Stearoyl-CoA desaturase is an essential enzyme for the parasitic protist Trypanosoma brucei

Trypanosoma brucei, the etiologic agent of sleeping sickness, is exposed to important changes in nutrients and temperature during its life cycle. To adapt to these changes, the fluidity of its membranes plays a crucial role. This fluidity, mediated by the fatty-acid composition, is regulated by enzymes named desaturases. We have previously shown that the oleoyl desaturase is essential for Trypanosoma cruzi and T. brucei. In this work, we present experimental support for the relevance of stearoyl-CoA desaturase (SCD) for T. brucei’s survival, in both its insect or procyclic-form (PCF) and bloodstream-form (BSF) stages. We evaluated this essentiality in two different ways: by generating a SCD knocked-down parasite line using RNA interference, and by chemical inhibition of the enzyme with two compounds, Isoxyl and a thiastearate with the sulfur atom at position 10 (10-TS). The effective concentration for 50% growth inhibition (EC₅₀) of PCF was 1.0 ± 0.2 μM for Isoxyl and 5 ± 2 μM for 10-TS, whereas BSF appeared more susceptible with EC₅₀ values 0.10 ± 0.03 μM (Isoxyl) and 1.0 ± 0.6 μM (10-TS). RNA interference showed to be deleterious for both stages of the parasite. In addition, T. brucei-infected mice were fed with Isoxyl, causing a reduction of the parasitemia and an increase of the rodents’ survival.     

Genetic and chemical evaluation of Trypanosoma brucei oleate desaturase as a candidate drug target

Background

Trypanosomes can synthesize polyunsaturated fatty acids. Previously, we have shown that they possess stearoyl-CoA desaturase (SCD) and oleate desaturase (OD) to convert stearate (C18) into oleate (C18:1) and linoleate (C18:2), respectively. Here we examine if OD is essential to these parasites.

Methodology

Cultured procyclic (insect-stage) form (PCF) and bloodstream-form (BSF) Trypanosoma brucei cells were treated with 12- and 13-thiastearic acid (12-TS and 13-TS), inhibitors of OD, and the expression of the enzyme was knocked down by RNA interference. The phenotype of these cells was studied.

Principal Findings

Growth of PCF T. brucei was totally inhibited by 100 µM of 12-TS and 13-TS, with EC₅₀ values of 40±2 and 30±2 µM, respectively. The BSF was more sensitive, with EC₅₀ values of 7±3 and 2±1 µM, respectively. This growth phenotype was due to the inhibitory effect of thiastearates on OD and, to a lesser extent, on SCD. The enzyme inhibition caused a drop in total unsaturated fatty-acid level of the cells, with a slight increase in oleate but a drastic decrease in linoleate level, most probably affecting membrane fluidity. After knocking down OD expression in PCF, the linoleate content was notably reduced, whereas that of oleate drastically increased, maintaining the total unsaturated fatty-acid level unchanged. Interestingly, the growth phenotype of the RNAi-induced cells was similar to that found for thiastearate-treated trypanosomes, with the former cells growing twofold slower than the latter ones, indicating that the linoleate content itself and not only fluidity could be essential for normal membrane functionality. A similar deleterious effect was found after RNAi in BSF, even with a mere 8% reduction of OD activity, indicating that its full activity is essential.

Conclusions/Significance

As OD is essential for trypanosomes and is not present in mammalian cells, it is a promising target for chemotherapy of African trypanosomiasis.     

Na(+)-ATPase and protein kinase C are targets to 1-O-hexadecylphosphocoline (miltefosine) in Trypanosoma cruzi

Miltefosine has been shown to be a very active compound against Trypanosoma cruzi. Here, we evaluated the effects of miltefosine on the activity of the Na⁺-ATPase and protein kinase C (PKC) present in the plasma membrane of T. cruzi. Furosemide (2 mM), a specific inhibitor of Na⁺-ATPase, abolished the growth of T. cruzi showing a crucial role of this enzyme to parasite growth. Miltefosine inhibited the Na⁺-ATPase activity with IC₅₀ = 18 ± 5 μg mL⁻¹. This effect was shown to be reversible, dependent on the pH and Ca²⁺. The inhibition was not observed when the membranes were solubilized with 0.1% deoxycholate, suggesting that the interaction between the enzyme and membrane phospholipids might be important for the drug effect. Miltefosine also inhibited the parasite PKC activity, but through a Na⁺-ATPase-independent way. Altogether the results indicate that miltefosine inhibits T. cruzi growth through, at least in part, the inhibition of both Na⁺-ATPase and PKC activities.     

Cardiodepressive effect elicited by the essential oil of Alpinia speciosa is related to L-type Ca current blockade

This study was undertaken to elucidate the effect of the essential oil from Alpinia speciosa (EOAs) on cardiac contractility and the underlying mechanisms. The essential oil was obtained from Alpinia speciosa leaves and flowers and the oil was analyzed by GC-MS method. Chemical analysis revealed the presence of at least 18 components. Terpinen-4-ol and 1,8-cineole corresponded to 38% and 18% of the crude oil, respectively. The experiments were conducted on spontaneously-beating right atria and on electrically stimulated left atria isolated from adult rats. The effect of EOAs on the isometric contractions and cardiac frequency in vitro was examined. EOAs decreased rat left atrial force of contraction with an EC₅₀ of 292.2 ± 75.7 μg/ml. Nifedipine, a well known L-type Ca²⁺ blocker, inhibited in a concentration-dependent manner left atrial force of contraction with an EC₅₀ of 12.1 ± 3.5 μg/ml. Sinus rhythm was diminished by EOAs with an EC₅₀ of 595.4 ± 56.2 μg/ml. Whole-cell L-type Ca²⁺ currents were recorded by using the patch-clamp technique. EOAs at 25 μg/ml decreased I_Ca,L by 32.6 ± 9.2% and at 250 μg/ml it decreased by 89.3 ± 7.4%. Thus, inhibition of L-type Ca²⁺ channels is involved in the cardiodepressive effect elicited by the essential oil of Alpinia speciosa in rat heart.     

Flow cytometric screening for anti-leishmanials in a human macrophage cell line

High-throughput drug screening methods against the intracellular stage of Leishmania have been facilitated by the development of in vitro models of infection. The use of cell lines rather than primary cells facilitates these methods. Peripheral blood mononuclear cell (PBMC) derived macrophages and THP-1 cells were infected with stationary phase egfp transfected Leishmania amazonensis parasites and then treated with anti-leishmanial compounds. Drug activity was measured using a flow cytometric approach, and toxicity was assessed using either the MTT assay or trypan blue dye exclusion. Calculated EC₅₀’s for amphotericin B, sodium stibogluconate, and miltefosine were 0.1445 ± 0.0005 μg/ml, 0.1203 ± 0.018 mg/ml, and 26.71 μM using THP-1 cells, and 0.179 ± 0.035 μg/ml, 0.1948 ± 0.0364 mg/ml, and 13.77 ± 10.74 μM using PBMC derived macrophages, respectively. We conclude that a flow cytometric approach using egfp transfected Leishmania species can be used to evaluate anti-leishmanial compounds against the amastigote stage of the parasite in THP-1 cells with excellent concordance to human PBMC derived macrophages.     

Neolignan Licarin A presents effect against Leishmania (Leishmania) major associated with immunomodulation in vitro

Leishmaniasis’ treatment is based mostly on pentavalent antimonials or amphotericin B long-term administration, expensive drugs associated with severe side effects. Considering these aforementioned, the search for alternative effective and safe leishmaniasis treatments is a necessity. This work evaluated a neolignan, licarin A anti-leishmanial activity chemically synthesized by our study group. It was observed that licarin A effectively inhibited Leishmania (Leishmania) major promastigotes (IC₅₀ of 9.59 ± 0.94 μg/mL) growth, by inducing in these parasites genomic DNA fragmentation in a typical death pattern by apoptosis. Additionally, the neolignan proved to be even more active against intracellular amastigotes of the parasite (EC₅₀ of 4.71 ± 0.29 μg/mL), and significantly more effective than meglumine antimoniate (EC₅₀ of 216.2 ± 76.7 μg/mL) used as reference drug. The antiamastigote activity is associated with an immunomodulatory activity, since treatment with licarin A of the infected macrophages induced a decrease in the interleukin (IL)-6 and IL-10 production. This study demonstrates for the first time the antileishmanial activity of licarin A and suggests that the compound may be a promising in the development of a new leishmanicidal agent.     

First total synthesis and antiprotozoal activity of (Z)-17-methyl-13-octadecenoic acid, a new marine fatty acid from the sponge Polymastia penicillus

The first total synthesis for the (Z)-17-methyl-13-octadecenoic acid was accomplished in seven steps and in a 45% overall yield. The use of (trimethylsilyl)acetylene was key in the synthesis. Based on a previous developed strategy in our laboratory the best synthetic route towards the title compound was first acetylide coupling of (trimethylsilyl)acetylene to the long-chain protected 12-bromo-1-dodecanol followed by a second acetylide coupling to the short-chain 3-methyl-1-bromobutane, which resulted in higher yields. Complete spectral data is also presented for the first time for this recently discovered fatty acid. The title compound displayed antiprotozoal activity against Leishmania donovani (EC₅₀ = 19.8 μg/ml) and inhibited the leishmania DNA topoisomerase IB at concentrations of 50 μM.     

3-Hydroxy-2-methylene-3-(4-nitrophenylpropanenitrile): A new highly active compound against epimastigote and trypomastigote form of Trypanosoma cruzi

We have synthesized the Morita-Baylis-Hillman adduct (MBHA) 3-hydroxy-2-methylene-3-(4-nitrophenyl)-propanenitrile (3) in quantitative yield and evaluated on Trypanosoma cruzi epimastigote and bloodstream trypomastigote forms. Compound 3 strongly inhibited epimastigote growth, with IC₅₀/72 h of 28.5 μM and also caused intense trypomastigotes lysis, with an IC₅₀/24 h of 25.5 μM. Ultrastructural analysis showed significant morphological changes on both parasite forms treated with 3, including increase of cell volume and rounding of cell body as well as intense intracellular disorganization. Morphological changes indicative of apoptosis, autophagy or necrosis were observed in most affected cells. Docking calculations of 1, 2 and 3 pointed out the possibility of T. cruzi Farnesyl Pyrophosphate Synthase (TcFPPS) enzyme inhibition in 3 mechanism of action.     

Neolignans from plants in northeastern Brazil (Lauraceae) with activity against Trypanosoma cruzi

Trypanosoma cruzi is the ethiological agent for Chagas disease in Latin America. This study aimed to test the trypanocidal effect of licarin A and burchellin isolated from plants in northeastern Brazil. These neolignans were tested on T. cruzi and on peritoneal macrophages, to evaluate drug toxicity. Epimastigote growth was inhibited in 45% with licarin A and 20% with burchellin with an IC₅₀/96 h of 462.7 μM and 756 μM, respectively. Epimastigotes treated with licarin A presented swollen mitochondria and disorganized mitochondrial cristae, kDNA and Golgi complex. When treated with burchellin, they presented enormous autophagosomes and chromatin disorganization. Licarin A and burchellin were able to induce trypomastigote death with IC₅₀/24 h of 960 μM and 520 μM, respectively. Although licarin A presented an IC₅₀ for trypomastigotes higher than for epimastigotes, both substances acted as therapeutic trypanocidal agents, because they were able to kill parasites without affecting macrophages. Due to our results, burchellin and licarin A need to be further analysed to observe if they may be used as alternative blood additive prophylaxis against Chagas disease, since it has been established that blood transfusion is an important mechanism in the transmission process.     

A comparison of galvanic skin conductance and skin wettedness as indicators of thermal discomfort during moderate and high metabolic rates

The relationship between local thermal comfort, local skin wettedness (w_local) and local galvanic skin conductance (GSC) in four body segments during two different exercise intensities was compared in 10 males. In a balanced order, participants walked at 35% VO_2max for 45 min (WALK) (29.0±1.9°C, 29.8±3.6% RH, no wind) in one test and in a separate test ran at 70% VO_2max for 45 min (RUN) (26.2±2.1°C, 31.1±7.0% RH, no wind). During both tests, participants wore a loose fitting 100% polyester long sleeve top and trouser ensemble with a low resistance to heat and vapour transfer (total thermal resistance of 0.154 m² K W⁻¹ and total water vapour resistance of 35.9 m² Pa W⁻¹). w_local, change from baseline in GSC (ΔGSC) and local thermal comfort were recorded every 5 min. The results suggest that both w_local and ΔGSC are strong predictors of thermal comfort during the WALK when sweat production is low and thermal discomfort minimal (r²>0.78 and r²>0.71, respectively). Interestingly, during the RUN w_local plateaued at ~0.6 to 0.8 due to the high sweat production, whilst ΔGSC gradually increased throughout the experiment. ΔGSC had a similar relationship with thermal comfort to w_local during the RUN (r²>0.95 and r²>0.94, respectively). Despite the strength of these relationships, the ability of w_local to predict local thermal comfort accurately dramatically reduces in the exponential part of the curve. In a situation of uncompensated heat stress such as high metabolic rate in hot climate, where sweat production is high, ΔGSC shows to be a better predictor of local thermal comfort than w_local. The w_local data shows regional differences in the threshold which triggers local discomfort during the WALK than RUN; lower values are found for upper arms (0.22±0.03 and 0.28 ±0.22) and upper legs (0.22±0.11 and 0.22±0.10), higher values for upper back (0.30±0.12 and 0.36 ±0.10) and chest (0.27±0.10 and 0.39 ±0.32), respectively. However, no regional differences in the threshold of discomfort are found in the ?GSC data. Instead, the data suggests that the degree of discomfort experienced appears to be related to the amount of sweat within and around the skin (as indirectly measured by ΔGSC) at each body site.     

Isolation and characterization of a novel protein toxin from fire coral

Fire corals (Millepora spp.) cause severe pain and inflammatory effects in humans upon contact, and the organs responsible for these effects are called nematocysts. Here, we isolated an active cytotoxin of ca. 18 kDa (MCTx-1) from nematocysts of Millepora dichotoma var. tenera. MCTx-1 was potently cytotoxic (EC₅₀ value 79 ng/mL) towards L1210 mouse leukemia cells, hemagglutinated a 0.8% suspension of sheep erythrocytes (0.2 μg protein/mL) and was lethal in crayfish (LD₅₀, 106 μg/kg). We deduced the primary structure of MCTx-1 from the corresponding cDNA sequence and found that MCTx-1 is a novel dermatopontin that is an extracellular matrix protein in mammals. This is the first characterization of a proteinaceous toxin from fire coral.     

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.     

Cytotoxic and antioxidant activities of diterpenes and sterols from the Vietnamese soft coral Lobophytum compactum

Two new diterpenes, lobocompactols A (1) and B (2), and five known compounds (3-7) were isolated from the methanol extract of the soft coral Lobophytum compactum using combined chromatographic methods and identified based on NMR and MS data. Each compound was evaluated for cytotoxic activity against A549 (lung) and HL-60 (acute promyelocytic leukemia) human cancer cell lines. Among them, compound 5 exhibited strong cytotoxic activity against the A549 cell line with an IC₅₀ of 4.97 ± 0.06 μM. Compounds 3, 4, and 7 showed moderate activity with IC₅₀ values of 23.03 ± 0.76, 31.13 ± 0.08, and 36.45 ± 0.01 μM, respectively. The cytotoxicity of 5 on the A549 cells was comparable to that of the positive control, mitoxantrone (MX). All compounds exhibited moderate cytotoxicity against the HL-60 cell line, with IC₅₀ values ranging from 17.80 ± 1.43 to 59.06 ± 2.31 μM. Their antioxidant activity was also measured using oxygen radical absorbance capacity method, compounds 1 and 2 exhibiting moderate peroxyl radical scavenging activity of 1.4 and 1.3 μM Trolox equivalents, respectively, at a concentration of 5 μM.     

A continuous assay for α-methylacyl-coenzyme A racemase using circular dichroism

α-Methylacyl-coenzyme A racemase (AMACR) catalyzes the epimerization of (2R)- and (2S)-methyl branched fatty acyl-coenzyme A (CoA) thioesters. AMACR is a biomarker for prostate cancer and a putative target for the development of therapeutic agents directed against the disease. To facilitate development of AMACR inhibitors, a continuous circular dichroism (CD)-based assay has been developed. The open reading frame encoding AMACR from Mycobacterium tuberculosis (MCR) was subcloned into a pET15b vector, and the enzyme was overexpressed and purified using metal ion affinity chromatography. The rates of MCR-catalyzed epimerization of either (2R)- or (2S)-ibuprofenoyl-CoA were determined by following the change in ellipticity at 279 nm in the presence of octyl-β-d-glucopyranoside (0.2%). MCR exhibited slightly higher affinity for (2R)-ibuprofenoyl-CoA (K_m = 48 ± 5 μM, k_cat = 291 ± 30 s⁻¹), but turned over (2S)-ibuprofenoyl-CoA (K_m = 86 ± 6 μM, k_cat = 450 ± 14 s⁻¹) slightly faster. MCR expressed as a fusion protein bearing an N-terminal His₆-tag had a catalytic efficiency (k_cat/K_m) that was reduced 22% and 47% in the 2S → 2R and 2R → 2S directions, respectively, relative to untagged enzyme. The continuous CD-based assay offers an economical and efficient alternative method to the labor-intensive, fixed-time assays currently used to measure AMACR activity.     

Kinetic mechanism and catalysis of Trypanosoma cruzi dihydroorotate dehydrogenase enzyme evaluated by isothermal titration calorimetry

Trypanosoma cruzi dihydroorotate dehydrogenase (TcDHODH) catalyzes the oxidation of l-dihydroorotate to orotate with concomitant reduction of fumarate to succinate in the de novo pyrimidine biosynthetic pathway. Based on the important need to characterize catalytic mechanism of TcDHODH, we have tailored a protocol to measure TcDHODH kinetic parameters based on isothermal titration calorimetry. Enzymatic assays lead to Michaelis-Menten curves that enable the Michaelis constant (K_M) and maximum velocity (V_max) for both of the TcDHODH substrates: dihydroorotate (K_M = 8.6 ± 2.6 μM and V_max = 4.1 ± 0.7 μM s^-1) and fumarate (K_M = 120 ± 9 μM and V_max = 6.71 ± 0.15 μM s^-1). TcDHODH activity was investigated using dimethyl sulfoxide (10%, v/v) and Triton X-100 (0.5%, v/v), which seem to facilitate the substrate binding process with a small decrease in K_M. Arrhenius plot analysis allowed the determination of thermodynamic parameters of activation for substrates and gave some insights into the enzyme mechanism. Activation entropy was the main contributor to the Gibbs free energy in the formation of the transition state. A factor that might contribute to the unfavorable entropy is the hindered access of substrates to the TcDHODH active site where a loop at its entrance regulates the open-close channel for substrate access.     

Antibacterial and antioxidant activities of four kaempferol methyl ethers isolated from Dodonaea viscosa Jacq. var. angustifolia leaf extracts

Fractionation of dichloromethane and acetone fractions obtained by serial extraction from the leaf powder of Dodonaea viscosa Jacq. var. angustifolia (Sapindaceae) resulted in the isolation of four kaempferol methyl ethers. The compounds were identified by spectral data (¹H NMR, ¹³C NMR and MS) as: 3, 5, 7-trihydroxy-4'-methoxyflavone (1); 5, 7, 4'-trihydroxy-3, 6-dimethoxyflavone (2); 5, 7-dihydroxy-3, 6, 4'-trimethoxyflavone (santin) (3); and 5-hydroxy -3, 7, 4'-trimethoxyflavone (4) together with 3,4',5,7-tetrahydroxy flavone (kaempferol) (5). Antioxidant potential of the compounds was evaluated using a DPPH spectrophotometric assay, while antibacterial activity was determined using a serial dilution microplate technique. The isolates demonstrated varying degrees of antioxidant and antibacterial activities. Of all the compounds investigated, compounds 1 and 5 demonstrated some antioxidant activity (EC₅₀ = 75.49 ± 1.76 µM and 35.06 ± 0.85 respectively) but lower than l-ascorbic acid (EC₅₀ = 13.55 ± 0.28 µM) used as a standard antioxidant agent. The minimum inhibitory concentration (MIC) of isolated compounds against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa varied from 16 µg/ml to more than 250 µg/ml. Some structure activity relationships could be established for these compounds.     

Antimicrobial peptides isolated from Phyllomedusa nordestina (Amphibia) alter the permeability of plasma membrane of Leishmania and Trypanosoma cruzi

Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC₅₀ values in the range concentration of 0.25–0.68 μM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC₅₀ value of 10 μM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC₅₀ = 34 μM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX® Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies.     

Unfolding thermodynamics of the Delta-domain in the prohead I subunit of phage HK97: determination by factor analysis of Raman spectra

An early step in the morphogenesis of the double-stranded DNA (dsDNA) bacteriophage HK97 is the assembly of a precursor shell (prohead I) from 420 copies of a 384-residue subunit (gp5). Although formation of prohead I requires direct participation of gp5 residues 2-103 (Δ-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and DNA packaging. The prohead I Δ-domain is thought to resemble a phage scaffolding protein, by virtue of its highly α-helical secondary structure and a tertiary fold that projects inward from the interior surface of the shell. Here, we employ factor analysis of temperature-dependent Raman spectra to characterize the thermostability of the Δ-domain secondary structure and to quantify the thermodynamic parameters of Δ-domain unfolding. The results are compared for the Δ-domain within the prohead I architecture (in situ) and for a recombinantly expressed 111-residue peptide (in vitro). We find that the α-helicity (∼ 70%), median melting temperature (T_m = 58 °C), enthalpy (ΔH_m = 50 ± 5 kcal mol^− 1), entropy (ΔS_m = 150 ± 10 cal mol^− 1 K^− 1), and average cooperative melting unit (〈n_c〉 ∼ 3.5) of the in situ Δ-domain are altered in vitro, indicating specific interdomain interactions within prohead I. Thus, the in vitro Δ-domain, despite an enhanced helical secondary structure (∼ 90% α-helix), exhibits diminished thermostability (T_m = 40 °C; ΔH_m = 27 ± 2 kcal mol^− 1; ΔS_m = 86 ± 6 cal mol^− 1 K^− 1) and noncooperative unfolding (〈n_c〉 ∼ 1) vis-à-vis the in situ Δ-domain. Temperature-dependent Raman markers of subunit side chains, particularly those of Phe and Trp residues, also confirm different local interactions for the in situ and in vitro Δ-domains. The present results clarify the key role of the gp5 Δ-domain in prohead I architecture by providing direct evidence of domain structure stabilization and interdomain interactions within the assembled shell.     

Tyrosinase activity of Greyia flanaganii (Bolus) constituents

Hyper-pigmentation of the skin is a common problem that is prevalent in middle aged and elderly people. It is caused by over production of melanin. Tyrosinase is known to be the key enzyme in melanin production. Ethanolic extract of Greyia flanaganii leaves showed significant (P < 0.05) antityrosinase activity exhibiting the IC₅₀ of 32.62 μg/ml. The total extract was further investigated for its toxicity and effect on melanin production by melanocytes cells, and showed significant inhibition (P < 0.05) (20%) of melanin production at 6.25 μg/ml and low levels of cytotoxicity (IC₅₀ < 400 μg/ml). The amount of antioxidants necessary to decrease the initial DPPH absorbance by 50% (EC₅₀) by the total ethanolic extract was found to be 22.01 μg/ml. The effect of G. flanaganii against acne causing bacteria, Propionibacterium acnes, was investigated using microdilution assay. The MIC of the extract of G. flanaganii was found to be 250 μg/ml. Bioassay-guided fractionation led to the isolation of (3S)-4-hydroxyphenethyl 3-hydroxy-5-phenylpentanoate (1), 2′,4′,6′-trihydroxydihydrochalcone (2), 2′,6′,4-trihydroxy-4′-methoxydihydrochalcone (3), 2′,6′-dihydroxy-4′-methoxydihydrochalcone (4), 5,7-dihydroxyflavanone [(2S)-pinocembrin] (5), 2′,6′-dihydroxy-4′,4-dimethoxy dihydrochalcone (6) and (2R,3R)-3,5,7-trihydroxy-3-O-acetylflavanone (7). The isolated compounds were tested for their antioxidant, cytotoxicity, tyrosinase inhibition and antibacterial activities. Compound 2 exhibited significant (P < 0.05) antityrosinase activity exhibiting the IC₅₀ of 69.15 μM. The isolated compounds showed low toxicity of the cells with reduction of melanin content of the cells. All compounds tested showed good radical scavenging activity. These data indicates that G. flanaganii extract and its isolated phenolic constituents could be possible skin lightening agents.     

Using S-adenosyl-l-homocysteine capture compounds to characterize S-adenosyl-l-methionine and S-adenosyl-l-homocysteine binding proteins

S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH–CC with biotin used in conjunction with streptavidin–horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the K_d values for COMT, SAHH, and PRDM2 (24.1 ± 2.2 μM, 6.0 ± 2.9 μM, and 10.06 ± 2.87 μM, respectively) and found them to be close to previously established K_d values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs.