Modulation of alfa-adrenoceptor activity by beta-adrenoceptor agonists and antagonists in rat brain cortex membranes

The influence of β-adrenoceptor activation and inhibition by isoprenaline and propranolol on the specific binding of nonselective α₁- and α₂-adrenoceptor antagonists [³H]prazosin and [³H]RX821002 in rat cerebral cortex subcellular membrane fractions was studied. It was established that for the α₁- and α₂-adrenoceptors the ligand–receptor interaction corresponds to the model of one affinity pool of receptors and binding of two ligand molecules by one dimer receptor. The parameters of [³H]prazosin binding to α₁-adrenoceptors were: K _d = 1.85 ± 0.16 nM, B _max = 31.14 ± 0.35 fmol/mg protein, n = 2. The parameters of [³H]RX821002 binding to α₂-adrenoceptors were: K _d = 1.57 ± 0.27 nM, B _max = 7.2 ± 1.6 fmol/mg protein, n = 2. When β-adrenoceptors were activated by isoprenaline, the binding of radiolabelled ligands with α₁- and α₂-adrenoceptors occurred according to the same model. The affinity to [³H]prazosin and the concentration of active α₁-adrenoceptors increased by 27% (K _d = 1.36 ± 0.03 nM) and 84% (B _max = 57.37 ± 0.28 fmol/mg protein), respectively. The affinity of α₂-adrenoceptors to [³H]RX821002 decreased by 56% (K _d = 3.55 ± 0.02 nM), and the concentration of active receptors increased by 69% (B _max = 12.24 ± 0.06 fmol/mg protein). Propranolol alters the binding character of both ligands. For [³H]prazosin and [³H]RX821002, two pools of receptors were detected with the following parameters: K _d1 = 1.13 ± 0.09, K _d2 = 6.07 ± 1.06 nM, B _m1 = 11.36 ± 1.77, Bm2 = 51.09 ± 0.41 fmol/mg protein, n = 2 and K _d1 = 0.61 ± 0.02, K _d2 = 3.41 ± 0.13 nM, B _m1 = 1.88 ± 0.028, B _m2 = 9.27 ± 0.08 fmol/mg protein, n = 2, respectively. The concentration of active receptors (B _max) increased twofold for both ligands. It was suggested that α₁- and α₂-adrenoceptors in rat cerebral cortex subcellular membrane fractions exist as dimers. A modulating influence of isoprenaline and propranolol on the specific binding of the antagonists to α₁- and α₂- adrenoceptors was revealed, which was manifested in the activating effect on the [³H]prazosin binding parameters, in the inhibitory effect on the [³H]RX821002 binding parameters, and in a change of the general character of binding for both ligands.     

Comparative characteristics of binding kinetics of specific antagonists by α<Subscript>1</Subscript>- and α<Subscript>2</Subscript>-adrenoceptors in rat cerebral cortex membranes

The binding of specific nonselective α₁- and α₂-adrenoceptor antagonists [³H]prazosine and [³H]RX821002 has been studied on rat cerebral cortex synaptosomal membranes. It is shown that for α₁-adrenoceptors the ligand-receptor interaction corresponds to the model assuming the presence of one pool of receptors and binding of two ligand molecules to the receptor. The parameters of [³H]prazosine binding to α₁-adrenoceptors were: K _d= 1.56 ± 0.17 nM, B _max = 30.25 ± 1.78 fmol/mg protein, n = 2. The parameters of [³H]RX821002 binding to α₂-adrenoceptors were: K _d = 1.94 ± 0.08 nM, B _max = 12.77 ± 3.17 fmol/mg protein, n = 2. For α₂ -adrenoceptors the ligand-receptor interaction corresponded to the same model. For α₁ - and α₂-adrenoceptor antagonists the dissociation constants (K _d) are approximately equal (1.56 ± 0.17 and 1.94 ± 0.08 nM, respectively), but the concentration of α₂-adrenoceptors is two times lower than that of α₁-adrenoceptors ( 12.77 ± 3.17 and 30.25 ± 1.78 fmol/mg protein, respectively). The efficiency (E = B _max/2K _d) of the ligand binding to α₁-adrenoceptors is 2.3 times higher than that to α₂-adrenoceptors (7.46 ± 1.32 and 3.29 ± 0.68 fmol/mg protein/nM, respectively. The data suggest that α₁- and α₂ -adrenoceptors in rat cerebral cortex exist as dimers.     

Surface Plasmon Resonance Immunosensor for the Detection of <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis>

Environmental surveillance of the Gram-negative bacterium Burkholderia pseudomallei is important in order to define human populations at risk of acquiring the infection; hence, in this study, we developed a method for the detection of B. pseudomallei based on surface plasmon resonance (SPR) using 4-mercaptobenzoic acid (4-MBA) modified gold SPR chip by monitoring the interaction of rpGroEL antigen (rpGroEL Ag) with immobilized rabbit antibody (anti-rpGroEL rAb). Affinity constant (K _D ) and maximum binding capacity of analyte (B _max) values for the interaction of rpGroEL Ag with the immobilized anti-rpGroEL rAb were calculated by using kinetic evaluation software and found to be 14.7 7 pM and 105.40 m^o, respectively. In addition, thermodynamic parameters such as ?G (Gibb’s free energy change), ?H (change in the enthalpy), and ?S (change in the entropy) were determined for the interaction between rpGroEL Ag and immobilized anti-rpGroEL rAb, and the values revealed that the interaction is spontaneous, exothermic, and driven by entropy.     

Synthesis of oligosaccharide fragments of mannan from <Emphasis Type="Italic">Candida albicans</Emphasis> cell wall and their BSA conjugates

3-Aminopropyl glycosides of α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose, α-D-mannopyranosyl-(1→3)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose, and α-D-mannopyranosyl-(1→2)-[α-D-mannopyranosyl-(1→3)]-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose were efficiently synthesized starting from ethyl 2-O-acetyl(benzoyl)-3,4,6-tri-O-benzyl-1-thio-α-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2-O-benzoyl-1-thio-α-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2,3-di-O-benzoyl-1-thio-α-D-mannopyranoside, and 2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl bromide. The oligosaccharide chains synthesized correspond to the three structural types of side chains of mannan from Candida albicans cell wall. A conjugate of the third pentasaccharide with bovine serum albumin was prepared using the squarate method.     

Calponin-like protein from mussel smooth muscle is a competitive inhibitor of actomyosin ATPase

The goal of this work was to elucidate the mechanism of inhibition of the actin-activated ATPase of myosin subfragment-1 (S1) by the calponin-like protein from mussel bivalve muscle. The calponin-like protein (Cap) is a 40-kDa actin-binding protein from the bivalve muscle of the mussel Crenomytilus grayanus. Kinetic parameters V_max and K_ATPase of actomyosin ATPase in the absence and the presence of Cap were determined to investigate the mechanism of inhibition. It was found that Cap mainly causes increase in K_ATPase value and to a lesser extent the decrease in V_max, which indicates that it is most likely a competitive inhibitor of actomyosin ATPase. Analysis of V_max and K_ATPase parameters in the presence of tropomyosin revealed that the latter is a noncompetitive inhibitor of the actomyosin ATPase.     

Purification,characterization and gene analysis of a new α-glucosidase from <Emphasis Type="Italic">shiraia</Emphasis> sp. SUPER-H168

A new α-glucosidase from Shiraia sp. SUPER-H168 under solid-state fermentation was purified by alcohol precipitation and anion-exchange and by gel filtration chromatography. The optimum pH and temperature of the purified α-glucosidase were 4.5 and 60 °C, respectively, using p-nitrophenyl-α-glucopyranoside (α-pNPG) as a substrate. Ten millimoles of sodium dodecyl sulfate, Fe²⁺, Cu²⁺, and Ag⁺ reduced the enzyme activity to 0.7, 7.6, 26.0, and 6.2 %, respectively, of that of the untreated enzyme. The K _m, V _max, and k _cat/K _m of the α-glucosidase were 0.52 mM, 3.76 U mg^?1, and 1.3?×?10⁴ L s^?1 mol^?1, respectively. K _m with maltose was 0.62 mM. Transglycosylation activities were observed with maltose and sucrose as substrates, while there was no transglycosylation with trehalose. DNA and its corresponding full-length cDNA were cloned and analyzed. The α-glucosidase coding region consisted of a 2997-bp open reading frame encoding a 998-amino acid protein with a 22-amino acid signal peptide; one 48-bp intron was located. The α-glucosidase was a monomeric protein with a predicted molecular mass of 108.2 kDa and a predicted isoelectric point of 5.08. A neighbor-joining phylogenetic tree demonstrated that Shiraia sp. SUPER-H168 α-glucosidase is an ascomycetes α-glucosidase. This is the first report of α-glucosidase from a filamentous fungus that had good glycoside hydrolysis with maltose and α-pNPG, transglycosylation and conversion activity of maltose into trehalose.     

Stomatal regulation in Douglas fir following a fungal-mediated chronic reduction in leaf area

Pathogens can cause chronic premature needle abscission in coniferous species. To assess the potential impacts on tree productivity, stomatal regulation was investigated in Douglas fir with chronic stomatal occlusion and defoliation from varying levels of the Swiss needle cast (SNC) fungus, Phaeocryptopus gaeumannii. Levels of SNC disease and subsequent defoliation were manipulated by choosing six sites with varying levels of disease and by foliar applications of fungicides on six trees per site. Diurnal measurements of leaf water potential (Ψ_leaf), stomatal conductance (g _s) and vapor pressure deficit (D) were made on six fungicide treated and six control trees per site. In addition, leaf specific hydraulic conductance was calculated on a single branch (K _{L_B}) from three trees per treatment per site. Stomatal conductance at D=1 kPa (g _sref) was negatively correlated with fungal colonization (number of fruiting bodies present in needle stomata) and positively correlated with K _{L_B}. Despite reduced needle retention in diseased trees, K _L declined due to a reduction in sapwood area and permeability (i.e., increasing presence of latewood in functional sapwood). In general, stomatal sensitivity to D for all foliage was consistent with stomatal regulation based on a simple hydraulic model [g _s=K _L(Ψ_soil?Ψ_leaf)/ D], which assumes strict stomatal regulation of Ψ_leaf. However, when fungal presence reduced maximum g _s below the potential maximum supported by hydraulic architecture, stomatal sensitivity was lower than expected based on the theoretical relationship: dg _s/dlnD=0.6·g _sref. The results indicate that losses in productivity associated with physical blockage of stomata and defoliation are compounded by additional losses in K _L and a reduction in g _s in remaining functional stomata.     

Improving the acidic stability of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> α-acetolactate decarboxylase in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> by changing basic residues to acidic residues

The α-acetolactate decarboxylase (ALDC) can reduce diacetyl fleetly to promote mature beer. A safe strain Bacillus subtilis WB600 for high-yield production of ALDC was constructed with the ALDC gene saald from Staphylococcus aureus L3-15. SDS-PAGE analysis revealed that S. aureus α-acetolactate decarboxylase (SaALDC) was successfully expressed in recombinant B. siutilis strain. The enzyme SaALDC was purified using Ni-affinity chromatography and showed a maximum activity at 45 °C and pH 6.0. The values of K _m and V _max were 17.7 μM and 2.06 mM min^?1, respectively. Due to the unstable property of SaALDC at low pH conditions that needed in brewing process, site-directed mutagenesis was proposed for improving the acidic stability of SaALDC. Homology comparative modeling analysis showed that the mutation (K52D) gave rise to the negative-electrostatic potential on the surface of protein while the numbers of hydrogen bonds between the mutation site (N43D) and the around residues increased. Taken together the effect of mutation N43D-K52D, recombinant SaALDC_N43D-K52D showed dramatically improved acidic stability with prolonged half-life of 3.5 h (compared to the WT of 1.5 h) at pH 4.0. In a 5-L fermenter, the recombinant B. subtilis strain that could over-express SaALDC_N43D-K52D exhibited a high yield of 135.8 U mL^?1 of SaALDC activity, about 320 times higher comparing to 0.42 U mL^?1 of S. aureus L3-15. This work proposed a  strategy for improving the acidic stability of SaALDC in the  B. subtilis host.     

Polyhydroxylated steroid compounds from the Far Eastern starfish <Emphasis Type="Italic">Distolasterias nipon</Emphasis>

Two new steroid glycosides: distolasteroside D₆, (24S)-24-O-(β-D-xylopyranosyl)-5α-cholestane-3β,6α,8,15β,16β,24-hexaol, and distolasteroside D₇, (22E,24R)-24-O-(β-D-xylopyranosyl)-5α-cholest-22-ene-3β,6α,8,15β,24-pentaol were isolated along with the previously known distolasterosides D₁, D₂, and D₃, echinasteroside C, and (25S)-5α-cholestane-3β4β,6α,7α,8,15α,16β,26-octaol from the Far Eastern starfish Distolasterias nipon. The structures of new compounds were elucidated by NMR spectroscopy and MALDI TOF mass spectrometry. Like neurotrophins, distolasterosides D₁, D₂, and D₃ were shown to induce neuroblast differentiation in a mouse neuroblastoma C1300 cell culture.     

Characterization of Active Barley α-Amylase 1 Expressed and Secreted by Saccharomyces cerevisiae

Recombinant barley α-amylase 1 isozyme was constitutively secreted by Saccharomyces cerevisiae. The enzyme was purified to homogeneity by ultrafiltration and affinity chromatography. The protein had a correct N-terminal sequence of His-Gln-Val-Leu-Phe-Gln-Gly-Phe-Asn-Trp, indicating that the signal peptide was efficiently processed. The purified α-amylase had an enzyme activity of 1.9 mmol maltose/mg protein/min, equivalent to that observed for the native seed enzyme. The k_cat/K_m was 2.7 × 10² mM^?1.s^?1, consistent with those of α-amylases from plants and other sources.     

A chimeric α-amylase engineered from <Emphasis Type="Italic">Bacillus acidicola</Emphasis> and G<Emphasis Type="Italic">eobacillus thermoleovorans</Emphasis> with improved thermostability and catalytic efficiency

The α-amylase (Ba-amy) of Bacillus acidicola was fused with DNA fragments encoding partial N- and C-terminal region of thermostable α-amylase gene of Geobacillus thermoleovorans (Gt-amy). The chimeric enzyme (Ba-Gt-amy) expressed in Escherichia coli displays marked increase in catalytic efficiency [K _cat: 4 × 10⁴ s^?1 and K _cat/K _m: 5 × 10⁴ mL^?1 mg^?1 s^?1] and higher thermostability than Ba-amy. The melting temperature (T _m) of Ba-Gt-amy (73.8 °C) is also higher than Ba-amy (62 °C), and the CD spectrum analysis revealed the stability of the former, despite minor alteration in secondary structure. Langmuir–Hinshelwood kinetic analysis suggests that the adsorption of Ba-Gt-amy onto raw starch is more favourable than Ba-amy. Ba-Gt-amy is thus a suitable biocatalyst for raw starch saccharification at sub-gelatinization temperatures because of its acid stability, thermostability and Ca²⁺ independence, and better than the other known bacterial acidic α-amylases.     

Analysis of the <Emphasis Type="Italic">xynB5</Emphasis> gene encoding a multifunctional GH3-BglX β-glucosidase-β-xylosidase-α-arabinosidase member in <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted β-glucosidase-β-xylosidase enzyme that was amplified by polymerase chain reaction; the product was cloned into the blunt ends of the pJet1.2 plasmid. Analysis of the protein sequence indicated the presence of conserved glycosyl hydrolase 3 (GH3), β-glucosidase-related glycosidase (BglX) and fibronectin type III-like domains. After verifying its identity by DNA sequencing, the xynB5 gene was linked to an amino-terminal His-tag using the pTrcHisA vector. A recombinant protein (95 kDa) was successfully overexpressed from the xynB5 gene in E. coli Top 10 and purified using pre-packed nickel-Sepharose columns. The purified protein (BglX-V-Ara) demonstrated multifunctional activities in the presence of different substrates for β-glucosidase (pNPG: p-nitrophenyl-β-D-glucoside) β-xylosidase (pNPX: p-nitrophenyl-β-D-xyloside) and α-arabinosidase (pNPA: p-nitrophenyl-α-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 °C for β-glucosidase and α-l-arabinosidase and 60 °C for β-xylosidase. BglX-V-Ara predominantly presented β-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (K_m 0.24 ± 0.0005 mM, V_max 0.041 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.27 mM^?1 s^?1), followed by β-xylosidase (K_m 0.64 ± 0.032 mM, V_max 0.055 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.14 mM^?1s^?1) and finally α-l-arabinosidase (K_m 1.45 ± 0.05 mM, V_max 0.091 ± 0.0004 µmol min^?1 mg^?1 and K_cat/K_m 0.1 mM^?1 s^?1). To date, this is the first report to demonstrate the characterization of a GH3-BglX family member in C. crescentus that may have applications in biotechnological processes (i.e., the simultaneous saccharification process) because the multifunctional enzyme could play an important role in bacterial hemicellulose degradation.     

New paradoxical three-finger toxin from the cobra <Emphasis Type="Italic">Naja kaouthia</Emphasis> venom: Isolation and characterization

A new three-finger toxin nakoroxin was isolated from the cobra Naja kaouthia venom, and its complete amino acid sequence was established. Nakoroxin belongs to the group of “orphan” toxins, data on the biological activity of which are practically absent. Nakoroxin shows no cytotoxicity and does not inhibit the binding of α-bungarotoxin to nicotinic acetylcholine receptors of muscle and α7 types. However, it potentiates the binding of α-bungarotoxin to the acetylcholine-binding protein from Lymnaea stagnalis. This is the first toxin with such an unusual property.     

Characterization of the interactions between coumarin-derivatives and acetylcholinesterase: Examination by NMR and docking simulations

Alzheimer’s disease (AD) is one of the most common forms of dementia and a significant threat to the elderly populations, especially in the Western world. The rapid hydrolysis of the principal neurotransmitter into choline and acetate by acetylcholinesterase (AChE) at synapses causes the loss of cognitive response that becomes the real cause of AD. Therefore, inhibition of AChE is the most fundamental therapy among currently available treatments for AD. In this context, we designed and performed molecular recognitions studies of coumarin-based inhibitors towards AChE. STD NMR and Tr-NOESY applications were utilized to evaluate the binding epitope, the dissociation constant (K_D) and bound conformations of these inhibitors within this inhibitor-AChE complex. Compound 1, which has a similar inhibition activity to tacrine (a current drug) led in this study as a stronger binder with K_D?=?30 μM ,even greater than tacrine (K_D?=?140 μM). Moreover, docking simulations mimic NMR results and provided evidence of synchronizing binding of compound 1 with three sites; the peripheral anionic site, the bottom of the gorge, and the catalytic site. Therefore, we envisioned from our experimental and theoretical results that coumarin-based inhibitors containing a piperidinyl scaffold might be a potential drug candidates for AD in the future.     

Study of Surface Plasmon Resonances of Core-Shell Nanosphere: A Comparison between Numerical and Analytical Approach

An investigation of the wavelength dependent extinction spectra of coated sphere with different core@shell compositions based on discrete dipole approximation technique has been presented in this paper. We have used combinations of A g, A u, and S i O ₂ for this analysis. Specifically, we study the impact of spherical core-shell thickness on its surface plasmon resonance (SPR) peak positions and corresponding spectral widening in distinct regimes of the spectrum. We observe that SPR peak of core-shell nanoparticle(CSNP) can be tuned over the visible to near-infrared spectrum region by manipulating the core/shell ratio and composition of core and shell. Specifically, for dielectric@metal (core@shell) nanoparticle, SPR peak position shifted towards lower wavelength as we increase the shell thickness, which is opposite to the SPR behavior of metal@dielectric. The extinction spectrum shows linear relation between SPR position and thickness of the shell. Further, we observed two resonant peaks for the case of metal@metal CSNP. The SPR peak of Au@Ag (a _eff 100 nm with shell thickness 8 nm) reveals two resonant peak corresponding to Au (594 nm) in red domain, while the peak in blue domain corresponds to Ag (402 nm). We also observe that optical resonance of CSNP can be tuned across the near-infrared region by changing the surrounding medium of higher refractive index. Further, near field pattern of core@shell geometry at resonance wavelength is also shown in the present study. We have also compared the numerical and analyticalmethod for smaller size CSNP with varying thickness and the results show good agreement.     

How Common Are Intragene Windows with <Emphasis Type="Italic">K</Emphasis><Subscript>A</Subscript> > <Emphasis Type="Italic">K</Emphasis><Subscript>S</Subscript> Owing to Purifying Selection on Synonymous Mutations?

One method for diagnosing the mode of sequence evolution considers the ratio of nonsynonymous substitutions per nonsynonymous site (K _A) to the corresponding figure for synonymous substitutions (K _S). A ratio (K _A/K _S) greater than unity is taken as evidence for positive selection. This, however, need not necessarily be the case. Notably, there is one instance of a high intragenic K _A/K _S peak, revealed by sliding window analysis and observed in two pairwise comparisons, better accounted for by localised purifying selection on synonymous mutations that affect splicing. Is this example exceptional? To address this we isolate intragenic domains with K _A/K _S > 1 from more than 1000 long mouse-rat orthologues. Approximately one K _A/K _S > 1 peak is found per 12–15 kb of coding sequence. Surprisingly, low synonymous substitution rates underpin more incidences than do high nonsynonymous rates. Several reasons, however, prevent us from supposing that the low synonymous rates reflect purifying selection on synonymous mutations. First, for many peaks, the null that the peak is no higher than expected given the underlying rates of evolution, cannot be rejected. Second, of 18 statistically significant incidences with unusually low K _S values, only 3 are repeatable across independent comparisons. At least two of these are within alternatively spliced exons. We conclude that repeatable statistically significant intragenic domains of low intragenic K _S are rare. As so few K _A/K _S peaks reflect increased rates of protein evolution and so few hold statistical support, we additionally conclude that sliding window analysis to infer domains of positive selection is highly error-prone.     

<Emphasis Type="Italic">CENTB5</Emphasis> gene expression in humans and mice

Centaurin β5, a protein with a yet unknown function, belongs to the centaurin family. It is encoded by CENTB5, whose expression pattern has been studied insufficiently. Intron 14–15 of human CENTB5 contains a lowly variable minisatellite repeat UPS29, while the mouse Centb5 contains an imperfect microsatellite repeat (CATG)₁₉. The shorter UPS29 alleles have previously been associated with certain forms of Parkinson’s disease and epilepsy. Moreover, both human and murine CENTB5 are syntenic with SCNN1D and ACOT7, which are active primarily in the nervous system, and whose aberrations are associated with epilepsy and neurodegenerative processes. As intronic sequences can modulate the expression of not only those genes that harbor them, but also of neighboring and remote genes, the CENTB5, SCNN1D, and ACOT7 expression levels were all analyzed by RT-PCR. The potential of intronic tandem repeats UPS29 and (CATG)₁₉ to regulate/modulate the expression of CENTB5, SCNN1D, and ACOT7 has been assessed in silico. CENTB5, SCNN1D, and ACOT7 expression was detected in all human and murine tissues studied, suggestive of their physiologic importance. The putative role of UPS29 in the regulation of CENTB5, SCNN1D, and ACOT7 activity in the nerve tissue is discussed.     

High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension

Background

Dicer is a 219-kDa protein that plays key roles in gene regulation, particularly as the ribonuclease III enzyme responsible for cleaving precursor miRNA substrates. Its enzymatic activity is highly regulated by protein factors, and this regulation can impact on the levels of miRNAs and modulate the behavior of a cell. To better understand the underlying mechanisms of regulation, detailed enzymatic and structural characterization of Dicer are needed. However, these types of studies generally require several milligrams of recombinant protein, and efficient preparation of such quantities of pure human Dicer remains a challenge. To prepare large quantities of human Dicer, we have optimized transfection in HEK293-6E cells grown in suspension and streamlined a purification procedure.

Results

Transfection conditions were first optimized to achieve expression levels between 10 and 18?mg of recombinant Dicer per liter of culture. A three-step purification protocol was then developed that yields 4–9?mg of purified Dicer per liter of culture in a single day. From SEC-MALS/RI analysis and negative stain TEM, we confirmed that the purified protein is monomerically pure ( ≥ 98%) and folds with the characteristic L-shape geometry. Using an electrophoretic mobility shift assay, a dissociation constant (K_d) of 5?nM was measured for Dicer binding to pre-let-7a-1, in agreement with previous reports. However, when probing the cleavage activity of Dicer for pre-let-7a-1, we measured k_cat (7.2?±?0.5?min^??1) and K_M (1.2?±?0.3?μM) values that are much higher than previously reported due to experimental conditions that better respect the steady-state assumption.

Conclusions

The expression and purification protocols described here provide high yields of monomerically pure and active human Dicer. Cleavage studies of a pre-let-7 substrate with this purified Dicer reveal higher k_cat and K_M values than previously reported and support the current view that conformational changes are associated with substrate binding. Large quantities of highly pure Dicer will be valuable for future biochemical, biophysical and structural investigations of this key protein of the miRNA pathway.

     

Kinetic characterization of a novel acid ectophosphatase from <Emphasis Type="Italic">Enterobacter asburiae</Emphasis>

Expression of acid ectophosphatase by Enterobacter asburiae, isolated from Cattleya walkeriana (Orchidaceae) roots and identified by the 16S rRNA gene sequencing analysis, was strictly regulated by phosphorus ions, with its optimal activity being observed at an inorganic phosphate concentration of 7 mM. At the optimum pH 3.5, intact cells released p-nitrophenol at a rate of 350.76 ± 13.53 nmol of p-nitrophenolate (pNP)/min/10⁸ cells. The membrane-bound enzyme was obtained by centrifugation at 100,000 × g for 1 h at 4°C. p-Nitrophenylphosphate (pNPP) hydrolysis by the enzyme follows “Michaelis-Menten” kinetics with V = 61.2 U/mg and K_0.5 = 60 μM, while ATP hydrolysis showed V = 19.7 U/mg, K_0.5 = 110 μM, and n_H = 1.6 and pyrophosphate hydrolysis showed V = 29.7 U/mg, K_0.5 = 84 μM, and n_H = 2.3. Arsenate and phosphate were competitive inhibitors with Ki = 0.6 mM and K_i = 1.8 mM, respectively. p-Nitrophenyl phosphatase (pNPPase) activity was inhibited by vanadate, while p-hydroxymercuribenzoate, EDTA, calcium, copper, and cobalt had no inhibitory effects. Magnesium ions were stimulatory (K_0.5 = 2.2 mM and n_H = 0.5). Production of an acid ectophosphatase can be a mechanism for the solubilization of mineral phosphates by microorganisms such as Enterobacter asburiae that are versatile in the solubilization of insoluble minerals, which, in turn, increases the availability of nutrients for plants, particularly in soils that are poor in phosphorus.     

Interaction of the synthetic immunomodulatory dipeptide bestim with murine macrophages and thymocytes

The tritium-labeled dipeptide bestim (γ-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by high-temperature solid-state catalytic isotope exchange. It was found that [³H]bestim binds with a high affinity to murine peritoneal macrophages (K _d 2.1 ± 0.1 nM) and thymocytes (K _d 3.1 ± 0.2 nM), as well as with plasma membranes isolated from these cells (K _d 18.6 ± 0.2 and 16.7 ± 0.3 nM, respectively). The specific binding of [³H]bestim to macrophages and thymocytes was inhibited by the unlabeled dipeptide thymogen (L-Glu-L-Trp) (K _i 0.9 ± 0.1 and 1.1 ± 0.1 nM, respectively). After treatment with trypsin, macrophages and thymocytes lost the ability to bind [³H]bestim. Bestim in the concentration range of 10^?10 to 10^?6 M reduced the adenylate cyclase activity in the membranes of murine macrophages and thymocytes.