure and Functional Characterization of a Novel Human Low-Voltage Activated Calcium Channel

Abstract : We have isolated and characterized overlapping cDNAs encoding a novel, voltage-gated Ca²⁺ channel α₁ subunit, α_1H, from a human medullary thyroid carcinoma cell line. The α_1H subunit is structurally similar to previously described α₁ subunits. Northern blot analysis indicates that α_1H mRNA is expressed throughout the brain, primarily in the amygdala, caudate nucleus, and putamen, as well as in several nonneuronal tissues, with relatively high levels in the liver, kidney, and heart. Ba²⁺ currents recorded from human embryonic kidney 293 cells transiently expressing α_1H activated at relatively hyperpolarized potentials (-50 mV), rapidly inactivated (τ = 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing α_1H. Singlechannel measurements in human embryonic kidney 293 cells revealed a single-channel conductance of ~9 pS. These channels are blocked by Ni²⁺ (IC₅₀ = 6.6 μ M ) and the T-type channel antagonists mibefradil (~50% block at 1 μ M ) and amiloride (IC₅₀ = 167 μ M ). Thus, α_1H-containing channels exhibit biophysical and pharmacological properties characteristic of low voltage-activated, or T-type, Ca²⁺ channels.     

Anti-Candida activity of four antifungal benzothiazoles

Abstract Anti- Candida activity of 6-amino-2- n -pentylthiobenzothiazole (I), benzylester of (6-amino-2-benzothiazolylthio)acetic acid (II) and of 3-butylthio-(1,2,4-triazolo)-2,3-benzothiazole (III) was followed and compared to that of 2-mercaptobenzothiazole (IV). I and II exhibited good activity against the C. albicans yeast form, similar to IV. They were inhibitorily active against other Candida strains, IC₅₀ values being of the order of 10⁻⁵ M, which means better activity than IV. Compound I also exhibited inhibitory activity on germ-tube formation and mycelial growth in the C. albicans strains, while II, III and IV were not active in these tests. III was the least active form of the compounds tested, IC₅₀ values being of the order of 10⁻⁴ M. All the compounds tested were highly active on a nystatin-resistant C. albicans mutant, with IC₅₀s of the order of 10⁻⁶ M−10⁻⁵ M.     

Interaction of α-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor

α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7 nAChR-targeting α-conotoxin ImI, blocked α7 and muscle nAChRs without displacing α-bungarotoxin ( Ellison et al. 2003, 2004 ), suggesting binding at a different site. We synthesized α-conotoxin ImII, its ribbon isomer (ImII iso ), 'mutant' ImII(W10Y) and found similar potencies in blocking human α7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [¹²⁵I]-α-bungarotoxin from human α7 nAChRs in the cell line GH₄C₁ (IC₅₀ 17 and 23 μM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC₅₀ 2.0–9.0 μM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized α-bungarotoxin ( K _d and IC₅₀ 2.5–8.2 μM). On Torpedo nAChR, α-conotoxin [¹²⁵I]-ImII(W10Y) revealed specific binding ( K _d 1.5–6.1 μM) and could be displaced by α-conotoxin ImII, ImII iso and ImII(W10Y) with IC₅₀ 2.7, 2.2 and 3.1 μM, respectively. As α-cobratoxin and α-conotoxin ImI displaced [¹²⁵I]-ImII(W10Y) only at higher concentrations (IC₅₀≥ 90 μM), our results indicate that α-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.     

Spiroethers of German chamomile inhibit production of aflatoxin G1 and trichothecene mycotoxin by inhibiting cytochrome P450 monooxygenases involved in their biosynthesis

The essential oil of German chamomile showed specific inhibition toward aflatoxin G₁ (AFG₁) production, and ( E )- and ( Z )-spiroethers were isolated as the active compounds from the oil. The ( E )- and ( Z )-spiroethers inhibited AFG₁ production of Aspergillus parasiticus with inhibitory concentration 50% (IC₅₀) values of 2.8 and 20.8 μM, respectively, without inhibiting fungal growth. Results of an O- methylsterigmatocystin (OMST) conversion study indicated that the spiroethers specifically inhibited the OMST to AFG₁ pathway. A cytochrome P450 monooxygenase, CYPA, is known as an essential enzyme for this pathway. Because CYPA has homology with TRI4, a key enzyme catalyzing early steps in the biosynthesis of trichothecenes, the inhibitory actions of the two spiroethers against TRI4 reactions and 3-acetyldeoxynivalenol (3-ADON) production were tested. ( E ) - and ( Z ) - spiroethers inhibited the enzymatic activity of TRI4 dose-dependently and interfered with 3-ADON production by Fusarium graminearum , with IC₅₀ values of 27.1 and 103 μM, respectively. Our results suggest that the spiroethers inhibited AFG₁ and 3-ADON production by inhibiting CYPA and TRI4, respectively.     

Inhibition of heat shock proteins (HSP) expression by quercetin and differential doxorubicin sensitization in neuroblastoma and Ewing's sarcoma cell lines

Neuroblastoma (NB) and Ewing's sarcoma (ES) represent the most common extracranial solid tumors of childhood. Heat shock proteins (HSP) are elevated in cancer cells and their over-expression was correlated to drug-resistance. In this work we identified the HSP by a sensitive proteomic analysis of NB and ES cell lines, then, we studied the HSP response to doxorubicin. Some identified HSP were constitutively more expressed in NB than in ES cells. Doxorubicin-stimulated HSP response only in NB cells. Quercetin was found to inhibit HSP expression depleting heat shock factor 1 (HSF1) cellular stores. Quercetin caused a higher anti-proliferative effect in NB (IC₅₀: 6.9 ± 5.8 μmol/L) than in ES cells (IC₅₀: 85.5 ± 53.1 μmol/L). Moreover, quercetin caused a very pronounced doxorubicin sensitizing effect in NB cells (241 fold IC₅₀ decrease) and a moderate effect in ES cells. HSP involvement in NB cells sensitization was confirmed by the silencing of HSF1. Quercetin treatment and HSF1 silencing increased the pro-apoptotic effect of doxorubicin. In conclusion, the higher HSP levels, observed in NB cells, did not confer increased resistance to doxorubicin; on the contrary, HSP inhibition by quercetin or gene silencing caused higher sensitization to doxorubicin. These results may have a potential application in the treatment of NB.     

Image analysis for the automated estimation of clonal growth and its application to the growth of smooth muscle cells

Abstract. Image analysis was used for the automated measurement of colony frequency ( f ) and colony diameter ( d ) in cultures of smooth muscle cells, Initial studies with the inverted microscope showed that number of cells ( N ) in a colony varied directly with d : log N = 1.98 log d - 3.469 Image analysis generated the complement of a cumulative distribution for f as a function of d . The number of cells in each segment of the distribution function was calculated by multiplying f and the average N for the segment. These data were displayed as a cumulative distribution function. The total number of colonies ( f_T ) and the total number of cells ( N_T ) were used to calculate the average colony size ( N_A ). Population doublings (PD) were then expressed as log₂ N_A . Image analysis confirmed previous studies in which colonies were sized and counted with an inverted microscope. Thus, image analysis is a rapid and automated technique for the measurement of clonal growth.     

Toxicity of organotins towards cyanobacterial photosynthesis and nitrogen fixation

Abstract Inhibition of photosynthesis by a range of organotin compounds in Plectonema boryanum was concentration-dependent and decreased in the order tributyltin (Bu₃SnCl) > tripropyltin (Pr₃SnCl) ≥ dibutyltin (Bu₂SnCl₂) ≥ triphenyltin (Ph₃SnCl) > triethyltin (Et₃SnCl) > trimethyltin (Me₃SnCl) > monobutyltin (BuSnCl₃). IC₅₀ values were determined for the most toxic organotin species and varied from approximately 1.2 μM for Bu₃SnCl to approximately 13 μM for Ph₃SnCl. A similar order of inhibition of photosynthesis was observed in Anabaena cylindrica , although here IC₅₀ values were slightly lower (e.g. approximately 1 μM for Bu₃SnCl and 5 μM for Ph₃SnCl).Nitrogenase activity was generally more sensitive to inhibition by organotin compounds than photosynthesis in A. cylindrica and this was particularlyy evident for Bu₂SnCl₂; approximate IC₅₀ values for Bu₂SnCl₂ were 3 and 9 μM, as estimated by nitrogenase activity and photosynthesis, respectively. These results indicate that organotin compounds have the potential to inhibit cyanobacterial metabolism in aquatic systems.     

Widespread gene flow and high genetic variability in populations of water voles Arvicola terrestris in patchy habitats

Theory predicts that the impact of gene flow on the genetic structure of populations in patchy habitats depends on its scale and the demographic attributes of demes (e.g. local colony sizes and timing of reproduction), but empirical evidence is scarce. We inferred the impact of gene flow on genetic structure among populations of water voles Arvicola terrestris that differed in average colony sizes, population turnover and degree of patchiness. Colonies typically consisted of few reproducing adults and several juveniles. Twelve polymorphic microsatellite DNA loci were examined. Levels of individual genetic variability in all areas were high ( H _O= 0.69–0.78). Assignments of juveniles to parents revealed frequent dispersal over long distances. The populations showed negative F _IS values among juveniles, F _IS values around zero among adults, high F _ST values among colonies for juveniles, and moderate, often insignificant, F _ST values for parents. We inferred that excess heterozygosity within colonies reflected the few individuals dispersing from a large area to form discrete breeding colonies. Thus pre-breeding dispersal followed by rapid reproduction results in a seasonal increase in differentiation due to local family groups. Genetic variation was as high in low-density populations in patchy habitats as in populations in continuous habitats used for comparison. In contrast to most theoretical predictions, we found that populations living in patchy habitats can maintain high levels of genetic variability when only a few adults contribute to breeding in each colony, when the variance of reproductive success among colonies is likely to be low, and when dispersal between colonies exceeds nearest-neighbour distances.     

Multiparameter analysis of progeny of individual cells by laser scanning cytometry

BACKGROUND: Effectiveness of antitumor drugs to suppress unrestricted proliferation of cancer cells is commonly measured by cell clonogenicity assays. Assays of clonogenicity are also used in studies of stem/progenitor cells and in analysis of carcinogenic transformation. The conventional assays are limited to providing information about frequency of colonies (cloning efficiency) and do not reveal the qualitative (phenotype) attributes of individual colonies that may yield clues on mechanisms by which cell proliferation was affected by the studied agent. METHODS: Laser scanning cytometry (LSC) was adapted to identify and characterize size and phenotype of colonies of MCF-7 cells growing in microscope slide chambers, untreated and treated with the cytotoxic ribonuclease, onconase (Onc). Individual colonies were located and data representing each colony were segmented based on >650-nm fluorescence excited by a He-Ne laser of the cells whose protein was stained with BODIPY 630/650-X. The DNA of the cells was stained with propidium iodide (red fluorescence) whereas specific proteins (estrogen receptor [ER] or tumor suppressor p53) were detected immunocytochemically (green fluorescence), each excited by an Ar ion laser. RESULTS: A plethora of attributes of individual colonies were measured, such as (a) morphometric features (area, circumference, area/circumference ratio, DNA or protein content per area ratio), (b) number of cells (nuclei), (c) DNA content, (d) protein content and protein/DNA ratio, and (e) expression of ER or p53 per colony, per total protein, per nucleus or per DNA, within a colony. Also cell cycle distribution within individual colonies and heterogeneity of colonies with respect to all the measured features could be assessed. The colonies growing in the presence of Onc had many of the above attributes different than the colonies from the untreated cultures. CONCLUSIONS: Analysis of the features of cell colonies by LSC provides a wealth of information about the progeny of individual cells. Changes in colony size and phenotype, reflecting altered cell shape, cell size, colony protein/DNA ratio, and expression of individual proteins, may reveal mechanisms by which drugs suppress the proliferative capacity of the cells. This may include inducing growth imbalance and differentiation and modulating expression of the genes that may be associated with cell cycle, apoptosis, or differentiation in a progeny of individual cells. Extensions of LSC may make it applicable for automatic analysis of cloning efficiency and multiparameter analysis of cell colonies in soft agar. Such analyses may be useful in studies of the mechanisms and effectiveness of antitumor drugs, in the field of carcinogenesis, and for analyzing primary cultures and assessing tumor prognosis and drug sensitivity. The assay can also be adapted to analysis of microbial colonies.     

Aurintricarboxylic Acid Prevents NMDA-Mediated Excitotoxicity: Evidence for Its Action as an NMDA Receptor Antagonist

Abstract: The effect of the endonuclease inhibitor aurintricarboxylic acid (ATA) versus NMDA-mediated delayed cell death was examined in an ex vivo chick retinal preparation. Transient exposure to 100 μM NMDA for 60 min followed by a 24-h recovery period resulted in a sevenfold increase in lactate dehydrogenase (LDH) release into the medium. ATA at 100 μM significantly reduced NMDA-mediated LDH release by 60%. In clarifying the mechanism of protection versus NMDA, ATA was found to inhibit several acute NMDA-mediated effects: ATA attenuated NMDA-mediated GABA release in a dose-dependent manner (IC₅₀= 29.5 μM ), prevented NMDA-stimulated cyclic GMP formation, and blocked NMDA-mediated ²²Na⁺ influx. These acute inhibitory effects of ATA were overcome by increasing the NMDA concentration, which suggested a competitive interaction between NMDA and ATA. In a binding assay using membranes prepared from adult rat forebrain, ATA displaced the competitive NMDA receptor ligand [³H]CGS 19755 with an IC₅₀ of 26.9 μM. Maximal displacement was 88% with 100 μM ATA. These studies demonstrate that ATA protected neurons from NMDA-mediated cell death upstream of endonuclease inhibition, i.e., by antagonizing NMDA receptor activity in a manner consistent with competitive antagonism.