Potentiation of the reductase activity of protein disulphide isomerase (PDI) by 19-nortestosterone, bacitracin, fluoxetine, and ammonium sulphate

Protein disulphide isomerase (PDI) in the endoplasmic reticulum catalyzes the rearrangement of disulphide bridges during folding of secreted proteins. It binds various molecules that inhibit its activity. But here, we looked for molecules that would potentiate its activity. PDI reductase activity was measured in vitro using di-eosin-oxidized glutathione as substrate. Its classical inhibitor bacitracin was found to exert a biphasic effect: stimulatory at low concentrations (～10(-6) M) and inhibitory only at higher concentrations (～10(-4)-10(-3) M). The weak oestrogenic molecule bisphenol A was found to exert a weak inhibitory effect on PDI reductase activity relative to the strong oestrogens, ethynylestradiol, and diethylstilbestrol. Like 19-nortestosterone, fluoxetine was found to exert a potentiating effect on PDI reductase activity and their potentiating effects could be reversed by increasing concentrations of oestrogens. In conclusion, this paper provides the first identification of potentiators of PDI activity that are potential pharmaceuticals against pathologies affecting protein folding such as Alzheimer's disease.     

Relation between the structure of alliin analogues and their inhibitory effect on platelet aggregation

Alliin analogues have been synthesized and tested for their inhibitory activity on platelet aggregation. It is found that only allicin, the S-oxodiallyl disulphide, has a strong inhibitory effect, comparable to that of alliin, while all the other tested compounds do not show any inhibitory effect even at concentrations of 10^?3 M.     

Blocking action of adenosine and ATP on synaptic transmission in isolated rat brain slices

The effect of ATP and adenosine on spontaneous activity and orthodromic responses of single neurons and on global evoked potentials was investigated in surviving slices of rat neocortex, hippocampus, dentate fascia, and cerebellumin vitro. ATP and adenosine, added to the incubation medium, had a twofold action on neurons: excitatory and inhibitory. Excitation was observed only if high concentrations of the substances (10^?2, less frequently 10^?3 M) were used, and in the case of adenosine it was very weak. The excitatory effect is evidently due to the direct depolarizing action of these substances on the cell membrane. The inhibitory action of both ATP and adenosine was manifested even in low concentrations (10^?6–10^?7 M) and was expressed as inhibition of postsynaptic responses of neurons at the presynaptic level and of their spontaneous activity. Hippocampal neurons were most sensitive to these substances, cerebellar neurons least. Apamine was found to have no effect on the inhibitory action of ATP. The results do not support the view that ATP and adenosine may be classed as CNS neurotransmitters. The possible role of these drugs as neuromodulators of synaptic transmission in the CNS is discussed.     

Juglone prevents human platelet aggregation through inhibiting Akt and protein disulfide isomerase

Background/PurposeJuglone, a natural compound widely found in Juglandaceae plants, has been suggested as a potential drug candidate for treating cancer, inflammation, and diabetic vascular complications. In the present study, the antiplatelet effect and underlying mechanisms of juglone were investigated for the first time.Study design/methodsHuman platelet aggregation and activation were measured by turbidimetric aggregometry, flow cytometry, and Western blotting. In vitro antithrombotic activity of juglone was assessed using collagen-coated flow chambers under whole-blood flow conditions. The effect of juglone on protein disulfide isomerase (PDI) activity was determined by the dieosin glutathione disulfide assay.ResultsJuglone (1 – 5 μM) inhibited platelet aggregation and glycoprotein (GP) IIb/IIIa activation caused by various agonists. In a whole blood flow chamber system, juglone reduced thrombus formation on collagen-coated surfaces under arterial shear rates. Juglone abolished intracellular Ca²⁺ elevation and protein kinase C activation caused by collagen, but had no significant effect on that induced by G protein-coupled receptor agonists. In contrast, Akt activation caused by various agonists were inhibited in juglone-treated platelets. Additionally, juglone showed inhibitory effects on both recombinant human PDI and platelet surface PDI at concentrations similar to those needed to prevent platelet aggregation.ConclusionJuglone exhibits potent in vitro antiplatelet and antithrombotic effects that are associated with inhibition of Akt activation and platelet surface PDI activity.     

Purification and properties of a peptic heme peptide from cytochrome c1.

Highly purified mouse liver plasma membranes have been used to define the properties of an NADH dehydrogenase activity associated with plasma membrane. The NADH indophenol reductase activity is two-fold stimulated at 5 × 10^?8 M glucagon and the stimulation is inhibited by atebrin. Corresponding activity in endoplasmic reticulum is not stimulated by glucagon. The NADH indophenol reductase is 90% inhibited by insulin at 7 × 10^?11M and shows a return to the original activity at higher insulin concentrations. NADH dehydrogenase activity in endoplasmic reticulum is inhibited up to 50% by insulin at a similar concentration. Triiodothyronine at 10^?7M also inhibits the plasma membrane dehydrogenase whereas thyroxine has little effect. The response of this dehydrogenase to hormones suggests a role in regulation of cellular function.     

Exopeptidase activity in eggs of Trichostrongylus colubriformis (Nematoda)

Summary

A supernatant from eggs of the ruminant nematode Trichostrongylus colubriformis contained an enzyme that was similar to leucine aminopeptidase (LAP), based on hydrolysis of the substrate L-leucine β-naphthylamide to β-naphthylamine. A Michaelis-Menten constant (K _m) of 0.155 mM was obtained. Rate of hydrolysis of 16 substrates revealed that L-phenylalanine and L-tyrosine β-naphthylamides were hydrolyzed most readily while seven additional substrates were hydrolyzed at lesser rates. The optimum pH for enzymatic activity was 6.75–7.5. Enzymatic activity was lost by heating the egg supernatant to 60°C for 5 min or freezing at 0°C for 28 days. Addition of millimolar concentrations of the chlorides of zinc, manganese and magnesium to the egg supernatant had no stimulatory effect on enzyme activity while 10 and 100 mM concentrations significantly reduced activity. Ethylenediamine tetraacetic acid at 10^?4 M had no effect on enzymatic activity. Activity was inhibited by 10^?4 M 1,10-phenanthroline, but the inhibition was reversed by zinc chloride at 10^?3 M. Di-isopropylphosphofluoridate at 10^?3 M reduced enzymatic activity moderately. Enzyme activity in egg supernatant increased 2.2-fold from 21 days to 60–90 days of a primary infection in the host while a 3.3-fold increase was found in primary versus secondary infections.     

Synthesis of N-substituted indole-2-carboxamides and investigation of their biochemical responses against free radicals

The antioxidant role of novel N-substituted indole-2-carboxamides (I2CDs) was investigated for their inhibitory effects on superoxide anion (O₂^? ) and lipid peroxidation (LP). Among the synthesized I2CDs, 3, 4, 6, 8 and 9 significantly inhibited O₂^{· ?} with an inhibition range at 70–98%. Examination of substituent effects on activity showed that both the ortho- and para-positions of the benzamide residue needs to be dichlorinated in order to get a maximum inhibitory effect on superoxide anion. In general, halogenated derivatives were found more active then the non-halogenated ones. However, none of the I2CDs had a significant inhibitory effects on the level of lipid peroxidation; only compounds 7 and 10 moderately decreased LP levels by over 50% at 10^{? 3} M concentrations.     

The Effect of Exogenous IAA and Kinetin on Nitrate Reductase,Nitrite Reductase and Glutamate Dehydrogenase Activities in Excised Pea Roots

Nitrate reductase (NO₃R) activity, nitrite reductase (NO₂R) activity and NADH₂ dependent glutamate dehydrogenase (GDH) activity were followed in extracts from excised pea roots incubated under aseptic conditions for 9 and 24 h in nitrate containing nutrient medium to which IAA was added in concentrations promoting lateral root formation (1 × 10^?5; 3 × 10^?5; 5 × 10^?5 M) and kinetin in concentrations which reduce lateral root formation (0.1; 1; 5 mg 1^?1, that is 4.65 × 10^?7;4.65 × 10^?6 and 2.3 × 10^?5 M). NO₃R activity was not influenced by IAA, NO₂R activity was slightly depressed by IAA after 24 h incubation and GDH activity was slightly increased after 24 h incubation in the presence of IAA. Kinetin decreased NO₃R activity significantly both after 9 h and 24 h incubation, slightly increased NO₂R activity after 9 h incubation but slightly decreased it after 24 h incubation, and did not affect GDH activity after 24 h incubation. However, when applied together with IAA, kinetin abolished the promoting effect of IAA on GDH activity. IAA neither reversed nor accentuated the effect of kinetin on NO₂R activity. Nevertheless the depressing effect of kinetin on NO₃R activity was emphasized by the presence of IAA after 9 h incubation. The results obtained indicate that reduced nitrate assimilation due to the depression of nitrate reductase activity caused by kinetin probably contributes to the negative growth effect of kinetin in pea root segments grown in nitrate medium.     

Kinetics of Inhibition of Horse Liver Alcohol Dehydrogenase byp-Methylbenzyl Hydroperoxide

Abstract

The complex kinetic behaviour of p-methylbenzyl hydroperoxide in its inhibitory action on horse liver alcohol dehydrogenase was examined. The kinetic patterns are markedly different at very low (<10^?8 M) and high (> 10^?7 M) hydroperoxide concentrations. In both cases very low inhibition constants (4nM and 14nM, respectively) were found. A possihle mechanistic model based on these results is discussed.     

Inhibition of sterol biosynthesis by 14α-hydroxymethyl sterols

14α-Hydroxymethyl-5α-cholest-7-en-3β-ol (I) and 14α-hydroxymethyl-5α-cholest-6-en-3β-ol (II) have been prepared by chemical synthesis from 3β-acetoxy-7α,32-epoxy-14α-methyl-5α-cholestane. Compound I, previously shown to be efficiently convertible to cholesterol upon incubation with rat liver homogenate preparations, has been found to be a potent inhibitor of sterol synthesis in animal cells in culture. Compound I caused a 50% reduction of the levels of HMG-CoA reductase activity in cultures of L cells and fetal liver cells at concentrations of 3 × 10^?6 M and 8 × 10^?6 M, respectively. Compound II, the Δ⁶-analogue of I, caused a 50% suppression of the enzyme activity in the two cell types at even lower concentrations, 5 × 10^?7 M and 2 × 10^?6 M, respectively. Concentrations of I and II required to specifically inhibit sterol synthesis from acetate were similar to those required to suppress the levels of HMG-CoA reductase activity.     

Spontaneous bioelectric activity of cultured purkinje cells during exposure to glutamate,glycine, and strychnine

The addition of glutamate to the bathing medium increased the average firing rate of cerebellar rat Purkinje cells in vitro. At concentrations lower than 10^?6 M, there was no deviation from controls in the firing pattern or rate that was detectable. At 10^?3 M glutamate, the amplitude of the action potentials was gradually decreased until all activity was abolished. The action of glutamate was rapid in onset and reversible. Glycine produced sustained depression of firing at concentrations higher than 10^?3 M. This inhibition was strychnine-insensitive and considered nonspecific. Strychnine, on the other hand, exerted an excitatory influence on Purkinje cells when applied at low concentrations (10^?8 to 10^?6 M). The firing became more irregular and complex discharges appeared. Higher concentrations of strychnine (>10^?5 M) inhibited the spontaneous activity. The effect of strychnine was partly reversible. The data suggest that low concentrations of strychnine lower the threshold for inputs at excitatory as well as inhibitory synapses.     

Interaction of Growth Retardants and Temperature in Growth, Flowering, Regeneration, and Auxin Activity of Begonia × cheimantha Everett

Soil application of CCC reduced stem and leaf growth in Begonia plants. This effect was evident with all concentrations tested at 18°C, whereas at 21 and 24°C no growth–retarding effect was observed with 2 × 10^?2 M CCC, and with 5 × 10^?3 M growth was even stimulated. Flowering was promoted by CCC in long day and neur–critical temperature, particularly under low light intensity in the winter. The formation of adventitious buds in leaves of plants grown at 21 and 24°C was stimulated when the plants received 5 × 10^?2 and 2 × 10^?2 M CCC, while 8 7times; 10^?2 M was inhibitory. In plants grown at 18°C bud formation was inhibited by all CCC concentrations. Root formation in the the leaves was usually stimulated by high CCC concentrations, while root elongation was reduced. The level of ether–extractable. acidic auxin (presumably IAA) in the leaves was lowered by CCC treatment of the plants, hut this required higher CCC concentrations at higt than at low temperature. When applied to detached leaves CCC stimulated bud formation at concentrations ranging from 10^?4 to 10^?2 M in leaves planted at 18 and 21°C. At 24°C budding was inhibited by 10^?2 M CCC, the lower concentrations being stimulatory also at this temperature. Root formation and growth were not much affected by CCC treatment of the leaves, but increased with the temperature. Soil application of Phosfon (4 × 10^?4 M) had no effect on growth and flowering, nov did it affect the subsequent regeneration of buds and roots in the leaves. In detached leaves Phosfon stimulated bud formation with au optimum at 10^?6 M. Root formation was stimulated by Phosfon at all temperatures, the optimal concentration being 10^?5 M, whereas root length was conversely affected. Foliar application of B-995 to intact plants and treatment of detached leaves greatly inhibited the formation of buds and had little effect on root formation. B-99D reduced the growth and delayed flowering in the plants.     

Estrone and progesterone inhibit the growth of murine MC38 colon cancer line

The unsatisfactory effectiveness of reference chemotherapy in colon cancer (fluorouracil – FU) results in continuous search for agents, which could enhance the action of FU. Some epidemiological data such as a decreased risk of colorectal cancer among menopausal women receiving hormonal replacement therapy indicate the role of female sex hormones in the pathogenesis of this disease.The aim of this study was to examine the direct effects of various concentrations of estrone and progesterone (10^?4 to 10^?12 M) applied alone or together with FU on the growth of murine MC38 colon cancer in vitro.Estrone inhibited MC38 cancer growth in a wide range of concentrations (10^?12 to 10^?4 M) with similar potency and at some concentrations (10^?6 and 10^?4 M) augmented also the cytotoxic action of FU. Progesterone induced MC38 cancer growth inhibition at high concentrations (10^?5 to 10^?4 M) in dose- and time-dependent manner but it did not intensify antineoplastic effect of FU. A weak inhibitory effect of progesterone was also observed for lower concentrations (10^?5 to 10^?10 M) in long lasting cultures (72 h).The results indicate that estrone and progesterone inhibit the MC38 cancer growth and that estrone increases also the cytotoxic effect of FU, what confirms the role of female sex steroids in modulation of colon cancer growth.     

Presynaptic Control of the Synthesis and Release of Dopamine from Striatal Synaptosomes: A Comparison Between the Effects of 5-Hydroxytryptamine, Acetylcholine, and Glutamate

The effects of 5-HT and glutamate on dopamine synthesis and release by striatal synaptosomes were investigated and compared with the action of acetylcholine, which acts presynaptically on this system. 5-HT inhibited (28%) synthesis of [¹⁴C]dopamine from L-[U-¹⁴C]tyrosine, at 10-⁵M and above. This contrasts with the action of acetylcholine, which stimulated [¹⁴C]-dopamine synthesis by 24% at 10-⁴ M. Tissue levels of GABA were unaffected by either 5-HT or acetylcholine up to concentrations of 10-⁴ M. The inhibitory action of 5-HT (5 × 10^?5 M and 2 × 10^?4 M) on [¹⁹C]dopamine synthesis was completely abolished by methysergide (2 × 10^?6 M). Higher concentrations of methysergide (10^?4 M) or cyproheptadine (10^?5 M) inhibited [¹⁴C]dopamine synthesis by 28% and 25%, respectively, when added alone to synaptosomes. However, only methysergide prevented the further inhibition of synthesis caused by 5-HT. At concentrations of 2 × 10^?5 M and above, 5-HT stimulated [¹⁴C]dopamine release. This releasing action differed from that of acetylcholine, which occurred at lower concentrations (e.g., 10^?6 M). Methysergide (up to 10^?4 M) or cyproheptadine (2 × 10^?4 M) did not reduce the 5-HT (5 × 10^?5 M)-induced release of [¹⁴C]dopamine, but methysergide (10^?4 M) showed a potentiation (49%) of this increased release. The stimulatory effects of 5-HT (2 × 10^?5 M) and K⁺ (56 mM) on [¹⁴C]dopamine release were additive, indicating that two separate mechanisms were involved. However, when both agents were present the stimulatory effect of K⁺ (56 mM) on [¹⁴C]dopamine synthesis was not seen above the inhibitory effect of 5-HT. Glutamate (0.1-5 mM) did not affect [⁴C]dopamine release or its synthesis from L-[U-¹⁴C]tyrosine. It is concluded that 5-HT modulates the synthesis of dopamine in striatal nerve terminals through a presynaptic receptor mechanism, an action antagonised by methysergide. The releasing action of 5-HT apparently occurs through a separate mechanism which is also distinct from that involved in the response to K⁺ depolarisation.     

Stimulation of the activity of prolyl hydroxylase in 3T3 fibroblasts by 1,10-phenanthroline

In confluent cultures of 3T3 fibroblasts, incubated for 24 h with 1,10-phenanthroline at 10^?5–10^?9 M, the activity of prolyl hydroxylase was significantly increased. 1,10-Phenanthroline was inhibitory at concentrations greater than 10^?4 M. The stimulatory effect of 1,10-phenanthroline manifets itself after 6 h inhubation and increased with time up to 48 h. 2,2′-dipyridyl and 5,6-dimethyl-1-1,10-phemamthroline were also stimulatory; a nonchelating analog, 1,7-phenanthroline had no effect.Cycloheximide did not modify the 1,10-phenanthroline effect. The stimulatory effect does not seem to depend on the shift of an inactive precursor of prolyl hydroxylase to an active form because 1,10-phenanthroline was shown to be ineffective in logarithmically growing cells.While dialysis of washed and homogenized cells significantly increased prolyl hydroxylase activity in cell extracts, undialyzed 1,10-phenanthroline treated samples exhibited higher prolyl hydroxylase activity than dialyxed controls.These data suggested to us that 1,10-phenanthroline and other chelating agents may be forming complexes with certain metal ions or protein-metal ions which are inhibitory towards prolyl hydroxylase.     

Antioxidative iridoid glycosides from the sky flower (Duranta repens Linn)

Phytochemical investigations were performed on the EtOAc-soluble fraction of the whole plant of the sky flower (Duranta repens) which led to the isolation of the iridoid glycosides 1–6. Their structures were elucidated by both 1D and 2D NMR spectroscopic analysis. All the compounds showed potent antioxidative scavenging activity in four different tests, with half maximal inhibitory concentration (IC₅₀) values in the range 0.481–0.719?mM against DPPH radicals, 4.07–17.21 µM for the hydroxyl radical (^?OH) inhibitory activity test, 43.3–97.37 µM in the total reactive oxygen species (ROS) inhibitory activity test, and 3.39–18.94 µM in the peroxynitrite (ONOO^?) scavenging activity test. Duranterectoside A (1) displayed the strongest scavenging potential with IC₅₀ values of (0.481?±?0.06?mM, 4.07?±?0.03, 43.30?±?0.05, 3.39?±?0.02?µM) for the DPPH radicals, ^?OH inhibitory activity test, total ROS inhibitory activity test and the ONOO^? scavenging activity test, respectively.     

Pentobarbital sodium inhibits calcium uptake in vascular smooth muscle

This report demonstrates that the commonly used anesthetic agent, pentobarbital sodium, in concentrations of 1 · 10^?4 to 2 · 10^?3 M inhibits calcium (Ca²⁺) uptake in both rat aortic and portal venous smooth muscle. The data indicate that total exchangeable Ca²⁺ in portal vein is reduced by about 15% in 1 · 10^?4 M pentobarbital sodium, while the intracellular exchangeable Ca²⁺ is reduced by 24%. On the other hand, in aortic smooth muscle, while 5–20 · 10^?4 M pentobarbital sodium reduces total exchangeable Ca²⁺ by about 15%, intracellular Ca²⁺ is reduced by 22% in 5 · 10^?4 M pentobarbital sodium and by 38% in 2 · 10^?3 M pentobarbital sodium. The present studies thus reveal that concentrations of pentobarbital sodium known to be present during induction of surgical anesthesia can exert significant inhibitory effects on exchangeability and transmembrane movement of Ca²⁺ in at least two different types of blood vessels.     

Synthesis and biological evaluation of some new N4-substituted isatin-3-thiosemicarbazones

A new series of 12 N⁴-substituted isatin-3-thiosemicarbazones 2a-l has been synthesized, characterized and screened for in vitro cytotoxic, phytotoxic and urease inhibitory effects. All the compounds proved to be active in the brine shrimp bioassay; 2a, 2b, 2d, 2f and 2h-l exhibited a high degree of cytotoxic activity (LD₅₀ = 1.10 × 10^{? 5} M–3.10 × 10^{? 5} M). In urease-inhibition assay, compounds 2a, 2b, 2e, 2f, 2h-j and 2l proved to be potent inhibitors displaying relatively much greater inhibition of the enzyme with IC₅₀ values ranging from 20.6 μM to 50.6 μM. Amongst these, 2a and 2f were found to be the most potent ones exhibiting pronounced inhibition with IC₅₀ value 20.6 μM. All the synthetic compounds showed weak to moderate (10–40%) phytotoxicity at the highest tested concentration (500 μg/mL) indicating their usefulness as inhibitors of soil ureases.