Zn(II) and Cd(II) thiosemicarbazones for stimulation/inhibition of kojic acid biosynthesis from Aspergillus flavus and the fungal defense behavior against the metal complexes' excesses.

The complexes {[ZnL1Cl] C1, [ZnL2Cl].0.5H2O C2, [CdL1Cl] C3, and [CdL2Cl] C4} were prepared from tridentate thiosemicarbazones {HL1 = 4-(3-nitrophenyl)-1-((pyridin-2-yl)methylene) thiosemicarbazide and HL2 = 4-(2,4-dimethoxyphenyl)-1-((pyridin-2-yl)methylene)thiosemicarbazide} and identified by elemental CHNS, spectroscopic {IR and UV-Vis.}, thermal and DMF solution electrical conductivity data. On another hand, kojic acid (KA) which represents important secondary metabolite with numerous hot spot applications was successfully biosynthesized from Aspergillus flavus and structurally analyzed by single crystal analysis. The Zn(II) complexes C1&C2 (0.3 mM) enhanced the KA biosynthesis by 70.87% and 42.26%, while 76.09% of C1 and 72.78% of C2 were absorbed by the fungal cells. The Cd(II) complexes C3&C4 at 0.3 mM inhibited KA production by 87.95% and 97.03% with Cd(II) consumption reaching to 40.09% & 37.3%, while 0.4 mM of C3&C4 resulted in 100% inhibition of kojic acid biosynthesis. Light microscopic analysis showed the fungal structural abnormalities and the cell antioxidant behavior was detected. These complexes could be highly applicable as new stimulators and inhibitors of kojic acid production.

A new protein tyrosine phosphatase 1B inhibitory α-pyrone-type polyketide from Okinawan plant-associated Aspergillus sp. TMPU1623.

A new polyenyl-α-pyrone polyketide, aspopyrone A (1), was isolated from a culture broth of Okinawan plant-associated Aspergillus sp. TMPU1623 by solvent extraction, ODS column chromatography, and preparative HPLC (ODS). The structure of 1 was assigned based on NMR experiments. Compound 1 exhibited protein tyrosine phosphatase (PTP) 1B and T-cell PTP (TCPTP) inhibitory activities with IC50 values of 6.7 and 6.0 µM, respectively.

Characterization of three small molecule inhibitors of enterovirus 71 identified from screening of a library of natural products.

Enterovirus 71 (EV-A71) is a major cause of hand, foot, and mouth disease (HFMD). Infection with EV-A71 is more often associated with neurological complications in children and is responsible for the majority of fatalities, but currently there is no approved antiviral therapy for treatment. Here, we identified auraptene, formononetin, and yangonin as effective inhibitors of EV-A71 infection in the low-micromolar range from screening of a natural product library. Among them, formononetin and yangonin selectively inhibited EV-A71 while auraptene could inhibit viruses within the enterovirus species A. Time of addition studies showed that all the three inhibitors inhibit both attachment and postattachment step of entry. We found mutations conferring the resistance to these inhibitors in the VP1 and VP4 capsid proteins and confirmed the target residues using a reverse genetic approach. Interestingly, auraptene- and formononetin-resistant viruses exhibit cross-resistance to other inhibitors while yangonin-resistant virus still remains susceptible to auraptene and formononetin. Moreover, auraptene and formononetin, but not yangonin protected EV-A71 against thermal inactivation, indicating a direct stabilizing effect of both compounds on virion capsid conformation. Finally, neither biochanin A (an analog of formononetin) nor DL-Kavain (an analog of yangonin) exhibited anti-EV-A71 activity, suggesting the structural elements required for anti-EV-A71 activity. Taken together, these compounds could become potential lead compounds for anti-EV-A71 drug development and also serve as tool compounds for studying virus entry.

(+)-Altholactone exhibits broad spectrum immune modulating activity by inhibiting the activation of pro-inflammatory cytokines in RAW 264.7 cell lines.

An evaluation of Indonesian plants to identify compounds with immune modulating activity revealed that the methanolic extract of an Alphonsea javanica Scheff specimen possessed selective anti-inflammatory activity in a nuclear factor-kappa B (NF-κB) luciferase and MTT assay using transfected macrophage immune (Raw264.7) cells. A high-throughput LC/MS-ELSD based library approach of the extract in combination with the NF-κB and MTT assays revealed the styryl lactone (+)-altholactone (2) was responsible for the activity. Compound 2, its acetylated derivate (+)-3-O-acetylaltholactone (3), and the major compound of this class, (+)-goniothalmin (1), were further evaluated to determine their anti-inflammatory potential in the NF-κB assay. Concentration-response studies of 1-3 indicated that only 2 possessed NF-κB based anti-inflammatory activity. Compound 2 reduced the LPS-induced NO production, phosphorylation of IκBα, and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) using Western blot analysis. Further studies using qPCR indicated 2 reduced the expression of eight pro-inflammatory cytokines/enzymes (0.8-5.0µM) which included: COX-2, iNOS, IP-10, IL-1ß, MCP-1, GCS-F, IL-6 and IFN-ß. These results indicated that 2 displays broad spectrum immune modulating activity by functioning as an anti-inflammatory agent against LPS-induced NF-κB signaling. Conversely the selective cytotoxicity and in vivo anti-tumor and anti-inflammatory activity previously reported for 1 do not appear to arise from a mechanism that is linked to the NF-κB immune mediated pathway.

Herkinorin dilates cerebral vessels via kappa opioid receptor and cyclic adenosine monophosphate (cAMP) in a piglet model.

Since herkinorin is the first non-opioid mu agonist derived from salvinorin A that has the ability to induce cerebral vascular dilatation, we hypothesized that herkinorin could have similar vascular dilatation effect via the mu and kappa opioid receptors and the cAMP pathway. The binding affinities of herkinorin to kappa and mu opioid receptors were determined by in-vitro competition binding assays. The cerebral arteries were monitored in piglets equipped with a closed cranial window and the artery responses were recorded before and every 30s after injection of artificial cerebrospinal fluid (CSF) in the presence or absence of the investigated drugs: herkinorion, norbinaltorphimine (NTP), a kappa opioid receptor antagonist, ß-funaltrexamine (ß-FNA), a mu opioid receptor antagonist, or Rp-8-Br-cAMPS (Rp-cAMPS), an inhibitor of protein kinase A (PKA). CSF samples were collected before and 10 min after herkinorin and NTP administration for the measurement of cAMP levels. Data were analyzed by repeated-measures analysis of variance. Our results show that herkinorin binds to both kappa and mu opioid receptors. Its vasodilation effect is totally abolished by NTP, but is not affected by ß-FNA. The levels of cAMP in the CSF elevate after herkinorin administration, but are abolished with NTP administration. The cerebral vasodilative effect of herkinorin is also blunted by Rp-cAMPS. In conclusion, as a non-opioid kappa and mu opioid receptor agonist, herkinorin exhibits cerebral vascular dilatation effect. The dilatation is mediated though the kappa opioid receptor rather than the mu opioid receptor. cAMP signaling also plays an important role in this process.

The effects of herkinorin, the first mu-selective ligand from a salvinorin A-derived scaffold, in a neuroendocrine biomarker assay in nonhuman primates.

Herkinorin is the first mu-opioid receptor-selective ligand from the salvinorin A diterpenoid scaffold. Herkinorin has relative mu > kappa > delta binding selectivity, and it can act as an agonist at both mu- and kappa-receptors, in vitro. These studies were the first in vivo evaluation of the effects of herkinorin in nonhuman primates, using prolactin release, a neuroendocrine biomarker assay that is responsive to both mu- and kappa-agonists, as well as to compounds with limited ability to cross the blood-brain barrier. In cumulative dosing studies (0.01-0.32 mg/kg i.v.), herkinorin produced only small effects in gonadally intact males (n = 4), but a more robust effect in females (n = 4). Time course studies with herkinorin (0.32 mg/kg) confirmed this greater effectiveness in females and revealed a fast onset after i.v. administration (e.g., by 5-15 min). Antagonism experiments with different doses of nalmefene (0.01 and 0.1 mg/kg) caused dose-dependent and complete prevention of the effect of herkinorin in females. This is consistent with a principal mu-agonist effect of herkinorin, with likely partial contribution by kappa-agonist effects. The peripherally selective antagonist quaternary naltrexone (1 mg/kg s.c.) caused approximately 70% reduction in the peak effect of herkinorin (0.32 mg/kg) in females, indicating that this effect of herkinorin is prominently mediated outside the blood-brain barrier.

Syntheses of hydroxy substituted 2-phenyl-naphthalenes as inhibitors of tyrosinase.

Oxyresveratrol and resveratrol, with hydroxy substituted trans-stilbene structure, exert potent inhibitory effects on cyclooxygenase, rat liver mitochondrial ATPase activity, and tyrosinase. As the isosteres of oxyresveratrol, a new family of hydroxyl substituted phenyl-naphthalenes were synthesized to show excellent inhibition of mushroom tyrosinase. Compound 10, which is isostere of resveratrol, showed IC50 value of 16.52 microM in mushroom tyrosinase activity. As compared to this, the reference compound, resveratrol, showed IC50 value of 55.61 microM. Compound 4, which is isostere of oxyresveratrol, showed IC50 value of 0.49 microM. Among the other three derivatives, compound 13 showed IC50 value of 0.034 microM.

Contribution of lipid second messengers to the regulation of phosphatidylcholine synthesis during cell cycle re-entry.

During entry into the cell cycle a phosphatidylcholine (PC) metabolic cycle is activated. We have examined the hypothesis that PC synthesis during the G(0) to G(1) transition is controlled by one or more lipid products of PC turnover acting directly on the rate-limiting enzyme in the synthesis pathway, CTP: phosphocholine cytidylyltransferase (CCT). The acceleration of PC synthesis was two- to threefold during the first hour after addition of serum to quiescent IIC9 fibroblasts. The rate increased to approximately 15-fold above the basal rate during the second hour. The production of arachidonic acid, diacylglycerol (DAG), and phosphatidic acid (PA) preceded the second, rapid phase of PC synthesis. However, an increase in the cellular content of these lipid mediators was detected only for DAG. CCT activation and translocation to membranes accompanied the second phase of the PC synthesis acceleration. Bromoenol lactone (BEL), an inhibitor of calcium-independent phospholipase A(2) and PA phosphatase, blocked production of fatty acids and DAG, inhibited both phases of the PC synthesis response to serum, and reduced CCT activity and membrane affinity. The effect of BEL on PC synthesis was partially reversed by in situ generation of DAG via exogenous PC-specific phospholipase C to generate approximately 2-fold elevation in PC-derived DAG. Exogenous arachidonic acid also partially reversed the inhibition by BEL, but only at a concentration that generated a supra-physiological cellular content of free fatty acid. 1-Butanol, which blocks PA production, had no effect on DAG generation, or on PC synthesis. We conclude that fatty acids and DAG could contribute to the initial slow phase of the PC synthesis response. DAG is the most likely lipid regulator of CCT activity and the rapid phase of PC synthesis. However, processes other than direct activation of CCT by lipid mediators likely contribute to the highly accelerated phase during entry into the cell cycle.

Opposed regulation of aluminum-induced apoptosis by glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor in rat brains.

When intracisternally injected to rat brain, aluminum induced apoptosis as assessed by DNA fragmentation and activation of caspase-3 and caspase-12. Co-administration of glial cell line-derived neurotrophic factor (GDNF) effectively prevented aluminum-induced cell death through reduced apoptosis whereas brain-derived neurotrophic factor (BDNF) accelerated aluminum-induced apoptosis, suggesting that the extent of aluminum neurotoxicity in vivo may depend on the biological activity of the neurotrophic factors.

Brain-derived neurotrophic factor protects cultured rat hippocampal neurons from aluminum maltolate neurotoxicity.

Aluminum is environmentally abundant but not an essential trace element. Although there is increasing evidence suggesting the implication of aluminum in the pathogenesis of Alzheimer's disease, it is still controversial. We found and report here that aluminum maltolate, a stable and hydrophilic aluminum complex, causes death of primary cultured rat hippocampal neurons in a time- and dose-dependent manner. Degenerated neurons were TUNEL-positive. Immunohistochemical detection of synapsin I and microtubule associated protein 2 revealed the synapse loss between neurons intoxicated by aluminum maltolate. To explore the mechanism underlying its neurotoxicity, we administered various pharmacological compounds prior to the application of aluminum maltolate, and found that brain-derived neurotrophic factor (BDNF) markedly attenuated the neurotoxicity. Furthermore, aluminum maltolate inhibited the elevation of intracellular calcium levels caused by BDNF. Our results suggest the involvement of BDNF in the molecular mechanism underlying neurotoxicity induced by aluminum maltolate.

Lithium inhibits aluminum-induced apoptosis in rabbit hippocampus, by preventing cytochrome c translocation, Bcl-2 decrease, Bax elevation and caspase-3 activation.

A variety of studies on neuronal death models suggest that lithium has neuroprotective properties. In the present investigation, we have examined the effect of chronic lithium treatment on hippocampus, as monitored by changes at the subcellular level of apoptosis-regulatory proteins which have been induced by the neurotoxin, aluminum maltolate. Intracisternal administration of aluminum into rabbit brain induces cytochrome c release, decreases levels of the anti-apoptotic proteins Bcl-2 and Bcl-X(L), increases levels of the pro-apoptotic Bax, activates caspase-3, and causes DNA fragmentation as measured by the TUNEL assay. Pretreatment for 14 days with 7 mm of lithium carbonate in drinking water prevents aluminum-induced translocation of cytochrome c, and up-regulates Bcl-2 and Bcl-X(L,) down-regulates Bax, abolishes caspase-3 activity and reduces DNA damage. The regulatory effect of lithium on the apoptosis-controlling proteins occurs in both the mitochondria and endoplasmic reticulum. We propose that the neuroprotective effect of lithium involves the modulation of apoptosis-regulatory proteins present in the subcellular organelles of rabbit brain.

Inhibition of the catecholase activity of biomimetic dinuclear copper complexes by kojic acid.

The inhibition of the catechol oxidase activity exhibited by three dinuclear copper(II) complexes, derived from different diaminotetrabenzimidazole ligands, by kojic acid [5-hydroxy-2-(hydroxymethyl)-gamma-pyrone] has been studied. The catalytic mechanism of the catecholase reaction proceeds in two steps and for both of these inhibition by kojic acid is of competitive type. The inhibitor binds strongly to the dicopper(II) complex in the first step and to the dicopper-dioxygen adduct in the second step, preventing in both cases the binding of the catechol substrate. Binding studies of kojic acid to the dinuclear copper(II) complexes and a series of mononuclear analogs, carried out spectrophotometrically and by NMR, enable us to propose that the inhibitor acts as a bridging ligand between the metal centers in the dicopper(II) catalysts.

Photoinduction of strand scissions in DNA by kojic acid: role of transition metal ions and oxygen free radical intermediates in the reaction.

Kojic acid (5-hydroxy-2-hydroxymethyl-gamma-pyrone) is a bacterial metabolic product used intensively in the food industry. In the presence of visible light and molecular oxygen it was found to cause breakage of calf thymus DNA. Such degradation was considerably enhanced in the presence of the transition metal ions Fe(III), Fe(II) and Cu(II). The cleavage of DNA in the presence of Fe(III) did not appear to have any preferred site(s) or sequence(s) for strand scission. Kojic acid catalysed the reduction of transition metal which in the case of Cu(II) was found to play an essential role in the degradation of DNA. Kojic acid also reduced oxygen to superoxide and hydroxyl radicals were formed in the presence of metal ions. The involvement of these active oxygen species in the reaction was established by the inhibition of DNA breakage by superoxide dismutase, catalase, iodide, mannitol, formate and sodium azide.

Pharmacological analysis of the central cardiovascular effects of four GABA analogues.

The central cardiovascular effects of 4 structural analogues of GABA were investigated. The drugs were injected intracerebroventricularly (i.c.v.) in cumulative doses into pentobarbital-anaesthetized normotensive rats. Muscimol (0.01-10 micrograms/kg), THIP (0.01-100 micrograms/kg), kojic amine (0.1-100 micrograms/kg) and isoguvacine (0.1-100 micrograms/kg) produced dose-dependent hypotension and bradycardia. The maximal fall in the mean blood pressure was of about 35% of the initial values. These effects appears to be of central origin since the intravenous (i.v.) injection of the same doses of the drugs did not produce any similar cardiovascular modifications. The hypotensive effects of muscimol and kojic amine were antagonized partly by i.c.v. bicuculline. The combination of bicuculline and kainic acid almost completely prevented the blood pressure lowering effects of muscimol, kojic amine and isoguvacine. THIP however was only slightly antagonized by bicuculline and kainic acid. Atropine i.v. also prevented partly the cardiovascular effects of all these drugs. Thus, the mechanisms of the central cardiovascular actions of GABA analogues appear to be more complex than expected and variable from one drug to another. The involvement of GABA receptors of the A and B types and of cholinergic mechanisms in the hypotensive effect of the drugs is discussed.