Reduced cellular glutathione levels do not affect the cytotoxicity or photocytotoxicity of the cationic photosensitiser Victoria Blue BO

Victoria Blue BO (VBBO) is thought to exert its photocytotoxic effects via free radical generation. Glutathione and related enzymes are associated with the protection of normal tissues against free-radical damage and have also been implicated in multiple drug resistance. It might, therefore, be expected that cells containing higher levels of glutathione would be resistant to the cytotoxic effects of VBBO. The total glutathione content for a murine mammary tumour cell line, EMT6-S, was found to be lower than in a multi-drug resistant cell line, EMT6-R, 21.84+/-2.54 microg (mg protein)(-1) and 18.79+/-2.7 microg (mg protein)(-1), respectively; however, this was not found to be a significant difference (p > 0.05, Student t-test). Buthionine sulfoximine, a potent inhibitor of gamma-glutamyl cysteine synthetase, brought about a reduction in glutathione levels in both EMT6-S and EMT6-R cell lines in a concentration-dependent manner. Buthionine sulfoximine administration was effective in reducing intracellular glutathione levels by up to 90% in both types of cells. Interestingly, glutathione depletion of EMT6-S and EMT6-R cells did not enhance the photocytotoxic effect of VBBO, suggesting that the primary site of action of VBBO may be at an intracellular site not protected by glutathione or that the mechanism of action is not via the in situ generation of free radical species.     

The cross-resistance of mouse blasticidin S-resistant cell lines to puromycin and sparsomycin, inhibitors of ribosome function.

The antibiotic blasticidin S inhibits peptide-chain elongation in extracts of bacteria and mammalian cells. After spontaneous or nitrosoguanidine mutagenesis, we have isolated 46 blasticidin S-resistant (Blar) cell lines independently from mouse mammary carcinoma cells (FM3a). Among those Blar clones, we studied two clones, a spontaneously induced one (S501) and a nitrosoguanidine mutagenized one (N742) in more detail. The resistant phenotype of these Blar cells is retained without change for at least four months in the absence of the antibiotic. These Blar cells are 10- to 20-fold more resistant to the cytotoxic action of the antibiotic than their parental cells in vivo. Polyuridylate dependent polyphenylalanine synthesis in vitro with S-30 extracts either from N742 or S501 is 10- to 50-fold more resistant to the inhibitory action of blasticidin S compared to the parental FM3a cells. Ribosomes from FM3a and N742 are fractionated into 40-S and 60-S subunits, and polyphenylalanine synthesis by mixing them in various combinations with S-100 fraction from mouse leukemia L5178Y cells indicating that the resistant phenotype of Blar cells is due to the alteration of 60 S ribosomal subunit. We also found that these two Blar cell lines (N742 and S501) show cross-resistance to gougerotin, puromycin and sparsomycin, but not to emetine or cycloheximide. The polyribosomal pattern of FM3a (Blas) and N742 was compared when the cells were incubated with 3 microgram/ml puromycin for 6 h. Puromycin treatment of Blas cells induced accumulation of monosomes and ribosomal subunits, while little if any transition of polyribosomes into monosome and ribosomal subunits appeared in its counterpart N742 treated with the same dose of puromycin.     

THE EFFECT OF RADIO-ACTIVE RADIATIONS AND X-RAYS ON ENZYMES : I. THE EFFECT OF RADIATIONS FROM RADIUM EMANATION ON SOLUTIONS OF TRYPSIN.

A quantitative study has been made of the radiochemical decomposition of trypsin by the radiations from radium emanation. The following equation accounts quantitatively for the experimental results presented. See PDF for Equation It follows from this that the amount of trypsin decomposed by the radiations from radium emanation depends on the concentration of trypsin present and is proportional to the quantity of emanation expressed in millicuries and to the time of irradiation expressed in hours. It would seem that it is the active or undissociated trypsin that is affected. Evidence has been found which suggests that the beta radiations produce the decomposition observed for which the above statement holds. Qualitative evidence has been found which suggests that x-rays, gamma rays, and beta rays produce identical effects in dilute trypsin solutions.     

Rab11-FIP3 is a cell cycle-regulated phosphoprotein

Background

Rab11 and its effector molecule, Rab11-FIP3 (FIP3), associate with recycling endosomes and traffic into the furrow and midbody of cells during cytokinesis. FIP3 also controls recycling endosome distribution during interphase. Here, we examine whether phosphorylation of FIP3 is involved in these activities.

Results

We identify four sites of phosphorylation of FIP3 in vivo, S-102, S-280, S-347 and S-450 and identify S-102 as a target for Cdk1-cyclin B in vitro. Of these, we show that S-102 is phosphorylated in metaphase and is dephosphorylated as cells enter telophase. Over-expression of FIP3-S102D increased the frequency of binucleate cells consistent with a role for this phospho-acceptor site in cytokinesis. Mutation of S-280, S-347 or S-450 or other previously identified phospho-acceptor sites (S-488, S-538, S-647 and S-648) was without effect on binucleate cell formation and did not modulate the distribution of FIP3 during the cell cycle. In an attempt to identify a functional role for FIP3 phosphorylation, we report that the change in FIP3 distribution from cytosolic to membrane-associated observed during progression from anaphase to telophase is accompanied by a concomitant dephosphorylation of FIP3. However, the phospho-acceptor sites identified here did not control this change in distribution.

Conclusions

Our data thus identify FIP3 as a cell cycle regulated phosphoprotein and suggest dephosphorylation of FIP3 accompanies its translocation from the cytosol to membranes during telophase. S102 is dephosphorylated during telophase; mutation of S102 exerts a modest effect on cytokinesis. Finally, we show that de/phosphorylation of the phospho-acceptor sites identified here (S-102, S-280, S-347 and S-450) is not required for the spatial control of recycling endosome distribution or function.     

Antioxidant activity of thiosulfinates derived from garlic

Garlic extract significantly inhibited the oxidation of methyl linoleate in homogeneous acetonitrile solution, whereas the antioxidant effect of allicin-free garlic extract, prepared by removing allicin by prepared by removing allicin by preparative HPLC, was much lower than that of the garlic extract. These results suggest that the antioxidant properties are mostly attributed to the presence of allicin in the garlic extract. Allicin a major component of the thiosulfinates in garlic extract, was found to be effective for inhibiting methyl linoleate oxidation, but its efficiency was less than that of alpha-tocopherol. Next, the reactivity of allicin toward the peroxyl radical, which is a chain-propagating species, was investigated by direct ESR detection. The addition allicin to 2,2'-azobis(2,4-dimethylvaleronitrile)-peroxyl radical solution caused the signal intensity of the peroxyl radical to dose-dependently decrease, indicating that allicin is capable of scavenging the the peroxyl radical and acting as an antioxidant. Finally, we studied the structure-anioxidant activity relationship for thiosulfinates and suggested that the combination of the allyl group (-CH2CH=CH2) and the -S(O)S- group is necessary for the antioxidant action of thiosulfinates in the garlic extract. In addition, one of the two possible combinations, -S(O)S-CH2CH=CH2, was found to make a much larger contribution to the antioxidant activity of the thiosulfinates than the other, CH2=CH-CH2-S(O)S-.     

Effect of antibodies to S-100 group proteins on the electrical activity and chemical sensitivity of the cerebral cortical neurons

The influence of antibodies to proteins S-100 group on electrical activity and chemical sensitivity of rabbit cortical neurons has been investigated by means of extracellular recording and microiontophoresis. It has been found that anti-S-100 increase as a rule spontaneous discharge rate of the neurons, decrease, blockade or invert the neuronal responses to acetylcholine, noradrenaline and glutamate action. It is supposed that proteins S-100 group take part in regulation of eleitrogenic and postsynaptic (primarily glutamate- and GABA-ergic) processes of neural and glial cells in the brain.     

Mechanisms of inhibition of herpes simplex virus type 2 growth by 28-mer phosphorothioate oligodeoxycytidine

The 28-mer phosphorothioate oligodeoxycytidine (S-(dC)28) has been reported previously to be a strong inhibitor of herpes simplex virus type 2 (HSV-2) DNA polymerase and HSV-2 growth in cell culture. In this study, the mechanism of action of S-(dC)28 was studied. S-(dC)28 was found to interfere with the adsorption of HSV-1 and HSV-2 to HeLa cells. HSV-2 infection, but not HSV-1, was found to potentiate the uptake of S-(dC)28 into HeLa cells. The enhanced uptake reached a plateau at 6-9 h postinfection and appeared to be dose-dependent and saturable at concentrations higher than 1 microM. The amount of S-(dC)28 accumulated in HSV-2 infected cells was found to be 50 pmol/10(6) cells at 6 h postinfection, whereas no significant drug accumulation was found in uninfected cells. S-(dC)28 binding studies suggested that there are several types of tight binding sites associated with HSV-2 virions, which could play a role in the enhancement of S-(dC)28 uptake. Subcellular distribution studies showed that intracellular S-(dC)28 was associated with both nuclei and cytoplasm and remained intact. Mechanism studies suggested three different mechanisms which could be responsible for the anti-HSV-2 action of S-(dC)28; (i) S-(dC)28 could interfere with the uptake of HSV. (ii) HSV-2 infection enhances the uptake of S-(dC)28 into cells. (iii) S-(dC)28 inhibits HSV-2 DNA synthesis, possibly, by inhibiting the viral DNA polymerase. The unique mechanisms of anti-HSV action of S-(dC)28 suggest it could be a potential new agent in anti-HSV-2 chemotherapy.     

The effect of chelating agents on the absorption of radium by plants

Summary The absorption of radium from solution, by plants, has been compared with that of calcium, active strontium being used as a label for the calcium. It was found that radium was preferentially retained by the roots and discriminated against in passage to the shoots. However, the uptake and distribution of radium was influenced by ethylenediaminetetra acetic acid (EDTA) and citrate at the concentrations employed in water culture media to keep iron in solution. There was little discrimination against radium after plants had grown for a week in active nutrient solution when EDTA was present, but in the presence of citrate radium moved less rapidly by a factor of about 0.3. In the early stages of treatment, less than a day, the results with citrate were comparable with those of EDTA, whence it is inferred that the decrease in transfer to the shoots is dependent upon the relative rates at which the two complexes decompose.     

Studies on the antigenic determinants of the Thy-1.2 alloantigen as expressed by the murine lymphoblastoid line S-49.1 TB-2-3.

Sialic acid has been shown to be part of the antigenic determinant of the Thy-1.2 alloantigen as expressed by the murine cell line S-49.1 TB-2-3 (S-49). This conclusion is based on the loss of cytotoxic inhibitory activity by the action of neuraminidase on the Thy-1.2 alloantigen. Also, sialic acid has the ability to inhibit the cytotoxic assay of AKR anti-C3H Thy-1.2 serum for S-49 cells. Further evidence for the protein nature of the Thy-1.2 alloantigen is apparent by trypsin digestion. Possibly the Thy-1.2 alloantigen as expressed on S-49 cells is a glycoprotein.     

A comparative study of the inducing effect of homoserine lactone and hexylresorcinol on phenotypic dissociation in bacteria

It has been shown that the phenotypic dissociation of Bacillus subtilis SK1 and S. typhimurium TA100 is induced by hexylresorcinol, an exogenous non-species-specific autoregulator of pleiotropic action, which is genotoxic for both pro- and eukaryotes. Nongenotoxic homoserine lactone, a chemical analogue of cell-density-responsive species-specific regulators, does not induce bacterial dissociation. The phage resistance of the S- and R-type variants of S. typhimurium TA100 induced by hexylresorcinol has been found to be the same as that of the S- and R-type salmonella variants obtained by the routine subculturing method.     

Interaction of smooth muscle caldesmon with S-100 protein

The interaction of caldesmon with certain Ca-binding proteins was investigated by means of electrophoresis under non-denaturating conditions. In the presence of Ca2+ calmodulin, troponin C and S-100 protein form a complex with caldesmon. No complex formation takes place in the absence of Ca2+. Lactalbumin and pike parvalbumin (pI4.2) do not interact with caldesmon independently of Ca-concentration. Both S-100 protein and calmodulin effectively inhibit phosphorylation of caldesmon by Ca-phospholipid-dependent protein kinase. At low ionic strength S-100 protein reverses the inhibitory action of caldesmon on the skeletal muscle acto-heavy meromyosin ATPase more effectively than calmodulin. It is supposed that in certain tissues and cell compartments the proteins belonging to the S-100 family are able to substitute for calmodulin in the caldesmon-dependent regulation of actin and myosin interaction.     

Expression of Biologically Active Basic Fibroblast Growth Factor by Genetically Modified Rat Primary Skin Fibroblasts

Abstract: Basic fibroblast growth factor (FGF-2) is normally expressed as a cell-associated protein, and accordingly it is not clear how it exerts its action on target cells in vivo. It has been proposed that cells release, by death or other mechanisms, small amounts of FGF-2 that then acts in an autocrine manner. To address the question of whether it is necessary that FGF-2 remain cell associated or needs to be secreted from cells to have biological activity, we expressed the 18-kDa form of FGF-2 in primary fibroblasts as a cell-associated (FGF-2-B) or as a secreted (FGF-2-S) protein. FGF-2 protein is detected in cell lysates and membrane fractions of both cell types, whereas it is present in significant amounts only in the conditioned medium of FGF-2-S cells. No FGF-2 is detected in control (untransfected) cells. FGF-2-S cells also grow faster than the control or FGF-2-B cells. Yet, when evaluated for their ability to promote the survival of embryonic hippocampal neurons in vitro, both the cell types are active, establishing the activity of the transgene product. We conclude that FGF-2 is active when engineered to be expressed as a cell-associated form or secreted from cells.     

Thyroid Hormone Actions on a Cholinergic Neuroblastoma Cell Line (S-20Y)

Thyroid hormone (T3) has a multiplicity of effects on the developing nervous system. We have investigated T3 action using a cholinergic neuroblastoma cell line (S-20Y) as a model. S-20Y contains a nuclear receptor for T3 with binding properties similar to those of other T3 target tissues. In addition, these cells can carry out 5'-deiodination, which is necessary to produce active thyroid hormone in vivo. The enzyme involved in this process appears to be a type I deiodinase, based on its reaction kinetics and its susceptibility to inhibition by propylthiouracil. S-20Y cells maintained in T3-depleted medium showed decreased choline acetyltransferase (ChAT) activity. ChAT activity was restored to the control level in a dose-dependent manner by T3 repletion. Neither cell density nor viability was influenced by the hypothyroid state. The presence of a T3 receptor and the enzyme activity for T3 production, together with an effect of T3 on ChAT activity, demonstrate that S-20Y cells are a target for T3 action and suggest that these cells represent an excellent model system for studies of T3 effects on nervous tissues.     

Effects of the bioreductive alkylating agent 2,3-bis(chloromethyl)-1,4-naphthoquinone on coupled mitochondria isolated from sarcoma 180 ascites cells.

The effect of CMNQ was studied on mitochondria isolated from S-180 ascites tumor cells. It was found that the primary metabolic event upon addition of CMNQ to S-180 mitochondria was a stimulation of oxygen uptake. The oxygen utilization rate was maximized at about 50 nmoles CMNQ/mg protein; at doses higher than this, inhibition of respiration was observed relative to the stimulation of respiration produced by CCCP. It was also up to 50 nmoles CMNQ/mg protein. S-180 ATPase activity is stimulated maximally by 125 nmoles CMNQ/mg protein; at doses higher than this, slight inhibition of the ATPase activity relative to the stimulation produced by CCCP is seen. In vivo treatment of CMNQ to tumor bearing animals leads to a significant reduction of in vitro S-180 cellular respiration rates. The data presented in this work coupled with previously published reports involving CMNQ support the proposal for a mitochondrial level of action for this bioreductive alkylating antineoplastic agent.     

Studies on Temperate Phages of Lactobacillus salivarius

Comparative studies were carried out on the mode of action of the defective temperate phage 208 against the homologous lysogenic strain S-208 and a nonlysogenic strain S-161 of Lactobacillus salivarius. Treatment of both strains with phage 208 led to a specific inhibition of protein synthesis, cell killing without any reversion of protein synthesis, and of viable counts by treatment with trypsin. Killing action of phage 208 followed a single hit kinetics for nonlysogenic S-161 and S-208 No. 006, which is a cured strain of S-208, whereas, two to five hit kinetics was obtained for lysogenic S-208. Phage particles exposed to ultraviolet light (5.7 kergs/cm²) also killed S-161 with a single hit kinetics and S-208 with a 3-hit kinetics. However, the kinetics of killing approached a single hit when the protein synthesis of S-208 was inhibited by chloramphenicol prior to the phage addition. Based upon these results, the possible mechanisms of immunity breakdown and of subsequent cell killing were discussed.     

Binding of labeled initiation factor IF-1 to ribosomal particles and the relationship to the mode of IF-1 action in ribosome dissociation.

The binding of labeled initiation factor IF-1 to ribosomal particles has been studied in relation to the mode of action of this factor in the dissociation of 70-S ribosomes. It is demonstrated that IF-1 interacts specifically with active 70-S tight couples and free 30-S subunits. The binding of IF-1 to both 70-S and 30-S particles is not influenced by the Mg2+ concentration and the affinity of the factor for both particles is about the same. The interaction of IF-1 with these particles is highest at low Tris-HCl concentrations. Under these conditions IF-1 shows a slight dissociating activity. Using 3H-labeled IF-1 and 14C-labeled IF-3 the formation of a 30-S-subunit.IF-1 . IF-3 complex from 70-S ribosomes is demonstrated. Our studies show that IF-3 enhances the binding of IF-1 to the 30-S subunit. In contrast to IF-1, which binds about equally well to 70-S and 30-S particles in the absence of IF-3, 14C-labeled IF-3 binds predominantly to the 30-S subunit. This finding confirms the view that IF-3 acts as an anti-association factor. On the other hand, IF-1 enhances the supply of 30-S subunits in the presence of IF-3 by acting on the 30-S moiety of the 70-S ribosome.     

Effect of protein S-100 on phosphorylation of nuclear proteins of cells of rat brain and liver

The effect of acidic neurospecific protein S-100 on the phosphorylation of brain and liver nuclear proteins with 1 and 10 microM ATP was investigated. It was shown that protein S-100 increases the phosphorylation of brain nuclear proteins, while antigen D, another acidic neurospecific protein half-identical to 14-3-2 protein, inhibits this process. Ca2+ and cAMP at concentration of 10(-6) M do not affect the phosphorylation of brain nuclear proteins. In control assays the tracer 32P is presumably incorporated into high molecular weight nuclear protein fractions (Mr greater than 40000). After addition of protein S-100 the tracer is mainly incorporated into these proteins as well independently of ATP concentration (1 or 10 microM). The phosphorylation of nuclear proteins with molecular weights above 100000 is mostly increased in this case. At ATP concentration of 1 microM protein S-100 decreases histone phosphorylation 2.3 times but does not affect that of non-histone proteins. However, at 10 microM ATP the inhibitory action of this protein on histone phosphorylation is absent. The possible mechanisms of protein S-100 action on nuclear proteins phosphorylation are discussed.     

Dual Functions for Connexins: Cx43 Regulates Growth Independently of Gap Junction Formation

Connexins, the family of proteins that form vertebrate gap junctions, have key roles during development and in the adult. Previously, the physiological actions of connexins have been ascribed solely to formation of gap junction channels and thought to be mediated by the transfer of small molecules between neighboring cells. In conflict with this hypothesis here we demonstrate that Cx43 can affect cell growth independently of gap junction formation. This conclusion is based on four findings: (1) There is a lack of correlation between the action of Cx43 mutants Cx43-S255A, Cx43-S279A, and Cx43-S282A on growth and cell coupling in 3T3 A31 fibroblasts. (2) Blockade of gap junction formation, by either heptan-1-ol treatment or culturing cells at low density, had no effect on the ability of the Cx43 mutants to control growth. (3) Wildtype Cx43 inhibited growth of Neuro2a cells under conditions where gap junctions were unable to form. (4) The CT domain of Cx43, which lacks intrinsic gap junction activity, is as effective as the wildtype molecule in suppressing the growth of Neuro2a cells. These observations demonstrate that Cx43 has dual functions: first, its well-accepted role in forming a gap junction channel and, second, a direct action of the connexin protein on growth that is mediated via the cytoplasmic carboxyl domain.     

Mechanisms of 2''-deoxyguanosine toxicity in mouse T-lymphoma cells with purine nucleoside phosphorylase deficiency and resistance to inhibition of ribonucleotide reductase by dGTP.

Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) deficiency is thought to cause T-lymphocyte depletion by accumulation of dG and dGTP, resulting in feedback inhibition of ribonucleotide reductase (RR; EC 1.17.4.1) and hence DNA synthesis. To test for additional toxic mechanisms of dG, we selected a double mutant of the mouse T-lymphoma S-49 cell line, dGuo-L, which is deficient in PNP and partially resistant to dGTP feedback inhibition of RR. The effects of dG on dGuo-L cells (concn. causing 50% inhibition, IC50 = 150 microM) were compared with those on the wild-type cells (IC50 = 30 microM) and the NSU-1 mutant with PNP deficiency only (IC50 = 15 microM). Fluorescence flow cytometry showed that equitoxic dG concentrations arrested wild-type and NSU-1 cells at the G1-S interface while allowing continued DNA synthesis in the S-phase, whereas the double mutant dGuo-L cells progressed through the cell cycle normally. dGuo-L cells accumulated high levels of dGTP in G1-phase, but not in S-phase cells, because of the utilization of dGTP for DNA synthesis and limited capacity to synthesize dGTP from dG. These results support the hypothesis that dG/dGTP toxicity occurs in the G1-phase or at the G1-S interface. Failure of dG to arrest the double mutant dGuo-L cells at the G1-S interface allows these cells to escape into S-phase, with an accompanying drop in dGTP levels. Thus the partial resistance of dGuo-L cells to dG toxicity may result from their shorter residence time in G1, allowing them to sustain higher dGTP levels. Hence RR inhibition by dGuo may not be the primary toxic mechanism in S-49 cells; rather, it may serve as an accessory event in dG toxicity by keeping the cells in the sensitive phase of the cell cycle. Among the possible targets of dG toxicity is RNA synthesis, which was inhibited at an early stage in dGuo-L cells.     

Somatostatin receptors on rat pancreatic acinar cells. Pharmacological and structural characterization and demonstration of down-regulation in streptozotocin diabetes

The binding of somatostatin-14 (S-14) to rat pancreatic acinar cell membranes was characterized using [125I-Tyr11]S-14 as the radioligand. Maximum binding was observed at pH 7.4 and was Ca2+-dependent. Such Ca2+ dependence of S-14 receptor binding was not observed in other tissues. Scatchard analysis of the competitive inhibition by S-14 of [125I-Tyr11]S-14 binding revealed a single class of high affinity sites (Kd = 0.5 +/- 0.07 nM) with a binding capacity (Bmax) of 266 +/- 22 fmol/mg of protein. [D-Trp8]S-14 and structural analogs with halogenated Trp moiety exhibited 2-32-fold greater binding affinity than S-14, [D-F5-Trp8]S-14 being the most potent. [Tyr11]S-14 was equipotent with S-14. The affinity of somatostatin-28 for binding to these receptors was 50% of that of S-14. Cholecystokinin octapeptide (CCK-8) inhibited the binding of [125I-Tyr11]S-14, but its inhibition curve was not parallel to that of S-14. In the presence of 1 nM CCK-8, the Bmax of S-14 receptors was reduced to 150 +/- 17 fmol/mg of protein. Dibutyryl cyclic GMP, a CCK receptor antagonist, partially reversed the inhibitory action of CCK-8, suggesting that CCK receptors mediate the inhibition of S-14 receptor binding. GDP, GTP, and guanyl-5'-yl imidodiphosphate inhibit S-14 receptor binding in this tissue. The inhibition was shown to be due to decrease in binding capacity and not due to change in affinity. Specifically bound [125I-Tyr11]S-14 cross-linked to the S-14 receptors was found associated with three proteins of approximate Mr = 200,000, 80,000, and 70,000 which could be detected under both reducing and nonreducing conditions. Finally, pancreatic acinar cell S-14 receptors were shown to be down-regulated by persistent hypersomatostatinemia 1 week after streptozotocin-induced diabetes characterized by decreased Bmax (105 +/- 13 fmol/mg of protein) without any change in affinity. We conclude that pancreatic acinar cell membrane S-14 receptors require Ca2+ for maximal binding and thus differ from S-14 receptors in other tissues, S-14 receptors in this tissue also exhibit selective ligand specificities, these receptors are regulated by CCK-8 and guanine nucleotides, three receptor proteins of apparent Mr = 200,000, 80,000, and 70,000 specifically bind S-14, and (v) these receptors are regulated by S-14 in vivo as evidenced by decreased binding in streptozotocin diabetic rats characterized by hypersomatostatinemia.