Interaction of cholera toxin B-subunit with human T-lymphocytes

In this work, ¹²⁵I-labeled cholera toxin B-subunit (CT-B) (specific activity 98 Ci/mmol) was prepared, and its high-affinity binding to human blood T-lymphocytes (K _d = 3.3 nM) was determined. The binding of the ¹²⁵I-labeled CT-B was inhibited by unlabeled interferon-α₂ (IFN-α₂), thymosin-α1 (TM-α₁), and by the synthetic peptide LKEKK, which corresponds to sequences 16-20 of human TM-α₁ and 131-135 of IFN-α₂ (K _i 0.8, 1.2, and 1.6 nM, respectively), but was not inhibited by the unlabeled synthetic peptide KKEKL with inverted sequence (K _i > 1 μM). In the concentration range of 10-1000 nM, both CT-B and peptide LKEKK dose-dependently increased the activity of soluble guanylate cyclase (sGC) but did not affect the activity of membrane-bound guanylate cyclase. The KKEKL peptide tested in parallel did not affect sGC activity. Thus, the CT-B and peptide LKEKK binding to a common receptor on the surface of T-lymphocytes leads to an increase in sGC activity.     

α<Subscript>1</Subscript>-Thymosin, α<Subscript>2</Subscript>-interferon,and the LKEKK syntetic peptide inhibit the binding of the B subunit of the cholera toxin to intestinal epithelial cell membranes

The ¹²⁵I-labeled B-subunit of the cholera toxin ([125I]CT-B, specific activity of 98 Ci/mmol) was prepared. This subunit was shown to be bound to the membranes which were isolated from epithelial cells of a mucous tunic of the rat thin intestine with high affinity (K _d = 3.7 nM). The binding of the labeled protein was inhibited by the unlabeled α₂-interferon (IFN-α₂), α₁-thymosin, (TM-α₁), and the LKEKK synthetic peptide corresponding to the 16–20 sequence of TM-α₁ and the 131–135 sequence of human IFN-α₂ (Ki 1.0, 1.5, and 2.0 nM, respectively), whereas the KKEKL unlabeled synthetic peptide did not inhibit the binding (K _i > 100 μМ). The LKEKK peptide and CT-B were shown to dose-dependently increase an activity of the soluble guanylate cyclase (sGC) in the concentration range from 10 to 1000 nM. Thus, the binding of TM- α₁, IFN-α₂, and the LKEKK peptide to the CT-B receptor on a surface of the epithelial cells of the mucous tunic of the rat thin intestine resulted in an increase in the intracellular level of cGMP.     

Binding of synthetic LKEKK peptide to human T-lymphocytes

The synthetic peptide LKEKK corresponding to sequence 16-20 of human thymosin-α₁ and 131-135 of human interferon-α₂ was labeled with tritium to specific activity 28 Ci/mol. The [³H]LKEKK bound with high affinity (K_d = 3.7 ± 0.3 nM) to donor blood T-lymphocytes. Treatment of cells with trypsin or proteinase K did not abolish [³H]LKEKK binding, suggesting the non-protein nature of the peptide receptor. The binding was inhibited by thymosin-α₁, interferon-α₂, and cholera toxin B subunit (K_i = 2.0 ± 0.3, 2.2 ± 0.2, and 3.6 ± 0.3 nM, respectively). Using [3H]LKEKK, we demonstrated the existence of a non-protein receptor common for thymosin-α₁, interferon-α₂, and cholera toxin B-subunit on donor blood T-lymphocytes.     

The LKEKK synthetic peptide as a ligand of rat intestinal epithelial cell membranes

A tritium-labeled synthetic LKEKK pentapeptide corresponding to the sequences 16–20 of human thymosin-α₁ and 131–135 of human interferon-α₂ was obtained with a specific activity of 28 Ci/mmol. [³H]LKEKK was found to bind with high affinity (K _d 3.7 ± 0.3 nM) to the membranes isolated from epithelial cells of rat small intestinal mucosa. The trypsin treatment of the membranes did not affect the binding, thus supporting the nonprotein nature of the peptide receptor. The binding of the labeled peptide was inhibited by unlabeled thymosin-α₁, interferon-α₂, and cholera toxin B subunit (K _i 4.2 ± 0.4, 3.5 ± 0.3, and 4.7 ± 0.3 nM respectively). The pentapeptide did not affect the adenylate cyclase activity within the concentration range of 1–1000 nM.     

Binding of β-endorphin and its fragments to the nonopiod receptor of murine peritoneal macrophages

The tritium-labeled selective agonist of the nonopioid β-endorphin receptor the decapeptide immunorphin ([³H]SLTCLVKGFY) with a specific activity of 24 Ci/mmol was prepared. It was shown that [³H]immunorphin binds with a high affinity to the non-opioid β-endorphin receptor of mouse peritoneal macrophages (K _d 2.4 ± 0.1 nM). The specific binding of [³H]immunorphin to macrophages was inhibited by unlabeled β-endorphin (K _i of the [³H]immunorphin-receptor complex 2.9 ± 0.2 nM) and was not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin, and [Met⁵]enkephalin (K _i > 10 μM). Thirty fragments of β-endorphin were synthesized, and their ability to inhibit the specific binding of [³H]immunorphin to macrophages was studied. It was found that the shortest peptide having practically the same inhibitory activity as β-endorphin is its fragment 12–19 (K _i 3.1 ± 0.3 nM).     

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.     

Synthetic peptide KKRR corresponding to the human ACTH fragment 15–18 is an antagonist of the ACTH receptor

Tritium-labeled synthetic fragments of human adrenocorticotropic hormone (ACTH) [³H]ACTH (11–24) and [³H]ACTH (15–18) with a specific activity of 22 and 26 Ci/mmol, respectively, were obtained. It was found that [³H]ACTH-(11–24) binds to membranes of the rat adrenal cortex with high affinity and high specificity (K _d 1.8 ± 0.1 nM). Twenty nine fragments of ACTH (11–24) were synthesized, and their ability to inhibit the specific binding of [³H]ACTH (11–24) to adrenocortical membranes was investigated. The shortest active peptide was found to be an ACTH fragment (15–18) (KKRR) (K _i 2.3 ± 0.2 nM), whose [³H] labeled derivative binds to rat adrenocortical membranes (K _d 2.1 ± 0.1 nM) with a high affinity. The specific binding of [³H]ACTH-(15–18) was inhibited by 100% by unlabeled ACTH (11–24) (K _i 2.0 ± 0.1 nM). ACTH (15–18) in the concentration range of 1–1000 nM did not affect the adenylate cyclase activity of adrenocortical membranes and, therefore, is an antagonist of the ACTH receptor.     

Rational design and molecular engineering of peptide aptamers to target human pancreatic trypsin in acute pancreatitis

Human pancreatic trypsin (hPT) is an established target for acute pancreatitis (AP) therapeutics. Here, a bioinformatics protocol of protein docking, peptide refinement, dynamics simulation and affinity analysis was described to perform rational design and molecular engineering of hPT peptide aptamers. Protein docking was employed to model the intermolecular interactions between hPT and its cognate inhibitory protein, the human pancreatic trypsin inhibitor (hTI). A number of peptide fragments were cut out from the interaction sites of docked hPT–hTI complexes, from which a decapeptide fragment ¹³LNGCTLEYRP²² was found to exhibit potent inhibition against hPT (K _i = 5.3 ± 0.8 μM). We also carried out alanine scanning and virtual mutagenesis to systematically examine the independent contribution of peptide residues to binding affinity, and the harvested knowledge were then used to guide modification and optimization of the decapeptide fragment. Subsequently, inhibition studies of nine promising candidates against recombinant hPT were conducted, from which four samples were successfully identified to have high or moderate potency (K _i < 10 μM). In particular, the peptides LQVCTLEYCN and LQICTLEYCT were found to inhibit hPT activity significantly (K _i = 0.23 ± 0.04 and 0.85 ± 0.18 μM, respectively). Structural analysis of hPT–peptide complex systems unraveled diverse chemical interactions such as hydrogen bonds, salt bridges and hydrophobic forces across the complex interfaces.     

Simulations of the Ion Current to a Probe in Plasma with Allowance for Ionization and Ion–Neutral Collisions: II. Cylindrical Probe

The ion current to a cylindrical probe is considered with allowance for volume ionization, ion–neutral collisions, and the ion orbital moment. A model based on the molecular dynamics method and applicable in a wide range of plasma parameters (r_p/λ_D= 0.01–100, r_i/λ_D= 0.002–200, ν_i/ω_0i= 0.01–0.05, and T_i/T_e = 0?0.01) is proposed A convenient representation of the dependence of the relative ion current density on the Langmuir coefficient β² and a technique for determining the plasma density from simulation results are offered.     

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.     

Assessing biodegradation of furfuryl alcohol using <Emphasis Type="Italic">Pseudomonas putida</Emphasis> MTCC 1194 and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> MTCC 1034 and its kinetics

The biodegradation of furfuryl alcohol (FA) in shake flask experiments using a pure culture of Pseudomonas putida (MTCC 1194) and Pseudomonas aeruginosa (MTCC 1034) was studied at 30 °C and pH 7.0. Experiments were performed at different FA concentrations ranging from 50 to 500 mg/l. Before carrying out the biodegradation studies, the bacterial strains were acclimatized to the concentration of 500 mg/l of FA by gradually raising 100 mg/l of FA in each step. The well acclimatized culture of P. putida and P. aeruginosa degraded about 80 and 66% of 50 mg/l FA, respectively. At higher concentration of FA, the percentage of FA degradation decreased. The purpose of this study was to determine the kinetics of biodegradation of FA by measuring biomass growth rates and concentration of FA as a function of time. Substrate inhibition was calculated from experimental growth parameters using the Haldane equation. Data for P. putida were determined as µ _max ?=?0.23 h^?1, K _s ?=?23.93 mg/l and K _i ?=?217.1 mg/l and for P. aeruginosa were determined as µ _max ?=?0.13 h^?1, K _s ?=?21.3 mg/l and K _i ?=?284.9 mg/l. The experimental data were fitted in Haldane, Aiba and Edwards inhibition models.     

Kinetic Analyses of the Substrate Inhibition of <Emphasis Type="Italic">Paramecium</Emphasis> Arginine Kinase

Paramecium tetraurelia expresses four types of arginine kinase (AK1–AK4). In a previous study, we showed that AK3 is characterized by typical arginine substrate inhibition, where enzymatic activity markedly decreases near a concentration of 1 mM of arginine substrate. This is in sharp contrast to the three other AK types, which obey the Michaelis–Menten reaction curve. Since cellular arginine concentration in another ciliate Tetrahymena is estimated to be 3–15 mM in vivo, Paramecium AK3 likely functions in conditions that are strongly affected by substrate inhibition. The purpose of this work is to find some novel aspect on the kinetic mechanism of the substrate inhibition of Paramecium AK3 enzyme. Substrate inhibition kinetics for AK3 were analyzed using three models and their validity were evaluated with three static parameters (R², AICc, and Sy.x). The most accurate model indicated that not only ES but also the SES complex reacts to form products, the latter being the complex with two substrates in the active center. The maximum reaction rate for the SES complex, V_max^SES?=?30.4 µmol Pi/min/mg protein, was one-eighth of the ES complex, V_max^ES?=?241.7. The dissociation constant for the SES complex (K_i^SES: 0.34 mM) was two times smaller than that of the ES complex (K_s^ES: 0.61 mM), suggesting that after the primary binding of the arginine substrate (ES complex formation), the binding of a second arginine to the secondarily induced inhibitory site is accelerated to form an SES complex with a lower V_max^SES. The same kinetics were used for the S79A, S80A, and V81A mutants. The results indicate that the S79 residue is significantly involved in the process of binding the second arginine substrate. Herein, the K_i^SES value was ten times (3.62 mM) the value for the wild-type (0.34 mM), weakening substrate inhibition. In contrast, V_max^ES and V_max^SES values for the mutants decreased by one-third, except for the V_max^SES of the S79A mutant, which had a value that was comparable with the value for the wild-type.     

Selenate reduction rates and kinetics across depth in littoral sediment of the Salton Sea,California

To predict selenium cycling in sediments, it is crucial to identify and quantify the processes leading to selenium sequestration in sediments. More specifically, it is essential to obtain environmentally-relevant kinetic parameters for selenium reduction and information on how they spatially vary in sediments. The Salton Sea (California, USA) is an ideal model system to examine selenium processes in sediments due to its semi-enclosed conditions and increasing selenium concentration over the last century. Selenium enters the Salton Sea mainly as selenate and might be sequestered in the sediment through microbial reduction. To determine the potential selenium sequestration of Salton Sea littoral sediments and which sediment properties are controlling selenate reduction kinetics, we determined the centimeter-scale vertical distribution of potential selenate reduction rates and apparent kinetic parameters (maximum selenate reduction rates, V_max, and selenate half-saturation concentration, K_m) using flow-through reactor (FTR) experiments. We compared sediments from two littoral sites (South and North) and four depth intervals (0–2, 2–4, 4–6 and 6–8 cm). Furthermore, we characterized the selenium fractions in the sediment recovered from the FTR experiments to identify the processes leading to the sequestration of selenium. Our results reveal higher potential for selenium reduction and sequestration in the topmost sediment (0–2 cm) suggesting that microorganisms inhabiting surface sediment are well adapted to reduce selenate entering the Salton Sea. As apparent K_m values (103–2144 µM) exceed the average selenium concentration in the overlying water (6–25 nM), in situ selenate reduction is limited by the low availability of selenate and the resident selenate-reducing microorganisms operate well below their V_max (11 and 43 nmol cm^?3 h^?1). Selenium speciation after FTR experiments confirms the primary sequestration of reduced biomass-associated and elemental selenium (68–99% of total selenium) in the sediment. Further, the absence of correlation between the tested sediment physical (porosity, bulk density, clay content), chemical (C_org, N_tot, total selenium content) and biological characteristics (abundance of culturable selenate-reducers) with the kinetic parameters of selenate reduction indicates that these sediment characteristics cannot be used as predictors of apparent V_max or K_m. Conclusively, microbial selenate reduction is an important, if not the primary process, leading to the sequestration of reduced selenium in the Salton Sea sediments and making the surficial Salton Sea sediments an important selenium sink.     

Purification and Characterization of a Novel (<Emphasis Type="Italic">R</Emphasis>)-1-Phenylethanol Dehydrogenase from <Emphasis Type="Italic">Lysinibacillus</Emphasis> sp. NUST506

A novel (R)-1-phenylethanol dehydrogenase was successfully purified from Lysinibacillus sp. NUST506 by preparative polyacrylamide gel electrophoresis. The enzyme is a NAD+-dependent oxidoreductase. The molecular weight of the (R)-1-phenylethanol dehydrogenase measured by SDS-PAGE was about 28 kDa. Furthermore, the optimal reaction conditions for the oxidative reaction were 70°C and pH 9.5 and for the reductive reaction were 65°C and pH 6.5. Under the optimal conditions, the K_M and k_cat values with (R)-1-phenylethanol as a substrate were found to be 0.78 mM and 123 s^–1 and with acetophenone they were 0.56 mM and 125 s^–1, respectively. The (R)-1-phenylethanol dehydrogenase became more stable at pH 9.5 in comparison with pH 5.0 and high stability was noticed at 4 and 37°C. Properties of the enzyme place it as a promising candidate for industrial applications.     

A serine protease inhibitor from <Emphasis Type="Italic">Musca domestica</Emphasis> larva exhibits inhibitory activity against elastase and chymotrypsin

Objective

Insect-derived serine protease inhibitors (serpins) exhibit multiple inhibitory activities, but so far, no functional roles for serpins of Musca domestica have been identified. Here, the functional features of M. domestica serine protease inhibitor (MDSPI16) were characterized.

Results

Hundred forty seven differentially expressed genes including the MDSPI16 gene were screened by constructing the subtractive cDNA library. The 1154-bp full-length MDSPI16 gene was cloned, and the recombinant MDSPI16 serpin protein was expressed as a 42.6 kDa protein in an Escherichia coli expression system. The recombinant MDSPI16 protein was purified using Ni–NTA affinity chromatography, and the inhibitory activity of MDSPI16 was assessed. MDSPI16 did not inhibit trypsin, papain, or proteinase K but strongly inhibited elastase (K_i = 2.8 nM) and chymotrypsin (K_i = 28 nM). The inhibitory activity of MDSPI16 remained stable over from 37 to 100 °C and from pH 2 to 12.

Conclusions

The MDSPI16 exhibited inhibitory activity against elastase and chymotrypsin and the inhibitory activity remained stable.

     

<Emphasis Type="Italic">Spirosoma metallicus</Emphasis> sp. nov., isolated from an automobile air conditioning system

A Gram-stain-negative and orangish yellow-pigmented bacterial strain, designated PR1014K^T, was isolated from an automobile evaporator core collected in Korea. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain PR1014K^T was related with the members of the genus Spirosoma (94.7–90.2%) and closely related with Spirosoma lacussanchae CPCC 100624^T (94.7%), Spirosoma knui 15J8-12^T (94.3%), and Spirosoma soli MIMBbqt12^T (93.3%). The strain grew at 15–40°C (optimum, 25°C), pH 6.5–7.0 (optimum, 6.5) and 0–1% (w/v) NaCl (optimum, 0%). The predominant fatty acids were summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c), C_16:0, iso-C_15:0, C_16:1 ω5c, and iso-C_17:0 3-OH. The major menaquinone was MK-7. The polar lipid profile of the strain indicated that the presence of one phosphatidylethanolamine, one unidentified aminolipid, two unidentified aminophospholipids, and three unidentified lipids. The DNA G+C content of the strain was 47.4 mol%. On the basis of the phenotypic, genotypic and chemotaxonomic characteristics, strain PR1014K^T represents a novel species in the genus Spirosoma, for which the name Spirosoma metallicus sp. nov. (=KACC 17940^T =NBRC 110792^T) is proposed.     

Analysis of applicability of triplet-state emission of molecular hydrogen for spectral diagnostics of a DC discharge

The applicability of emission of the N ³Λ_σ triplet states of molecular hydrogen for spectral diagnostics of the positive column of a dc glow discharge in hydrogen at translational gas temperatures of 360–600 K, specific absorbed powers of 0.8–4.25 W/cm, gas pressures of p = 0.3–15.0 Torr, reduced fields of E/N = 30–130 Td, and electron densities of n _e = 4.0 × 10⁹–6.5 × 10¹⁰ cm^–3 is analyzed by using an advanced level-based semi-empirical collisional?radiative model. It is found that secondary processes make the main contribution to the population and decay of the N ³Λ_σ = a ³Σ⁺ _g , c ³Π _u , g ³Σ⁺ _g , h ³Σ⁺ _g , and i ³Π _g triplet states. The dipole-allowed transitions e ³Σ⁺ _g → a ³Σ⁺ _g , f ³Σ⁺ _g → a ³Σ⁺ _g , g ³Σ⁺ _g and k ³Π _u → a ³Σ⁺ _g can be used for spectral diagnostics of a dc discharge within a simplified coronal model.