Antitumor effects of bis-thiosemicarbazones— inhibition of deoxyribonucleic acid synthesis in vitro

A group of bis-thiosemicarbazones was evaluated for potential antitumor activity, using the L1210 murine leukemia in cell culture. Drug levels required to inhibit DNA synthesis by 50 per cent, under standard conditions, were determined. The most potent of the agents examined had the structure X[CH₂CR₁=NNHCSNHR₂]₂ where X = C or S and R₁ = R₂ = CH₃. Optimal activity was also obtained with R₁ = H and R₂ = CH₃ only when X = S. The most potent derivatives inhibited DNA synthesis by 50 per cent within 10 min at 10^?6 M levels (id₅₀). Metal chelates of several compounds tested were extremely potent inhibitors of DNA synthesis ($\text{id}{}_{50}\text{= 10}{}^{\mathrm{?7}}\text{M or less}$). Insolubility in water and short duration of action in vivo may limit effectiveness of the bis-thiosemicarbazones.     

Antiviral activity of 5-thiocyanatopyrimidine nucleosides.

The antiviral activity of the 5-thiocyanatopyrimidine nucleosides 5-NCSrU¹, 5-NCSdU, 5-NCSaraU and tri-O′-acetyl-5-NCSrU has been evaluated in primary rabbit kidney (PRK) cell cultures challenged with either DNA (vaccinia, herpes simplex) or RNA (vesicular stomatitis) viruses. 5-NCSdU inhibited vaccinia virus multiplication at 10 μg/ml, and vaccinia and herpes simplex virus induced cytopathogenicity at 4 μg/ml. Tri-O′-acetyl-5-NCSrU inhibited vesicular stomatitis virus-induced cytopathogenicity at 1–10 μg/ml. None of the compounds had profound effects on host cell RNA or DNA synthesis, even at 200 μg/ml, as monitored by [³H]uridine and [³H]thymidine incorporation respectively, except 5-NCSdU, which brought about a 10–30-fold increase of [³H]thymidine incorporation at 200 μg/ml. The inhibitory effect of 5-NCSdU on vaccinia virus replication and its stimulatory effect on [³H]thymidine incorporation were almost completely reversed by thymidine at concentrations 100 times lower than that of the thiocyanato derivative. When treated with dithiothreitol, the 5-thiocyanatopyrimidine nucleosides also lost a significant part of their biological activity, presumably due to reduction to the corresponding 5-mercapto analogs.     

The effects of cadmium, manganese and aluminium on sodium-potassium-activated and magnesium-activated adenosine triphosphatase activity and choline uptake in rat brain synaptosomes

The effects of Cd²⁺, Mn²⁺ and Al³⁺ on rat brain synaptosomal sodium-potassium-activated and magnesium-activated adenosine triphosphatase (Na-K-ATPase and Mg-ATPase) activity and choline uptake were studied. All three types of metal ions inhibited Na-K-ATPase activity more markedly than Mg-ATPase activity. The rank order of inhibition of Na-K-ATPase was: $\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 5.4 μ}\text{M}\text{) >}\text{Mn}{}_{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 955 μ}\text{m}\text{) >}\text{Al}{}^{\mathrm{3+}}\text{(}\text{ic}{}_{50}\text{= 8.3}\text{mM}$). The rank order of inhibition of Mg- was:$\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 316 μ}\text{M}\text{>}\text{Mn}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 5.5}\text{mM}\text{>}\text{Al}{}^{\mathrm{3+}}\text{(}\text{ic}{}_{50}\text{= 21.9}\text{mM}\text{).}\text{Al}{}^{\mathrm{3+}}$ was most potent in inhibiting synaptosomal choline uptake ($\text{ic}{}_{50}\text{= 24μ}\text{M}$ in the absence of Ca²⁺ and 123 μ.M in the presence of 1 mM Ca²⁺). $\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 363 μ}\text{M}\text{)}$ was a more effective inhibitor of choline uptake than $\text{Mn}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 1.2?1.5}\text{mM}\text{)}$ . The presence of 1 mM Ca²⁺ did not alter choline uptake, nor did it antagonize the inhibitory actions of the three metals. Our observations that Cd²⁺ and Al³⁺ inhibited synaptosomal choline uptake, but did not show parallel inhibitory effects on Na-K-ATPase activity directly contradicts the ionic gradient hypothesis. These results are also discussed in relation to the in vivo neurotoxicity of cadmium, manganese and aluminium.     

Mechanisms by which quipazine,a putative serotonin receptor agonist,alters brain 5-hydroxyindole metabolism

Quipazine (2-[1-piperazinyl] quinoline maleate) was shown to increase serotonin and decrease 5-hydroxyindoleacetic acid concentrations in whole brain, several brain regions, and the spinal cord of rats 1 hr after its administration (10 mg/kg, i.p.). In animals with transected spinal cords, quipazine induced stronger activation of extensor reflexes than 5-hydroxytryptophan, chlorimipramine, or Lilly 110140. This response could be blocked by methiothepin. In slices of rat cerebral cortex, quipazine inhibited the uptakes of [³H]-serotonin (EC₅₀ = 10^?6 M) and [³H]-norepinephrine ($\text{EC}{}_{50}\text{= 2 × 10}{}^{\mathrm{?6}}\text{m}$); it was equipotent with Lilly 110140 in inhibiting serotonin uptake, but less potent than chlorimipramine ($\text{EC}{}_{50}\text{= 10}{}^{\mathrm{?7}}\text{m}$). Quipazine administration to rats did not inhibit monoamine oxidase activity, and actually elevated brain tryptophan levels. These observations suggest that the effects of quipazine on brain serotonin and 5-hydroxyindoleacetic acid concentrations could have been caused by direct activation of central serotonin receptors (which would secondarily decrease impulse flow along serotonergic neurones), or by the inhibition of serotonin reuptake, or by both mechanisms.     

Effects of bis(6-mercaptopurine-9-beta-D-ribofuranoside)-5',5"-phosphate and its butyryl derivative on mouse leukaemia L1210 and a 6-mercaptopurine-resistant subline in culture

Bis(6-mercaptppurine-9-β-d-ribofuranoside)-5′-5″′-monophosphate (bis(MPR)P) and its butyryl derivative, bis(O²,$\text{O}{}^3\text{-}\text{dibutyryl}\text{-6-}\text{mercaptopurine}\text{-9-β-}\text{d}\text{-}\text{ribofuranoside}$)-5′,5″′-monophosphate (bis(dibutyrylMPR)P) were synthesized from 6-mercaptopurine-9-β-d-ribofuranoside (MPR). Bis(MPR)P (ec₅₀ = 0.014 μM) and MPR (ec₅₀ = 0.022 μM) were essentially equivalent in their growth inhibitory activities against L1210/0 cell cultures, whilst bis(dibutyrylMPR)P (ec₅₀ = 1.1 μM) was considerably less effective. L1210/MPR cells grew normally in the presence of 1 mM MPR but were inhibited by bis(MPR)P (ec₅₀ = 580 μM) and (bis(dibutyrylMPR)P (ec₅₀ = 42 μM). Bis(dibutyrylMPR)P was less readily broken down to MPR by enzymes in the serum component of the culture medium than was bis(MPR)P, and leukaemia cells did not contribute to the extracellular degradation of the acylated derivative. The delayed cytotoxic effects of bis(MPR)P and bis(dibutyrylMPR)P on L1210/0 cells were those of the MPR breakdown product. Exposure to bis(MPR)P resulted in delayed cytotoxicity in L1210/MPR cultures whilst bis(dibutyrylMPR)P produced only acute growth inhibition and no delayed effect on the MPR-resistant subline. MPR was incorporated into DNA of L1210/0 cells as 6-thioguanine deoxyribonucleotide whilst bis(MPR)P was not incorporated into L1210/MPR cell DNA. These results suggested that the ultimate mechanisms of action of bis(MPR)P and bis(dibutyrylMPR)P in L1210/ MPR cells may have been different from that of MPR in sensitive L1210/0 cells and therefore might not represent true circumvention of resistance to MPR.     

Mechanism of action of 5'-methylthioadenosine in S49 cells

5'-Deoxy-5'-methylthioadenosine, a naturally occurring co-product of polyamine biosyn-thesis, has been shown to inhibit a variety of biological processes. To investigate the mode of action of this nucleoside and to assess the involvement of cAMP in this action, the effect of methylthioadenosine on S49 wild type and two cAMP-related mutant cells was examined. The sulfur-containing nucleoside potently inhibited the growth of the parental strain ($\text{IC}{}_{50}\text{= 50 μ}\text{M}$), whereas nearly 10-fold greater resistance was demonstrated by S49 adenylate cyclase deficient ($\text{IC}{}_{50}\text{= 420 μ}\text{M}$) and S49 cAMP-dependent protein kinase deficient ($\text{IC}{}_{50}\text{= 520 μ}\text{M}$) mutant cells. Methylthioadenosine was shown to competitively inhibit the S49-derived high-affinity cAMP phosphodiesterase (K_i = 62 μM) in vitro, whereas methylthioadenosine phosphorylase activity was equivalent in all three cell types. The intracellular levels of the regulatory nucleotide, cAMP, increased dramatically in the wild type (17-fold) and protein kinase deficient (6-fold) strains in response to 100 μM concentrations of the drug. It is concluded that the growth arrest produced by 5'-methylthioadenosine in S49 cells is primarily due to the inhibition of cAMP phosphodiesterase and the subsequent increase in cAMP levels that result.     

Uptake,distribution, and effects of carbon tetrachloride in rainbow trout (Salmo gairdneri)

Uptake, distribution, and effects of CCl₄ were studied in rainbow trout. Carbon tetrachloride (1 ml/kg, ip) produced 5- to 10-fold increases in serum GOT, GPT, and ICD activities, whereas exposure of trout to CCl₄ in the tank water (1–80 mg/liter) produced neither mortality nor significant changes in enzyme activities. CCl₄ residue(s) appeared highest in concentration in the adipose tissue, followed by liver, brain, and spleen, and was lowest in gill regardless of the administration route. The elimination rates of ¹⁴C residue(s) from the tissue samples were most rapid in muscle ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{β = 1.7}\text{hr}$) and relatively prolonged for liver ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{β = 38.9}\text{hr}$). Maximum liver concentrations of ¹⁴C residue(s) were reached at 2 hr by either ip (1 ml/kg) or water exposure (80 mg/liter) and were 4.8 μmol/g and 0.75 μmol/g, respectively. No increase in liver triglyceride (TG) concentrations were noted at liver CCl₄ concentrations that have been associated with increased TG levels in the rat. Histological examination of tissues revealed varying degrees of liver and splenic necrosis 6 hr after administration of CCl₄.     

The role of lipid,free radical initiator,and oxygen on the kinetics of lipid peroxidation

The relationship between the concentration of unsaturated lipid, free radical initiator, and oxygen concentration on the kinetics of lipid peroxidation was determined. The rate of lipid peroxidation was studied with the thiobarbituric acid (TBA), diene conjugation (DC), and ferrithiocyanate (Fe-SCN) methods. The rate of peroxidation was half-order with respect to unsaturated lipid, initiator, and oxygen. The half-order relationship could be expressed as: $\text{rate}\text{= (}\text{fk}{}_1\text{k}{}_2\text{k}{}_3\text{k}{}_6{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(}\text{azobisisobutyronitrile}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$$\text{(}\text{RH}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(}\text{O}{}_2\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$. The half-order relationship was found with linoleic (18:2), linolenic (18:3), and arachidonic (20:4) acids. A linear relationship existed between the logarithm of unsaturation and the rate of peroxidation. No peroxidation of linolenic acid was indicated when the DC method was employed, but was when the TBA and Fe-SCN methods were used.     

Antiherpes activity of [E]-5-(1-propenyl)-2′-deoxyuridine and 5-(1-propenyl)-1-β-d-arabinofuranosyluracil

5-(1-Propenyl)-1-β-d-arabinofuranosyluracil has been synthesized, and this compound and [E]-5-(1-propenyl)-2′-deoxyuridine have been tested for inhibition of herpes virus multiplication. Only [E]-5-(1-propenyl)-2′-deoxyuridine was found to be an active inhibitor reducing by 50% the plaque formation of herpes simplex virus type 1 (HSV-1) at about 1 μM. A comparison to the bromovinyl derivatives showed the following order of decreasing activity; $\text{[}\text{E}\text{]-5-(2-}\text{bromovinyl}\text{)-2′-}\text{deoxyuridine}\text{> 5-(2-}\text{bromovinyl}\text{)-1-β-}\text{d}\text{-}\text{arabinofuranosyluracil}\text{≥ [}\text{E}\text{]-5-(1-}\text{propenyl}\text{)-2′-}\text{deoxyuridine}\text{> 5-(1-}\text{propenyl}\text{)-1-β-}\text{arabinofuranosyluracil}$. HSV-1 mutants lacking thymidine kinase or resistant against acycloguanosine were resistant against [E]-5-(1-propenyl)-2′-deoxyuridine. All compounds seemed to be phosphorylated by HSV-1 thymidine kinase in a cell-free assay. The compounds were phosphorylated to a lower extent by cellular or HSV-2 thymidine kinase, and the HSV-2 strains tested were inhibited by less than 50% at 100 μM in plaque assays. A selective inhibition of HSV-1 DNA synthesis by [E]-5-(1-propenyl)-2′-deoxyuridine was observed in infected cells indicating an effect on viral DNA polymerase. [E]-5-(1-Propenyl)-2′-deoxyuridine had a low cellular toxicity and a therapeutic effect when applied topically to HSV-1-infected guinea pig skin.     

Antiviral activity of 5-methylthiomethyl-2'-deoxyuridine and other 5-substituted 2'-deoxyuridines.

Of a series of eight 5-substituted 2'-deoxyuridine (dUrd) derivatives, which were evaluated for their antiviral and antimetabolic activities in primary rabbit kidney or human skin fibroblast cell cultures, five dUrd derivatives, 5-dimethylaminomethyl-dUrd, 5-chloroacetamidomethyl-dUrd, 5-iodoacetamidomethyl-dUrd, 5-pyrrolidinylmethyl-dUrd and 5-N-methylpiperazinylmethyl-dUrd, showed little, if any, activity. The three others, 5-formyl-dUrd, 5-azidomethyl-dUrd and 5-methylthiom-ethyl-dUrd, were found to inhibit the replication of various HSV (herpes simplex virus) strains (whether type 1 or 2) at a concentration of approximately 1–10 μg/ml. The antiviral activity of 5-formyl-dUrd may be accounted for by an inhibition of thymidylate synthetase. The inhibitory effect of 5-formyl-dUrd on HSV multiplication was readily reversed by adddition of 2'-deoxythymidine (dThd), and, in analogy with other established thymidylate synthetase inhibitors, 5-formyl-dUrd blocked the incorporation of [2-¹⁴C]dUrd into cellular DNA to a markedly greater extent than the incorporation of [methyl-³H]dThd. Unlike 5-formyl-dUrd, 5-azidomethyl-dUrd and 5-methylthiomethyl-dUrd did not preferentially inhibit the incorporation of [2-¹⁴C]dUrd. Antiviral indexes, defined as the id₅₀ for [2-¹⁴C]dUrd incorporation divided by the id₅₀ for HSV (type 1, strain KOS) replication, were 0.25, 43 and > 100 for 5-formyl-dUrd, 5-azidomethyl-dUrd and 5-methylthiomethyl-dUrd, respectively. The latter two compounds may therefore be considered as rather selective anti-herpes agents.     

Similar activities of nerve growth factor and its homologue proinsulin in intracellular hydrogen peroxide production and metabolism in adipocytes: Transmembrane signalling relative to insulin-mimicking cellular effects

Generation of hydrogen peroxide in adipocyte plasma membrane and its intracellular metabolism and regulatory role have been shown by Mukherjee and co-workers to be a major effector system for insulin [Fedn Proc.35, 1694 (1976); Archs Biochem. Biophys.184, 69 (1977); Biochem. Pharmac.27, 2589 (1978); Fedn Proc.37, 1689 (1978); and Biochem. Pharmac.29, 1239 (1980)]. The possible involvement of this mechanism in the action of structurally similar polypeptides having some insulin-like metabolic effects was investigated. The β-subunit of nerve growth factor (2.5 S NGF, mol. wt 13,500) which has a striking structural homology with proinsulin and has been reported to exert certain insulin-like metabolic effects in its own target tissues (e.g. growing neurites and sympathetic ganglia), and the insulin-derived polypeptides, desalanine-insulin and desoctapeptide-insulin, as well as proinsulin, were examined for their effects on rat adipocytes, employing the technique of formate oxidation. Both NGF and proinsulin caused increased [¹⁴C]formate oxidation, showing similar intrinsic activities, up to a maximum of 140–160% of the basal rate; insulin increased the rate to 190–210% of the basal rate. The relative potencies of the hormones toward H₂O₂ formation and stimulation of the pentose phosphate pathway activity were: insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?11}}\text{M}\text{)}$, desalanine-insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?10}}\text{M}\text{)}$ , proinsulin $\text{(}\text{EC}{}_{50}\text{: 8 × 10}{}^{\mathrm{?9}}\text{M}\text{)}$, and NGF $\text{(}\text{EC}{}_{50}\text{: 10}{}^{\mathrm{?9}}\text{M}\text{)}$. The biologically inactive derivative, desoctapeptide-insulin, did not stimulate glucose oxidation, although it caused a small increase in formate oxidation, with an $\text{EC}{}_{50}\text{of}\text{5 × 10}{}^{\mathrm{?7}}\text{M}$, indicating a suboptimal level of H₂O₂ formation in the elevation of the hexose monophosphate shunt activity. 3-Amino-1,2,4,-triazole (50 mM), which irreversibly decomposes the peroxidatic compound II of the catalase: H₂O₂ complex, inhibited formate oxidation to a greater extent in the hormone-treated cells than in the control cells, whereas sodium azide, an inhibitor of the hemoprotein, catalase, completely inhibited it. The abilities of the polypeptides to stimulate H₂O₂ formation correlated with their abilities to promote lipogenesis from [U-¹⁴C]-D-glucose, as expected of insulin. The cellular GSH/GSSG ratio increased concomitantly with the stimulation of glucose oxidation via the shunt, indicating a tight coupling between these processes. The results confirm that the hydrogen peroxide production is a common basis of the metabolic actions of growth-promoting polypeptide hormones or mitogens beyond their respective receptors.     

Bradykinin receptor-like binding studied with iodinated analogues

The biological potencies of iodinated derivatives of Tyr¹ kallidin, Tyr⁵ bradykinin, and Tyr⁸ bradykinin were determined on isolated rat uteri and bovine uterine strips. Monoiodotyr¹ kallidin was the most potent of the derivatives (0.9 times as active as bradykinin on rat uterus). Monoiodotyr⁸ bradykinin was 0.2 and iodotyr⁵ bradykinin was less than 0.001 times as active as bradykinin. Using mono-[¹²⁵I]-Tyr¹ kallidin as a receptor probe, we demonstrated receptor-like binding in a subcellular fraction from bovine myometrium. Binding of bradykinin showed an apparent K_assoc. of 10¹⁰ M^?1, and was highly specific. The optimum pH for saturable binding of [¹²⁵I]-Tyr¹ kallidin to the homogenized myometrium was 6.0 to 6.5. Several cations inhibited binding, with calcium the most effective ($\text{id}{}_{50}\text{= 20}\text{mM}$), and potassium the least effective ($\text{id}{}_{50}\text{= 220}\text{mM}$). The problems encountered in using radioactive bradykinins to search for and study receptors in bradykinin target tissues are discussed.     

Antiviral activity of 5-ethyl pyrimidine deoxynucleosides.

The antiviral activity of the α- and β-anomers of 5-ethyl-2′-deoxyuridine (EtUdR) and 5-ethyl-2′-deoxycytidine (EtCdR) has been assessed in primary rabbit kidney (PRK) cell cultures. Cytosine arabinoside (ara-C) and 5-iodo-2′-deoxyuridine (IUdR) were included as reference materials. When inhibition of vaccinia virus multiplication was measured the following order of (decreasing) activity was established: ara-C > IUdR > (β-)EtUdR > (β-)EtCdR. The α-anomers of EtUdR and EtCdR were completely inactive.In marked contrast with ara-C and IUdR which were found to inhibit PRK cell growth and thymidine incorporation into cell DNA at relatively low doses (0.08–0.4 μg/ml and 8–40 μg/ml respectively), EtUdR did not inhibit cell growth or thymidine incorporation into DNA unless very high doses were used (200–500 μg/ml). At 0.4–200 μg/ml EtUdR had a stimulatory effect on the subsequent incorporation of thymidine and deoxycytidine into DNA, most probably due to a starvation of the cells for these precursors during their contact period with EtUdR. EtCdR did not markedly alter thymidine incorporation into DNA.Antiviral indices were calculated for EtUdR, IUdR and ara-C. These were defined as the ratios of the minimum toxic doses (inhibiting cell growth or thymidine incorporation by 50 per cent) to the minimum effective doses (inhibiting vaccinia virus induced cytopathogenicity by 50 per cent). The antiviral indices of EtUdR and IUdR were almost identical but considerably greater than that of ara-C.     

Effect of diphenylhydantoin on the uptake and catabolism of l-[3H]norepinephrine in vitro in rat cerebral cortex tissue

The effect of diphenylhydantoin on the accumulation of [³H]norepinephrine in vitro was examined in brain slices prepared from rat cerebral cortex. High concentrations of diphenylhydantoin (10^?3 M) caused a significant reduction in the 5-min accumulation of [³H]norepinephrine. On the other hand, 10^?5–10^?4 M diphenylhydantoin facilitated the 20-min accumulation of [³H]norepinephrine. This facilitative action of diphenylhydantoin was (1) associated with a reduction in oxidative catabolism of [³H]norepinephrine and (2) abolished by the 2-hr pretreatment of rats with 100 mg/kg of nialamide (i.p.). The inhibitory action of diphenylhydantoin on the oxidative catabolism of [³H]norepinephrine was observed in both whole and lyzed crude synaptosomal preparations. When diphenylhydantoin and pargyline were compared, it was found that pargyline $\text{(}\text{id}{}_{50}\text{= 1.5 × 10}{}^{\mathrm{?6}}\text{M}\text{)}$ was 37 times more effective than diphenylhydantoin $\text{(}\text{id}{}_{50}\text{= 5.5 × 10}{}^{\mathrm{?5}}\text{M}\text{)}$ in inhibiting the oxidative deamination of [³H]norepinephrine. These results suggest that diphenylhydantoin alters norepinephrine metabolism in cerebral cortex slices by an inhibitory action on (1) monoamine oxidase activity and (2) the neuronal uptake system.     

Nucleosides and Nucleotides,Part 144 Synthesis and Antiviral Activity of 5-Substituted (2′S)-2′-Deoxy-2′-C-Methylcytidines and -uridines[1]

Synthesis of several 5-substituted (2′S)-2′-deoxy-2′-C-methylcytidines ( 8 ) and -uridines ( 6, 11 ) has been accomplished using radical deoxygenation of the 2′-tert-alcohols via their methyl oxalyl esters as a key reaction. Anti-herpes simplex virus type-1 and -2, and anti-varicella-zoster virus activities of the newly synthesized nucleosides were evaluated. Among them, the 5-iodouracil derivative 6e showed the most potent activity against herpes simplex virus type-1, with an EC₅₀ of 0.14 μg/mL without showing cytotoxicity up to 100 μg/mL, but had a weak activity against herpes simplex virus type-2 and no activity against varicella-zoster virus up to 50 μg/mL in vitro. Although the 5-fluorocytosine derivative 8b had a potent anti-herpes simplex virus type-1 activity (EC₅₀ = 0.22 μg/mL), it was rather cytotoxic to the CCRF-HSB-2 human T-cell line (IC₅₀ ≥ 1.0 μg/mL).     

A fluorescent analogue of methotrexate as a probe for folate antagonist molecular receptors

A dansyl-l-lysine analogue of methotrexate, $\text{N}{}^{\mathrm{\alpha }}\text{-(}\text{4-amino-4-deoxy-10-methylpteroyl}\text{)-N}{}^{\mathrm{\epsilon }}\text{-(5-[N,N-}\text{dimethylamino]-1-naphthalenesulfonyl]-1-naphthalenesulfonyl)-}\text{l}\text{-lysine}$, is a potent inhibitor of murine L1210 dihydrofolate reductase. The dansyl fluorescence emission was enhanced approximately 3-fold with a 10 nm blue shift upon binding to L1210 dihydrofolate reductase. The fluorescent analogue was only 10-fold less potent than methotrexate in inhibiting the growth of methotrexate-sensitive and -resistant L1210 cells and competes effectively for [³H]methotrexate transport with a K_i of 7.02 μM, a value virtually identical to the K_t for methotrexate in both cell lines. In addition, strong dansyl fluorescence was found to be associated with dihydrofolate reductase from methotrexate-resistant, dihydrofolate reductase-overproducing L1210 cells following incubation of viable cells with the fluorescent methotrexate analogue for 4 hr. The results demonstrate that the dansyl-l-lysine analogue of methotrexate was rapidly transported into L1210 cells where it formed a high-affinity, fluorescent complex with intracellular dihydrofolate reductase.     

Determination of extraction constant,true partition coefficient and formation constant of ion-pair complexes of quaternary ammonium salts,tetrabutylammonium bromide and isopropamide iodide.with some organic anions by a solvent extraction technique

A new method of determining the extraction constant (K_e, the true partition coefficient (TPC) and the formation constant (K_f) of ion-pairs, was developed by the solvent extraction technique. K_e and TPC were estimated from the reciprocals of the intercept and the slope of the regression line obtained by plotting

$\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{APC ? dA}\text{vs}\text{B}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{dA}\text{APC ? dA}\text{+}\text{A}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{+}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}$

in the following equation.

$\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{APC ? dA}\text{=}\text{1}\text{K}{}_{\mathrm{e}}\text{+}\text{B}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{dA}\text{APC ? dA}\text{+}\text{A}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{+}\text{B}{}^{\mathrm{T}}{}_{\mathrm{W}}\text{x}\text{1}\text{TPC}$

where [A^T_W] and [B^T_W] are the total concentrations of the cationic compound A and that of the anionic compound B in the aqueous phase respectively, APC is the apparent partition coefficient of A, dA is the partition coefficient of cation A⁺. K_f, which is expressed by K_e/TPC, was then calculated. These constants were determined for the ion-pair extraction of tetrabutylammonium bromide and isopropamide iodide with 4 organic anions, i.e. benzoic acid, p-toluenesulfonic acid, salicylic acid and taurodeoxycholic acid. This new method might be applicable to other ion-pairs without further assumptions except that the molar ratio of the ion-pair formation be 1 : 1.     

Isolation of a soluble cadmium-binding protein from pulmonary macrophages

A soluble cadmium-binding protein, with properties similar to metallothionein, has been isolated from rabbit alveolar macrophages. The macrophages were cultured in Medium 199 with Earle's salts for 24 hr in the presence of 10 μm CdCl₂ and carrier-free ¹⁰⁹Cd as a tracer. The isolation procedure began with application of a 100,000 g cell supernatant to a column of Sephadex G-75 Fine. The fraction containing the greatest amount of cadmium was eluted at a relative elution volume, $\text{V}{}_{\mathrm{<mtext>e</mtext>}}\text{V}{}_{\mathrm{<mtext>o</mtext>}}$, of 1.87. A molecular weight determination performed following Sephadex chromatography indicated that the apparent molecular weight of the impure protein was approximately 11,000. The fractions containing cadmium were pooled and purification procedures were applied, including acetone fractionation, DEAE-cellulose chromatography, and polyacrylamide gel electrophoresis. DEAE-Cellulose chromatography following acetone fractionation indicated the presence of two forms of metalloprotein as has been demonstrated previously in the isolation of cadmium-thionein from liver and kidney. The two forms of metalloprotein were subjected to polyacrylamide gel electrophoresis and, although separation was incomplete, bands obtained corresponded to those typically observed in rat liver.