A novel neuroprotective agent with antioxidant and nitric oxide synthase inhibitory action

N(alpha)-vanillyl-N(omega)-nitroarginine (N - 1) that combines the active functions of natural antioxidant and nitric oxide synthase inhibitor was developed for its neuroprotective properties. N - 1 exhibited protective effects against hydrogen peroxide-induced cell damage and the inhibitory effect on nitric oxide 'NO' production induced by calcium ionophore in NG 108-15 cells. N - 1 inhibited the constitutive NOS isolated from rat cerebellar in a greater extent than constitutive NOS from human endothelial cells. Low binding energy (-10.2 kcal/mol) obtained from docking N - 1 to nNOS supported the additional mode of action of N - 1 as an nNOS inhibitor. The in vivo neuroprotective effect on kainic acid-induced nitric oxide production and neuronal cell death in rat brain was investigated via microdialysis. Rats were injected intra-peritonially with N - 1 at 75 micromol/kg before kainic acid injection (10 mg/kg). The significant suppression effect on kainic acid-induced NO and significant increase in surviving cells were observed in the hippocampus at 40 min after the induction.     

A broadly neuroprotective derivative of curcumin

The plant polyphenolic curcumin alters the response of nerve cells to some forms of toxic stress. The steroid-like compound, cyclohexyl bisphenol A, has broad neuroprotective properties that are very distinct from those of curcumin. To incorporate both families of biological activities into a single molecule, a pyrazole derivative of curcumin, called CNB-001, was synthesized. CNB-001 acquires a new activity and is far superior in neuroprotection assays to either parental molecule, but retains some of the properties of both. It is neuroprotective in cell culture assays for trophic factor withdrawal, oxidative stress, excitotoxicity, and glucose starvation, as well as toxicity from both intracellular and extracellular amyloid. While the creation of CNB-001 was based upon an uncommon approach to drug design, it has the potential of a lead drug candidate for treating multiple conditions involving nerve cell death.     

Subcellular localization of a fluorescent derivative of CuII(atsm) offers insight into the neuroprotective action of CuII(atsm)

Copper complexes of bis(thiosemicarbazone) (Cu(II)(btsc)s) have been studied as potential anti-cancer agents and hypoxia imaging agents. More recently, Cu(II)(btsc)s have been identified as possessing potent neuroprotective properties in cell and animal models of neurodegenerative disease. Despite their broad range of pharmacological activity little is known about how cells traffic Cu(II)(btsc)s and how this relates to potential anti-cancer or neuroprotective outcomes. One method of investigating sub-cellular localization of metal complexes is through confocal fluorescence imaging of the compounds in cells. Previously we harnessed the fluorescence of a pyrene group attached to diacetyl-bis(N4-methylthiosemicarbazonato)copper(ii)) (Cu(II)(atsm)), (Cu(II)L(1)). We demonstrated that Cu(II)L(1) was partially localized to lysosomes in HeLa cancer epithelial cells. Here we extend these studies to map the sub-cellular localization of Cu(II)L(1) in M17 neuroblastoma cells. Treatment of M17 or HeLa cells led to rapid association of the Cu-complex into distinct punctate structures that partially co-localized with lysosomes as assessed by co-localization with Lysotracker and acridine orange. No localization to early or late endosomes, the nucleus or mitochondria was observed. We also found evidence for a limited association of Cu(II)L(1) with autophagic structures, however, this did not account for the majority of the punctate localization of Cu(II)L(1). In addition, Cu(II)L(1) revealed partial localization with ER Tracker and was found to inhibit ER stress induced by tunicamycin. This is the first report to comprehensively characterize the sub-cellular localization of a Cu(II)(atsm) derivative in cells of a neuronal origin and the partial association with lysosome/autophagic structures and the ER may have a potential role in neuroprotection.     

Apoptotic action of estrogen

Estrogen as a mitogen stimulates cell proliferation and prevents cell death in many cell types. In patients, estrogen is known to stimulate breast and uterus cancer development. Ironically, high doses of estrogen can induce regression of hormone-dependent breast cancer in postmenopausal women. The comprehensive mechanism by which estrogen induces tumor recession in breast cancer is still unknown, but activation of the Fas/FasL pathways plays a key role in this process. Laboratory studies show that the apoptotic action of estrogen is the major factor leading to cell number decreases in several cell types. The effects of estrogen are estrogen-receptor dependent. In this mini review, we will focus on the latest findings regarding estrogen apoptotic effects in several cell models, including breast cancer cells, and summarize the possible mechanisms involved in these estrogen mediated processes. New potential implications for the pharmacological control of breast cancer with estrogen in post-menopausal women are also discussed.     

Mechanistic studies of cAMP-dependent protein kinase action

The details of the process by which protein kinase catalyzes phosphoryl group transfers are beginning to be understood. Early work that explored the primary specificity of cAMP-dependent protein kinase action enabled the synthesis of small peptide substrates for the enzyme. Enzyme-peptide interactions seem simpler to understand than protein-protein interactions, so peptide substrates have been used in most protein kinase studies. In most investigations the kinetics for the phosphorylation of small peptides have been interpreted as being consistent with mechanisms which do not invoke phospho-enzyme intermediates (see, for example, Bolen et al.). Protein kinase has been shown to bind two metal ions in the presence of a nucleotide. Using magnetic resonance techniques the binding of these ions has been utilized to elucidate the conformation of nucleotide and peptide substrates or inhibitors when bound in the enzymic active site. Also, two new peptides with the form Leu-Arg-Arg-Ala-Ser-Y-Gly, where Y was either Pro or (N-methyl)Leu, were synthesized and found not to be substrates, within the limits of detection, for protein kinase. The striking lack of affinity that protein kinase has for such peptides which are unlikely to form a beta 3-6 turn has not been reported before. Our results may indicate that this type of turn is a requirement for protein kinase catalyzed phosphorylation or that these peptides lack the ability to form a particular hydrogen bond with the enzyme. Magnetic resonance techniques have indicated that the distance between the phosphorous in the gamma-phosphoryl group of MgATP and the hydroxyl oxygen of serine in the peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly is 5.3 +/- 0.7 A. This, together with certain kinetic evidence, suggests that the mechanism by which protein kinase catalyzes phosphoryl group transfer has considerable dissociative character. Chemical modifications, including one using a peptide-based affinity label, have identified two residues at or near the active site, lysine-72 and cysteine 199. While neither of these groups has been shown to be catalytically essential, similar studies may help to identify groups that are directly involved in the catalytic process. Finally, a spectrophotometric assay for cAMP-dependent protein kinase has been described. Using this assay the preliminary results of an in-depth study of the pH dependence of protein kinase catalyzed phosphoryl group transfer have been obtained. This study shall aid in the identification of active site residues and should contribute to the elucidation of the enzyme's catalytic mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

The neuroprotective effects of estrogen in SK-N-SH neuroblastoma cell cultures

Estrogen has been considered to be a neuroprotectant and a neuromodulator in many neuronal cell lines and tissue preparations. The protective effects of estrogen may be mediated through classical estrogen receptors (ERs), or may be due to its anti-oxidant properties which are independent of receptors. The current studies show that 17beta-estradiol (E2) is neuroprotective against beta-amyloid protein 25-35 (Abeta)-, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-, high density culture condition-, and serum deprivation-induced neuronal death in SK-N-SH human neuroblastoma cells. SK-N-SH cells express ERbeta, but not ERalpha, as detected by Western blot analysis. Among all the insults, MPTP, high density culture and serum deprivation induce apoptotic cell death in this cell system as detected by ELISA determination of mono/oligonucleosomes and DNA laddering, while Abeta induces necrotic cell death. The protective effects of E2 are abolished by the addition of tamoxifen and ICI 182,780 in the MPTP treated cells, but not in the other models, suggesting that the effect of E2 in the MPTP model is probably associated with activation of ERbeta. The addition of ICI 182,780 shows a mitogenic effect in SK-N-SH cells in the presence of E2 in control culture or in the Abeta treated groups. Also, ICI 182,780 induced expression of ERalpha. Collectively, the current studies suggest that E2 is neuroprotective in apoptotic and necrotic death induced by multiple insults in SK-N-SH human neuroblastoma cells. Involvement of ER is insult type dependent. ICI 182,780 is able to influence the expression of ERs, probably through upregulation of ERalpha when ERbeta is totally antagonized.     

A ferrocenyl derivative of hydroxytamoxifen elicits an estrogen receptor-independent mechanism of action in breast cancer cell lines

The aim of this work was to investigate the mechanism of action of ferrocifen (Fc-OH-TAM), the ferrocenyl analog of 4-hydroxy-tamoxifen (OH-TAM), which is the active metabolite of tamoxifen, the drug most widely prescribed for treatment of hormone-dependent breast cancers. Fc-OH-TAM showed an anti-proliferative effect on the six breast cancer cell lines tested, 3 ERα positive (MCF-7, T-47D, ZR-75-1) and 3 ERα negative (MDA-MB-231, SKBR-3, Hs578-T) whatever their ER (estrogen receptor) status. However, the mechanism of action of the ferrocenyl derivative appeared to differ depending on the status of the ERα. Analysis of cell cycle distribution revealed that Fc-OH-TAM first recruits cells in the S phase in both ERα positive and ERα negative cells. In the presence of ERα, Fc-OH-TAM allowed cell cycle progression, with a subsequent blockade in G0/G1, whereas in the absence of ERα, cells remained in the S phase. Significant production of ROS was observed only in the presence of Fc-OH-TAM in both ERα positive and negative breast cancer cell lines. Within our experimental conditions, this ROS production is associated with cell cycle arrest and senescence rather than apoptosis. In the presence of ERα, Fc-OH-TAM seems to mainly act in the same way as OH-TAM but also induces an additional cytotoxic effect not mediated by the receptor. Our data suggest that this cytotoxic effect of Fc-OH-TAM is expressed via a mechanism of action distinct from the non-genomic pathway observed with high doses of OH-Tamoxifen.     

Mechanistic investigations of lipase-catalyzed degradation of polycarbonate in organic solvents

The biodegradation of an engineering thermoplastic, poly (bisphenol-A carbonate) (BPAPC), was carried out using three different lipases from Candida antarctica (CAL), Candida rugosa (CRL) and porcine pancreas (PPL) in water-miscible (tetrahydrofuran) and water-immiscible (chloroform) solvents for 10 days. The degradation was monitored by gel permeation chromatography and Fourier transform infrared spectroscopy. Maximum degradation (ca. 60% reduction in M(n)) of BPAPC was observed in THF with PPL when compared to the control without the enzyme. The degradation products in all the experiments were bisphenol-A and 4-α-cumyl phenol suggesting that the lipases act through an end-chain scission on the polymer. The degradation of BPAPC in THF was in the order of PPL>CAL>CRL, while in CHCl(3) it was CRL>CAL>PPL. To understand this disparity, and to probe the mechanistic aspects of degradation, molecular dynamics investigations were performed on the lipases with model BPAPC in both the solvents. The results also suggested that catalytic triad (Ser, His, Asp/Glu) was involved in the hydrolysis of carbonate bond leading to release of bisphenol-A. These data provide us the basic understanding of the degradation mechanism and a novel methodology for degrading polycarbonate.     

Mechanistic and biological aspects of helicase action on damaged DNA

Helicases catalytically unwind structured nucleic acids in a nucleoside-triphosphate-dependent and directionally specific manner, and are essential for virtually all aspects of nucleic acid metabolism. ATPase-driven helicases which translocate along nucleic acids play a role in damage recognition or unwinding of a DNA tract containing the lesion. Although classical biochemical experiments provided evidence that bulky covalent adducts inhibit DNA unwinding catalyzed by certain DNA helicases in a strand-specific manner (i.e., block to DNA unwinding restricted to adduct residence in the strand the helicase translocates), recent studies suggest more complex arrangements that may depend on the helicase under study, its assembly in a protein complex, and the type of structural DNA perturbation. Moreover, base and sugar phosphate backbone modifications exert effects on DNA helicases that suggest specialized tracking mechanisms. As a component of the replication stress response, the single-stranded DNA binding protein Replication Protein A (RPA) may serve to enable eukaryotic DNA helicases to overcome certain base lesions. Helicases play important roles in DNA damage signaling which also involve their partnership with RPA. In this review, we will discuss our current understanding of mechanistic and biological aspects of helicase action on damaged DNA.     

Study of the neuroprotective action of hybrid structures based on fullerene C60

The neuroprotective action of hybrid structures based on fullerene C₆₀ with attached proline amino acid has been studied. Hybrid structures contained natural antioxidant carnosine or addends with one or two nitrate groups. It has been shown that all studied compounds had antioxidant activity and decreased the concentration of malondialdehyde in homogenates of the rat brain. Compound I, which contained the antioxidant carnosine, has been found to be the most effective antioxidant. All compounds except IV and V inhibited the activity of monoamine oxidase B, while compounds I–IV increased the activity of monoamine oxidase A. All investigated compounds inhibited glutamate-induced Ca²⁺ uptake into synaptosomes of the rat brain cortex. Compound III, containing two nitrate groups, has been found to be the most effective inhibitor. This compound caused a significant increase of the currents of AMPA receptors (AMPA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid).     

Role of estrogen receptor-alpha in pharmacogenetics of estrogen action

PURPOSE OF REVIEW: To summarize recent data regarding the role of estrogen receptor-alpha polymorphisms in determining the response to estrogen therapy or the risk of clinical cardiovascular events. RECENT FINDINGS: Recent clinical trials of hormone replacement therapy for cardiovascular disease have yielded surprisingly negative results, shifting clinical opinions from a position of presumed cardiovascular benefit to one of confirmed harm. Understanding why hormone replacement therapy has beneficial effects on intermediate risk markers for cardiovascular disease, but produces an increase in cardiovascular events, is an important public health question with the potential to elucidate fundamentally important aspects on atherogenesis, cardiovascular disease, and the biology of estrogen action. One question concerning the cardiovascular effects of hormone replacement therapy is whether genetic factors can substantially modify individual responses to estrogen treatment. New clinical trial evidence is emerging that links the presence of particular variants in the estrogen receptor to the response of HDL and other intermediate endpoints to hormone replacement therapy. SUMMARY: One or more common variants in estrogen receptor-alpha are associated with a differential response to hormone replacement therapy in several domains of estrogen action. However, the effect of these variants on the risk of clinical cardiovascular events in the setting of hormone replacement therapy is not yet known. Additional research focusing on the clinical impact of common variants in estrogen receptor-alpha, estrogen receptor-beta and the progesterone receptor promise to improve clinical decision-making concerning the use of hormone replacement therapy and other novel estrogen agonists.     

Mechanistic investigations of Al(OH)3 oligomerization mechanisms

Aluminum aerogels have extremely low thermal conductivities, and are ideal candidates for use in thermal superinsulators, adsorbents, sensors, catalyst carriers, and inorganic fillers. In the present work, the oligomerization mechanisms of Al(OH)₃ were investigated systematically with the Gaussian 03 package at the B3LYP/6-311++G(d,p) level in combination with CPCM single-point energy calculations. The results of our theoretical model showed that: (1) the Al atoms are tetracoordinate and pentacoordinate; (2) in alkaline solution, Al(OH)₃ tends to condense into more soluble polyhydroxy compounds; (3) the neutral dimerization of Al(OH)₃ and the transfer of the hydrogen on the bridging hydroxyl are energetically favorable, but the most stable geometry is a four-membered Al–O ring structure linked by two bridging hydroxyls; (4) Al(OH)₃ is inclined to form tetracoordinate oligomers, which develop into three-dimensional structures connected by four-membered Al–O rings.     

Mechanistic investigations of the pseudouridine synthase RluA using RNA containing 5-fluorouridine

The pseuoduridine synthases (psi synthases) isomerize uridine (U) to pseudouridine (psi) in RNA, and they fall into five families that share very limited sequence similarity but have the same overall fold and active-site architecture, including an essential Asp. The mechanism by which the psi synthases operate remains unknown, and mechanistic work has largely made use of RNA containing 5-fluorouridine (f5U) in place of U. The psi synthase TruA forms a covalent adduct with such RNA, and heat disruption of the adduct generates a hydrated product of f5U, which was reasonably concluded to result from the hydrolysis of an ester linkage between the essential Asp and f5U. In contrast, the psi synthase TruB, which is a member of a different family, does not form an adduct with f5U in RNA but catalyzes the rearrangement and hydration of the f5U, which labeling studies with [18O]water showed does not result from ester hydrolysis. To extend the line of mechanistic investigation to another family of psi synthases and an enzyme that makes an adduct with f5U in RNA, the behavior of RluA toward RNA containing f5U was examined. Stem-loop RNAs are shown to be good substrates for RluA. Heat denaturation of the adduct between RluA and RNA containing f5U produces a hydrated nucleoside product, and labeling studies show that hydration does not occur by ester hydrolysis. These results are interpreted in light of a consistent mechanistic scheme for the handling of f5U by psi synthases.     

Experimental investigations on action of antihistaminic nasal solutions; a preliminary report

The effect of antihistaminic drugs in the form of solutions for nasal instillation on the mucous membranes of rabbits was investigated. So far as histologic observations would indicate, Antistine(R)-Privine(R) was the most harmful and caused almost complete necrosis throughout the entire nasal area. Pyribenzamine(R) was the least harmful. Allergan(R) caused more damage than did Pyribenzamine in the nasal mucosa of rabbits, but considerably less than Antistine-Privine.Clinically, the author has observed Antistine-Privine to be extremely irritating when employed as a topical application for symptomatic relief of nasal allergic disease. Pyribenzamine has caused considerable pain and discomfort in patients and has been least effective for relief of nasal congestion since it does not contain a vasoconstrictive agent. Allergan has not been observed by the author to be irritating to any degree in patients with nasal allergic disease unaccompanied by acute infection, and it has been noted to be the least irritating of these three antihistaminic solutions in nasal allergic disease complicated by infection.