A high-throughput colorimetric assay to characterize the enzyme kinetic and cellular activity of spermidine/spermine N-acetyltransferase 1

Spermidine/spermine N¹-acetyltransferase 1 (SSAT1) is a key enzyme that catalyzes the catabolism of polyamines. SSAT1 is a very important enzyme because it not only maintains the homeostasis of polyamines but also is involved in many physiological and pathological events. As such, a rapid assay of SSAT1 activity is valuable in drug screening and clinical diagnostics. Here, we report a novel colorimetric assay for monitoring SSAT1 activity in zebrafish (zSSAT1). In comparison with the available SSAT1 assays, this new method is cost-effective and simple. The optimal zSSAT1 activity was obtained below 55 °C in a mild alkaline environment. The K_m values of zSSAT1 for spermidine and spermine are 55 and 182 μM, respectively, whereas putrescine is not a good substrate for zSSAT1. In addition to enzyme kinetic studies, the colorimetric assay was also used to detect the cellular activity of SSAT1. Thus, the current method is a reliable assay for determining SSAT1 activity with many potential applications in medical biology.     

New bis(thiosemicarbazonate) gold(III) complexes inhibit HIV replication at cytostatic concentrations: potential for incorporation into virostatic cocktails

Four bis(thiosemicarbazonate)gold(III) complexes (1-4) with a general formula [Au(L)]Cl {L = L1, glyoxal-bis(N⁴-methylthiosemicarbazone); L2, glyoxal-bis(N⁴-ethylthiosemicarbazone); L3, diacetyl-bis(N⁴-methylthiosemicarbazone); L4, diacetyl-bis(N⁴-ethylthiosemicarbazone)} were synthesised and screened for activity against the human immunodeficiency virus (HIV). Complexes 1-4 were characterised using ¹H-NMR and IR spectroscopy; and their purity established by micronanalysis. Complex 3 inhibited viral infection of TZM-bl cells by 98% (IC₅₀ = 6.8 ± 0.6 μM) at a non toxic concentration of 12.5 μM while complex 4 inhibited infection of these cells by 72 and 98% (IC₅₀ = 5.3 ± 0.4 μM) at concentrations of 6.25 and 12.5 μM respectively. The mechanism of inhibition of infection in TZM-bl cells is presumably as a result of the cytostatic or anti-proliferative activity that was observed for complex 4 in real time cell electronic sensing (RT-CES) and carboxyflourescein succinimidyl ester (CFSE) analysis. Treatment of T lymphocytes from HIV infected individuals with 4 decreased CD4+ T cell expression (p = 0.0049) as demonstrated by multi-parametric flow cytometry without suppressing cytokine production. None of the ligands (L1-L4) demonstrated anti-viral activity, supporting the importance of metal (gold) complexation in these potential drugs. Complexes 3 and 4 were shown to have ideal lipophilicity values that were similar when shake flask (0.97 ± 0.5 and 2.42 ± 0.6) and in silico prediction (0.8 and 1.5) methods were compared. The activity and drug-like properties of complexes 3 and 4 suggests that these novel metal-based compounds could be combined with virus inhibitory drugs to work as cytostatic agents in the emerging class of anti-HIV drugs known as virostatics.     

Characterization of a new fluorogenic substrate for microsomal glutathione transferase 1

A new thiol-reactive electrophilic, disubstituted rhodamine-based fluorogenic probe (bis-2,4-dinitrobenzenesulfonyl rhodamine [BDR]) with very high quantum yield was synthesized and described recently [A. Shibata et al., Bioorg. Med. Chem. Lett. 18 (2008) 2246-2249]. Because hydrophobic electrophiles are often conjugated by glutathione transferases, the BDR or monosubstituted rhodamine derivatives (2,4-dinitrobenzenesulfonyl rhodamine [DR]) were tested with microsomal glutathione transferase 1 (MGST1) and shown to function as substrates. The kinetic parameters for purified enzyme and DR were k_cat = 0.075 ± 0.005 s⁻¹ and K_m = 21 ± 3 μM (k_cat/K_m = 3.6 × 10³ ± 5.6 × 10² M⁻¹ s⁻¹), giving a rate enhancement of 10⁶ compared with the nonenzymatic reaction. In cells overexpressing MGST1, the addition of BDR caused a time-dependent increase of fluorescence compared with control cells. Preincubating the cells with a thiol reagent (N-ethylmaleimide) abolished the fluorescent signal. By using DR, we could determine the MGST1 activity in whole cell extracts with high sensitivity. In addition, the activity could be increased by thiol reagents (a hallmark of MGST1). Thus, we have identified a new fluorogenic substrate for MGST1 that will be a useful tool in the study of this enzyme and related enzymes.     

A broad spectrum anticancer nucleoside with selective toxicity against human colon cells in vitro

2′,3′-Bis-O-tert-butyldimethylsilyl-5′-deoxy-5′-[N-(methylcarbamoyl)amino]-N⁶-(N-phenylcarbamoyl)adenosine, a new member of the N⁶,5′-bis-ureidoadenosine class of anticancer nucleosides, is found to exhibit broad spectrum antiproliferative activity. A majority of the cell lines in the NCI-60 are inhibited with an average GI₅₀ = 3.13 μM. Selective toxicity against human colon cancer cell lines (COLO 205, HCC-2998, HCT-116, HT29, KM12) was also exhibited (LC₅₀’s = 6-10 μM).     

Tissue-specific alternative splicing of spermidine/spermine N 1-acetyltransferase

The polyamines, spermidine and spermine, are abundant organic cations participating in many important cellular processes. We have previously shown that the rate-limiting enzyme of polyamine catabolism, spermidine/spermine N ¹-acetyltransferase (SSAT), has an alternative mRNA splice variant (SSATX) which undergoes degradation via nonsense-mediated mRNA decay (NMD) pathway, and that the intracellular polyamine level regulates the ratio of the SSATX and SSAT splice variants. The aim of this study was to investigate the effect of SSATX level manipulation on SSAT activity in cell culture, and to examine the in vivo expression levels of SSATX and SSAT mRNA. Silencing SSATX expression with small interfering RNA led to increased SSAT activity. Furthermore, transfection of SSAT-deficient cells with mutated SSAT gene (which produced only trace amount of SSATX) yielded higher SSAT activity than transfection with natural SSAT gene (which produced both SSAT and SSATX). Blocking NMD in vivo by protein synthesis inhibitor cycloheximide resulted in accumulation of SSATX mRNA, and like in cell culture, the increase of SSATX mRNA was prevented by administration of polyamine analog N ¹ ,N ¹¹ -diethylnorspermine. Although SSATX/total SSAT mRNA ratio did not correlate with polyamine levels or SSAT activity between different tissues, increasing polyamine levels in a given tissue led to decreased SSATX/total SSAT mRNA ratio and vice versa. Taken together, the regulated unproductive splicing and translation of SSAT has a physiological relevance in modulating SSAT activity. However, in addition to polyamine level there seems to be additional factors regulating tissue-specific alternative splicing of SSAT.     

Novel anti-apoptotic effect of the retinoblastoma protein: implications for polyamine analogue toxicity

The retinoblastoma protein (pRb) pathway is frequently altered in breast cancer cells. pRb is involved in the regulation of cell proliferation and cell death. The breast cancer cell line L56Br-C1 does not express pRb and is extremely sensitive to treatment with the polyamine analogue N ¹,N ¹¹-diethylnorspermine (DENSPM) which causes apoptosis. Polyamines are essential for the regulation of cell proliferation, cell differentiation and cell death. DENSPM depletes cells of polyamines, e.g., by inducing the activity of the polyamine catabolic enzyme spermidine/spermine N ¹-acetyltransferase (SSAT). In this study, L56Br-C1 cells were transfected with human pRb–cDNA. Overexpression of pRb inhibited DENSPM-induced cell death and DENSPM-induced SSAT activity. This suggests that the pRb protein level is a promising marker for polyamine depletion sensitivity and that there is a connection between pRb and the regulation of SSAT activity. We also show that SSAT protein levels and SSAT activity do not always correlate, suggesting that there is an unknown regulation of SSAT.     

The human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) is irreversibly inhibited by inorganic (Hg) and organic mercury (CH3Hg): Mechanism and kinetics

Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg²⁺) and organic (CH₃Hg⁺) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC₅₀) = 250 nM and k_inact = 1.4 × 10⁴ M⁻¹ s⁻¹ for Hg²⁺ and IC₅₀ = 1.4 μM and k_inact = 2 × 10² M⁻¹ s⁻¹ for CH₃Hg⁺). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC₅₀ = 3 and 20 μM for Hg²⁺ and CH₃Hg⁺, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1.     

Water column anammox and denitrification in a temperate permanently stratified lake (Lake Rassnitzer,Germany)

We studied microbial N₂ production via anammox and denitrification in the anoxic water column of a restored mining pit lake in Germany over an annual cycle. We obtained high-resolution hydrochemical profiles using a continuous pumping sampler. Lake Rassnitzer is permanently stratified at ca. 29 m depth, entraining anoxic water below a saline density gradient. Mixed-layer nitrate concentrations averaged ca. 200 μmol L⁻¹, but decreased to zero in the anoxic bottom waters. In contrast, ammonium was <5 μmol L⁻¹ in the mixed layer but increased in the anoxic waters to ca. 600 μmol L⁻¹ near the sediments. In January and October, ¹⁵N tracer measurements detected anammox activity (maximum 504 nmol N₂ L⁻¹ d⁻¹ in ¹⁵NH₄⁺-amended incubations), but no denitrification. In contrast, in May, N₂ production was dominated by denitrification (maximum 74 nmol N₂ L⁻¹ d⁻¹). Anammox activity in May was significantly lower than in October, as characterized by anammox rates (maximum 6 vs. 16 nmol N₂ L⁻¹ d⁻¹ in incubations with ¹⁵NO₃⁻), as well as relative and absolute anammox bacterial cell abundances (0.56% vs. 0.98% of all bacteria, and 2.7×10⁴ vs. 5.2×10⁴ anammox cells mL⁻¹, respectively) (quantified by catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) with anammox bacteria-specific probes). Anammox bacterial diversity was investigated with anammox bacteria-specific 16S rRNA gene clone libraries. The majority of anammox bacterial sequences were related to the widespread Candidatus Scalindua sorokinii/brodae cluster. However, we also found sequences related to Candidatus S. wagneri and Candidatus Brocadia fulgida, which suggests a high anammox bacterial diversity in this lake comparable with estuarine sediments.     

Sphingosine 1-phosphate lyase enzyme assay using a BODIPY-labeled substrate

Sphingosine 1-phosphate lyase (SPL) is responsible for the irreversible catabolism of sphingosine 1-phosphate, which signals through five membrane receptors to mediate cell stress responses, angiogenesis, and lymphocyte trafficking. The standard assay for SPL activity utilizes a radioactive dihydrosphingosine 1-phosphate substrate and is expensive and cumbersome. In this study, we describe an SPL assay that employs an ω-labeled BODIPY-sphingosine 1-phosphate substrate, allowing fluorescent product detection by HPLC and incorporating advantages of the BODIPY fluorophore. The major aldehyde product is confirmed by reaction with 2,4-dinitrophenylhydrazine. The SPL-catalyzed reaction is linear over a 30 min time period and yields a K_m of 35 μM for BODIPY-sphingosine 1-phosphate.     

Morphological and physiological adaptive traits of Mediterranean narrow endemic plants: The case of Centaurea gymnocarpa (Capraia Island,Italy)

A case study on Centaurea gymnocarpa Moris & De Not., a narrow endemic species, was carried out by analyzing its morphological, anatomical, and physiological traits in response to natural habitat stress factors under Mediterranean climate conditions. The results underline that the species is particularly adapted to the environment where it naturally grows. At the plant level, the above-ground/below-ground dry mass (1.73 ± 0.60) shows its investment predominately in the above-ground structure with a resulting total leaf area per plant of 1399 ± 94 cm². The senescent attached leaves at the base of the plant contribute to limit leaf transpiration by shading soil around the plant. Moreover, the dense C. gymnocarpa leaf pubescence, leaf rolling, the relatively high leaf mass area (LMA = 12.3 ± 1.3 mg cm⁻²) and leaf tissue density (LTD = 427 ± 44 mg cm⁻³) contribute to limit leaf transpiration, also postponing leaf death under dry conditions. At the physiological level, a relatively low respiration/photosynthesis ratio (R/P_N) in spring results from high R [2.26 ± 0.59 μmol (CO₂) m⁻² s⁻¹] and P_N [12.3 ± 1.5 μmol (CO₂) m⁻² s⁻¹]. The high photosynthetic nitrogen use efficiency [PNUE = 15.5 ± 0.4 μmol (CO₂) g⁻¹ (N) s⁻¹] shows the large amount of nitrogen (N) invested in the photosynthetic machinery of new leaves, associated to a high chlorophyll content (Chl = 35 ± 5 SPAD units). On the contrary, the highest R/P_N ratio (1.75 ± 0.19) in summer is due to a significant P_N decrease and increase of R in response to drought. The low PNUE [1.5 ± 0.2 μmol (CO₂) g⁻¹ (N) s⁻¹] in this season is indicative of a greater N investment in leaf cell walls which may contribute to limit transpiration. On the contrary, the low R/P_N ratio (0.05 ± 0.02) in winter is resulting from the limited enzyme activity of the respiratory apparatus [R = 0.23 ± 0.08 μmol (CO₂) m⁻² s⁻¹] while the low PNUE [3.5 ± 0.2 μmol (CO₂) g⁻¹ (N) s⁻¹] suggests that low temperatures additionally limit plant production. The experiment of the imposed water stress confirms that the C. gymnocarpa growth capability is in conformity with the severe conditions of its natural habitat, likewise as it may be the case with others narrow endemic species that have occupied niches with similar extreme conditions.     

A novel acetyl-CoA carboxylase inhibitor reduces de novo fatty acid synthesis in HepG2 cells and rat primary hepatocytes

To identify the novel inhibitor of de novo lipogenesis in hepatocytes, we screened for inhibitory activity of triglyceride (TG) synthesis using [¹⁴C]acetate in the human hepatoma cell line, HepG2. Using this assay system we discovered the novel compound, benzofuranyl α-pyrone (TEI-B00422). TEI-B00422 also inhibited the incorporation of acetate into the triglyceride (TG) fraction in rat primary hepatocytes. In HepG2 cells, the incorporation of oleate into TG was unaffected. TEI-B00422 inhibited rat hepatic acetyl-CoA carboxylase (ACC), K_i = 3.3 μM, in a competitive manner with respect to acety-CoA but not fatty acid synthase and acyl-CoA transferase/diacylglycerol. Thus, these results suggest that the inhibition of TG synthesis by TEI-B00422 is based on the inhibitory action of ACC. The structure of TEI-B00422 is totally different from the known inhibitors of ACC and may be useful in the development of therapeutic agents to combat a number of metabolic disorders.     

Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays

Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1×), tryptose phosphate broth (2.95 g l⁻¹), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 μg ml⁻¹ chloramphenicol, 100 μg ml⁻¹ streptomycin and 100 IU ml⁻¹ penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-2′-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24 h. Susceptibility of the cells to WSSV was confirmed by immunofluoresence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT₅₀/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC₅₀. The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals.     

Eurycomanone suppresses expression of lung cancer cell tumor markers, prohibitin, annexin 1 and endoplasmic reticulum protein 28

Bioactive compounds from the medicinal plant, Eurycoma longifolia Jack have been shown to promote anti-proliferative effects on various cancer cell lines. Here we examined the effects of purified eurycomanone, a quassinoid found in Eurycoma longifolia Jack extract, on the expression of selected genes of the A549 lung cancer cells. Eurycomanone inhibited A549 lung cancer cell proliferation in a dose-dependent manner at concentrations ranging from 5 to 20 μg/ml. The concentration that inhibited 50% of cell growth (GI₅₀) was 5.1 μg/ml. The anti-proliferative effects were not fully reversible following the removal of eurycomanone, in which 30% of cell inhibition still remained (p < 0.0001, T-test). At 8 μg/ml (GI₇₀), eurycomanone suppressed anchorage-independent growth of A549 cells by >25% (p < 0.05, T-test, n = 8) as determined using soft agar colony formation assay. Cisplatin, a chemotherapy drug used for the treatment of non small cell lung cancer on the other hand, inhibited A549 cells proliferation at concentrations ranging from 0.2 μg/ml to 15 μg/ml with a GI₅₀ of 0.58 μg/ml. The treatment with eurycomanone reduced the abundance expression of the lung cancer markers, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, p53 tumor suppressor protein and other cancer-associated genes including prohibitin (PHB), annexin 1 (ANX1) and endoplasmic reticulum protein 28 (ERp28) but not the house keeping genes. The mRNA expressions of all genes with the exception of PHB were significantly downregulated, 72 h after treatment (p < 0.05, T-test, n = 9). These findings suggest that eurycomanone at viable therapeutic concentrations of 5-20 μg/ml exhibited significant anti-proliferative and anti-clonogenic cell growth effects on A549 lung cancer cells. The treatment also resulted in suppression of the lung cancer cell tumor markers and several known cancer cell growth-associated genes.     

Inhibition of mammalian thioredoxin reductase by black tea and its constituents: Implications for anticancer actions

Black tea is recently reported to have anti-carcinogenic effects through pro-oxidant property, but the underlying mechanisms remain unclear. Mammalian cytosolic thioredoxin reductase (TrxR1) is well -known for its anti-oxidation activity. In this study, we found that black tea extract (BTE) and theaflavins (TFs), the major black tea polyphenols, inhibited the purified TrxR1 with IC₅₀ 44 μg/ml and 21 ± 1 μg/ml, respectively. Kinetics of TFs exhibited a mixed type of competitive and non-competitive inhibition, with K_is 4 ± 1 μg/ml and K_ii 26 ± 5 μg/ml against coenzyme NADPH, and with K_is 12 ± 3 μg/ml and K_ii 27 ± 5 μg/ml against substrate DTNB. In addition, TFs inhibited TrxR1 in a time-dependent manner. In an equilibrium step, a reversible TrxR1-TFs complex (E * I) forms, which is followed by a slow irreversible first-order inactivation step. Rate constant of the inactivation was 0.7 min⁻¹, and dissociation constant of E * I was 51.9 μg/ml. Treatment of NADPH-reduced TrxR1 with TFs decreased 5-(Iodoacetamido) fluorescein incorporation, a fluorescent thiol-reactive reagent, suggesting that Sec/Cys residue(s) in the active site may be involved in the binding of TFs. The inhibitory capacity of TFs depends on their structure. Among the TFs tested, gallated forms had strong inhibitory effects. The interactions between TFs and TrxR1 were investigated by molecular docking, which revealed important features of the binding mechanism of theaflavins. An inhibitory effect of BTE on viability of HeLa cells was observed with IC₅₀ 29 μg/ml. At 33 μg/ml of BTE, TrxR1 activity in HeLa cells was decreased by 73% at 22 h after BTE treatment. TFs inhibited cell viability with IC₅₀ 10 ± 4 μg/ml for HeLa cells and with IC₅₀ 20 ± 5 μg/ml for EAhy926 cells. The cell susceptibility to TFs was inversely correlated to cellular levels of TrxR1. The inhibitory actions of TFs on TrxR1 may be an important mechanism of their anti-cancer properties.     

Design of novel N-phenylnicotinamides as selective cyclooxygenase-1 inhibitors

A series of N-phenylnicotinamides (1-40) were designed and evaluated in vitro for their COX inhibitory activities. Most of the synthesized compounds were proved to be potent and selective inhibitors of COX-1. Compound 28 showed the most potent COX-1 inhibitory activity (COX-1 IC₅₀ = 0.68 ± 0.07 μM) and good selectivity (COX-2 IC₅₀ >100 μM). This compound may be useful as a lead compound for superior COX-1 inhibitors. On the basis of the biological results, structure-activity relationships for the COX-1-inhibitory activities of the synthesized N-phenylnicotinamides were discussed concisely.     

Dipentamethylene thiuram monosulfide is a novel inhibitor of Pin1

Pin1 is involved in eukaryotic cell proliferation by changing the structure and function of phosphorylated proteins. PiB, the Pin1 specific inhibitor, blocks cancer cell proliferation. However, low solubility of PiB in DMSO has limited studies of its effectiveness. We screened for additional Pin1 inhibitors and identified the DMSO-soluble compound dipentamethylene thiuram monosulfide (DTM) that inhibits Pin1 activity with an EC50 value of 4.1 μM. Molecular modeling and enzyme kinetic analysis indicated that DTM competitively inhibits Pin1 activity, with a K_i value of 0.05 μM. The K_D value of DTM with Pin1 was determined to be 0.06 μM by SPR technology. Moreover, DTM specifically inhibited peptidyl-prolyl cis/trans isomerase activity in HeLa cells. FACS analysis showed that DTM induced G0 arrest of the HCT116 cells. Our results suggest that DTM has the potential to guide the development of novel antifungal and/or anticancer drugs.