Guanidine HCl Research Articles

Properties of AlCl3 guanidine deep eutectic solvent with addition of polyethylene oxide of different molecular weights supported by quantum-chemical calculation

Deep eutectic solvent of AlCl3 with guanidine HCl was synthesized in the molar ratio 3:1 in the presence of polyethylene glycols of various molecular weights from 1500 to 5·106 g/mol. Concentration of polymer was 2.5 %, 5 % and 10 %. Temperature dependence between 25 and 70 °C of conductivity of each electrolyte was calculated from the cyclic voltammetry of aluminum deposition-stripping measurements. It was found that conductivity increases with molecular weight of polymer in the interval between 2 and 10 kD. This result was interpreted as binding of AlCl3 guanidine to two different sites which have hydroxyl (chain ends) and ether (middle chain) oxygen with relatively higher and lower binding energy respectively. Polymer with longer chain has more low energy binding sites which causes lesser binding and consequently higher conductivity of electrolyte. B3LYP-D3 and DLPNO-CCSD(T) computations support experimental results. DES are discussed in terms of Pearson HSAB principle.

HrpY protein of Ralstonia solanacearum exhibits spontaneous formation of pilus like assembly: analysis of its stability

Type 3 secretory system (T3SS), a complex protein machinery has a unique virulence mechanism that involves injecting effector proteins directly into host cells. The T3SS effector proteins are transported through an extracellular long hollow needle made up of multiple copies of a small protein. In T3SS of the plant pathogen Ralstonia solanacearum, the 8.6 kDa HrpY protein assembles into a large needle like apparatus (pilus) for transporting effector proteins. To study structural details of HrpY, we recombinantly expressed and purified HrpY in E. coli. The dynamic light scattering (DLS) analysis showed that rHrpY has spontaneously formed oligomers of large order (>100 nm). Transmission electron microscopy of rHrpY samples revealed that the large structures are tube like assembly having dimensions 86.3–166.6 nm and 5.8–6.8 nm in length and width respectively. Different molecular sizes of the purified rHrpY hindered the crystallization of the protein. The stability of oligomer assembly was studied with denaturants and surfactants. Denaturants like urea and guanidine HCl could not break them apart; however, detergents like SDS, sarkosyl, Octyl-β-Glucoside, CHAPS, Tween 20, Tween 80 and Triton X-100 showed disassembly of the oligomer. rHrpY assembly was found to withstand up to 50 °C and the circular dichroism analysis revealed that there is no significant change in the secondary structural composition with increase in temperature. However, change in the secondary structure was observed with the addition of SDS. Communicated by Ramaswamy H. Sarma

Influence of different chaotropic salts on etched mesoporous silica nanoparticles for the removal of bacteria DNA conveying antibiotic resistance genes from hospital wastewater

The adsorption of bacteria DNA onto mesoporous silica nanoparticles in their original state has been a great challenge due to the high negative charge exhibited by both the DNA and silica surface. The aim of this study is to mediate bacteria DNA onto synthesized mesoporous silica nanoparticles (E-MSN) in combination with different chaotropic salts. E-MSN was synthesized via chemical etching techniques using sodium dodecyl sulfate (SDS) as an etchant. SDS was used to remove unwanted layers and provides a convenient platform for underlining mesopore with different chaotropic salts. Scanning electron microscopy (SEM) coupled with energy-dispersive x-ray spectroscopy (EDX), Fourier-transformed infrared spectroscopy (FTIR), x-ray diffraction spectroscopy (XRD), and point of zero charges (PZC) results showed that synthesized etched mesoporous silica nanoparticles have a crystalline, non-spherical shape and elemental composition of silica at approximately 2 Kev, functional groups that depict silica particles. Molecular characterization of extracted genes showed that Enterococcus faecium harbors tetA, tetM, and ermB in 201bp, 158bp, and 320bp, with the DNA purity ranging from 1.7 to 1.9. DNA adsorption was studied as a function of operating parameters in different solutions of chaotropic salts (sodium chloride (NaCl), 2 M guanidine HCl (CH5N3.HCl), and urea ((NH3)2CO). Among the different chaotropic salts used to compliment the silica nanoparticles, 2 M guanidine HCl exhibited the highest percentage (%) removal efficiency (90%) compared to urea (75%) and sodium chloride (70%) in simulated water and hospital wastewater. Experimental results revealed that the pseudo-second-order kinetic and Sips isotherm is the best fit for the adsorption process. Therefore, mesoporous silica nanoparticles enhanced by different chaotropic salts in this study showed that this material might be promising and economical for the uptake of bacteria DNA conveying antibiotics resistance genes from hospital wastewater.

Biochemically prepared C-reactive protein conformational states differentially affect C1q binding

C-reactive protein (CRP) is commonly measured as an inflammatory marker in patient studies for coronary heart disease, autoimmune disease and recent acute infections. Due to a correlation of CRP to a vast number of disease states, CRP is a well-studied protein in medical literature with over 16000 references in PubMed [1]. However, the biochemical and structural variations of CRP are not well understood in regards to their binding of complement immune response proteins. Conformations of CRP are thought to affect disease states differently, with a modified form showing neoepitopes and activating the complement immune response through C1q binding. In this work, we compare the unfolding of CRP using chemical denaturants and identify which states of CRP bind a downstream complement immune response binding partner (C1q). We used guanidine HCl (GndHCl), urea/EDTA, and 0.01% SDS with heat to perturb the pentameric state. All treatments give rise to a monomeric state in non-denaturing polyacrylamide gel electrophoresis experiments, but only treatment with certain concentrations of denaturant or dilute SDS with heat maintains CRP function with a key downstream binding partner, C1q, as measured by enzyme-linked immunosorbent assays. The results suggest that the final form of modified CRP and its ability to mimic biological binding is dependent on the preparation method.

Novel Benzo[<i>f</i>]coumarin Derivatives as Probable Acetylcholinesterase Inhibitors: Synthesis, In Vitro, and In Silico Studies for Evaluation of Their Anti-AChE Activity

Novel benzo[f]coumarin derivatives bearing pyrimidine unit were successfully synthesized. The target is to develop novel acetylcholinesterase inhibitors. The benzo[f]coumarin chalcone 4 was prepared via Claisen-Schmidt condensation between 3-acetyl-5,6-benzocoumarin and 4-hydroxybenzaldehyde in the alkaline medium. Then, the cyclocondensation of chalcone 4 with urea, thiourea, and guanidine HCl in the presence of glacial acetic acid led to the formation of various pyrimidines. Structures of the newly synthesized compounds were characterized by FT-IR, 1H-NMR, 13C-NMR spectra, and elemental analysis. The acetylcholinesterase (AChE) inhibitory activity tests were carried out using Ellman's assay and donepezil as a reference drug. The biological activity results revealed that the derivatives 6 and 7 inhibit AChE activity in healthy samples showed that the greater inhibition percentage was found respectively at concentrations of 10–4 and 10–10 M while low inhibition percentage was obtained at 10–12 and 10–4 M. AChE showed inhibition constant Ki in the range of 10–4-10–12 M in the presence of maximum and minimum inhibitor concentrations, probably due to variant types of inhibition from non and uncompetitive. In addition, molecular modeling simulations of targeted compounds revealed their mechanism of action as potent inhibitors for the AChE enzyme.

Expression of HCV genotype-4 core antigen in prokaryotic E. coli system for diagnosis of HCV infection in Egypt

BackgroundEgypt has a high prevalence of hepatitis C virus (HCV) infection with 92.5% of genotype-4. AimThis study aimed to clone and express the core gene of HCV genotype-4 for using it to develop a highly sensitive, specific, and cost-effective diagnostic assay for detecting HCV infection. MethodsUsing synthetic HCV genotype-4 core gene, pET15b as E. coli expression vector, and 1 mM lactose as inducer, the HCV core protein (MW 17 kDa) was expressed in the form of inclusion bodies (IBs) that was purified and solubilized using 8 M guanidinium HCl. The recombinant core protein was in vitro refolded by a rapid dilution method for further purification using weak cation exchange liquid chromatography. The immunogenicity of the purified protein was tested by ELISA using 129 serum samples. ResultsThe recombinant core protein was successfully expressed and purified. The results also showed that the in-house anti-HCV core assay is accurate, specific (~96.6%), and highly sensitive (~100%) in accordance with the commercial ELISA kit. ConclusionThe sensitivity, specificity, and reproducibility of the developed assay were high and promising to be used as a screening assay for detecting HCV infection.

Purified recombinant human Chromogranin A N46 peptide with remarkable anticancer effect on human colon cancer cells

Human Chromogranin A N46 (CGA-N46) is a weak cationic α-helical peptide with wide-spectrum antibacterial, fungal, and anticancer activities. In this study, the recombinant human CGA-N46 peptide was expressed successfully in Escherichia coli. The gene of CGA-N46 was cloned into the expression vector pET-15b without a fusion tag at the N terminus and the peptide was expressed using Isopropyl-β-d-thiogalactoside (IPTG) as an inducer. Using 8 M guanidinium HCl, inclusion bodies containing the peptide were purified and solubilized. The rhCGA-N46 peptide was purified using Q-FF anion exchange column. The cytotoxicity of the purified rhCGA-N46 peptide was investigated on WI-38 human lung normal cell line. The anticancer activity was studied on human colon cancer cell line; HCT-116 cell line. Besides, the possible involvement of rhCGA-N46 peptide in regulating apoptotic signal pathways was analyzed by detecting the expression levels of BCL2, BID, and CAS-8 in the treated cells. The results concluded that the active peptide recovery was up to 41.98%. The purified rhCGA-N46 was safe on normal cells with IC50 = 227.74 µg/ml (40.67 µM) and had an obvious anticancer effect on colon cancer cells with IC50 = 1.997 µg/ml (356.6 nM). The expression level of BCL2 was down-regulated and BID and CAS-8 were up-regulated significantly in treated HCT-116 cells compared to untreated. In conclusion, the rhCGA-N46 peptide was produced successfully in the native form with promising anti-colon cancer activity.

Single-chain Fv antibody covalently linked to antigen peptides and its structural evaluation

The monoclonal antibody G2 specifically recognizes different peptides. The single-chain Fv (scFv) antibodies of G2 covalently linked to antigen peptides, Pep18mer and Pep395, via a flexible linker were expressed in Escherichia coli in the insoluble fraction, and were solubilized using guanidine HCl, followed by refolding. We analyzed the folding thermodynamics of the refolded proteins, purified as monomers using size-exclusion chromatography (SEC). The results of the differential scanning calorimetry (DSC) showed that the thermal stabilities of antigen peptide-linked G2 scFvs were higher than those of antigen-free G2 scFv in the absence or presence of antigen peptides. The folding thermodynamics further indicated how the antigen-antibody affinity affect the intramolecular interactions. The combination of SEC and DSC experiments could confirm the folding correctness of antigen peptide-linked G2 scFvs and could be applied for “structural screening” of refolded proteins in the case that the “functional screening” like antigen binding is difficult to apply. The present method to covalently link the peptide would contribute to the stable complex structure, and would be widely applied to other antibodies recognizing peptide antigens.

Consecutive alternating administration as an effective anti-coxsackievirus B3 in vivo treatment scheme.

Although chemotherapy is generally indicated for treatment of enterovirus infections, antivirals are currently not used in clinical practice. The use of monotherapy is the main reason for this unfavourable state. This is related to the fact that enterovirus progeny consist of innumerable quasispecies, allowing the virus to develop drug resistance quickly. Here, we present a consecutive alternating administration (CAA) treatment scheme for combining enterovirus inhibitors. Applying the CAA approach with a combination of pleconaril (capsid binder), guanidine HCl (viral 2C inhibitor), and oxoglaucine (PI4KB inhibitor) (PGO) was found to be effective in the treatment of newborn mice infected with a massive inoculum (20 MLD50) of the coxsackievirus B3 cardiotropic Woodruff or neurotropic Nancy strain. In addition to preventing drug resistance, the CAA approach resulted in the parallel development of increased susceptibility to the compounds in the PGO combination. These observations demonstrate the therapeutic potential of the CAA approach for treatment of enterovirus infections.

Growth factor profile in calcified cartilage from the metaphysis of a calf costochondral junction, the site of initial bone formation.

Endochondral bone formation is orchestrated by growth factors produced by chondrocytes and deposited in the cartilage matrix. Whilst some of these factors have been identified, the complete list and their relationship remains unknown. In the present study, the growth factors were isolated from non-calcified and calcified cartilage of costochondral junctions. Cartilage dissected from the ribs of 6-20-week-old calves was purchased from a local butcher within 24 h of the death of the animal. The isolation involved hyaluronidase digestion, guanidinium hydrochloride (GuHCl) extraction, HCl decalcification and GuHCl extraction of the decalcified matrix. Growth factors were purified by heparin chromatography and their quantities were estimated using ELISA. Decalcified cartilage was also used for protein sequence analysis (data are available via ProteomeXchange; ID, PXD021781). Bone morphogenetic protein-7 (BMP-7), growth/differentiation factor-5 (GDF-5) and NEL-like protein-1 (NELL-1), all known growth factors that stimulate bone formation, quantitatively accounted for the majority of the material obtained in all steps of isolation. Thus, cartilage serves as a store for growth factors. During initial bone formation septoclasts release osteoclastogenesis-stimulating factors deposited in non-calcified cartilage. Osteoclasts dissolve calcified cartilage and transport the released factors required for the stimulation of osteoprogenitor cells to deposit osteoid. High concentrations of BMP-7, GDF-5 and NELL-1 at the site of initial bone formation may suggest that their synergistic action favours osteogenesis.

Identification and Biochemical Characterization of some Strains of Clavibacter michiganensis subsp. Michiganensis, in Romania

Clavibacter michiganensis subsp. michiganensis is responsible for the ʻbacterial canker of tomato’. The study aimed to identify and characterize biochemically some strains of the bacteria. Five strains were used for biochemical identification and characterization and two reference strains were used as positive controls. The biochemical characterization was performed with Biolog Microbial Identification System. All the strains used many carbon sources and were positive for growth in the presence of: 1%NaCl, Nalidixic Acid, Aztreonam, Potassium Tellurite and pH 6, but negative for growth and sensitivite to: Troleandomycin, Lincomycin, Vancomycin, Fusidic Acid, Eimfamycin SV, Guanidine HCl, D-Serine, Minocycline and Niaproof 4. The differences between the Biolog system database and the studied strains did not change their classification at the level of genus and species, but modifed or did not allow the classification at the subspecies level. Furthermore, the analyzed strains had metabolic diversity.

Method of extraction and proteome profiling of mycobacteria using liquid chromatography-high resolution mass spectrometry

Advances in massively parallel sequencing, of complete bacterial genomes, have led to many novel findings in the field of genomics. However, these data often lack correlation with expressed protein profiles. It has been demonstrated that even very closely related genomes, such as in mycobacteria, express drastically different phenotypes. These phenotypes often have major roles in pathogenicity. Therefore, it is just as important to have a method for examining the proteome of a bacterium as well as its genome. These studies are further complicated in mycobacteria due to the cell wall and mycolic acid. A comprehensive method for the identification and characterization of the whole mycobacterium protein profile is needed. In the present study, a simple, sensitive, and specific liquid chromatography tandem mass spectrometry method was developed for the extraction, purification and profiling the mycobacterial proteome in various species. During development, sonication and bead-beating cell lysis protocol was tested using 15% Acetonitrile and 6 M guanidine-HCl (GuHCl) as extraction solvent. Sonication lysis in 6 M GuHCl with glass beads was the preferred method for cell lysis. This method was developed using reverse phase liquid chromatography and a Q Exactive ™ Plus Orbitrap™ mass spectrometer for peptide and protein identification. Bottom-up liquid chromatography-mass spectrometry LC–MS analysis resulted in identification of greater than 2500 proteins.

Measuring antibody avidity to Plasmodium falciparum merozoite antigens using a multiplex immunoassay approach

BackgroundAntibodies (Ab) play a significant role in immunity to Plasmodium falciparum malaria. Usually, following repeated exposure to pathogens, affinity maturation and clonal selection take place, resulting in increased antibody avidity. However, some studies suggest affinity maturation may not occur to malaria antigens in endemic areas. Information on development of antibody avidity is confusing and conflicting, in part, because different techniques have been used to measure avidity. Today, bead-based multiplex immunoassays (MIA) are routinely used to simultaneously quantitate antibody levels to multiple antigens. This study evaluated the feasibility of developing an avidity MIA with 5 merozoite antigens (AMA1, EBA-175, MSP1-42, MSP2, MSP3) that uses a single chaotropic concentration.MethodsThe most common ELISA protocols that used the chaotropic reagents guanidine HCl (GdHCl), urea, and ammonium thiocyanate (NH4SCN) were adapted to a multiplex MIA format. Then, different concentrations of chaotropes and incubation times were compared and results were expressed as an Avidity Index (AI), i.e., percentage of antibody remaining bound in the presence of chaotrope. Experiments were conducted to (i) identify the assay with the widest range of AI (discriminatory power), (ii) determine the amount of chaotrope needed to release 50% of bound Ab using plasma from adults and infants, and (iii) evaluate assay repeatability.ResultsOverall, 4 M GdHCl and 8 M urea were weaker chaotropes than 3 M NH4SCN. For example, they failed to release significant amounts of Ab bound to MSP1-42 in adult plasma samples; whereas, a range of AI values was obtained with NH4SCN. Titration of NH4SCN revealed that 2 M NH4SCN gave the widest range of AI for the 5 antigens. Binding studies using plasma from 40 adults and 57 1-year old infants in Cameroon showed that 2.1 M ± 0.32 (mean ± SD) NH4SCN (adults) and 1.8 M ± 0.23 M (infants) released 50% of bound Ab from the merozoite antigens.ConclusionsAn avidity MIA is feasible for the 5 merozoite antigens that uses a single concentration (2 M) of NH4SCN. The assay provides a simple method to quickly obtain information about Ab quantity and quality in the acquisition of immunity to malaria in endemic populations.

Refolding, characterization, and dye decolorization ability of a highly thermostable laccase from Geobacillus sp. JS12

A putative laccase gene (lacG) from Geobacillus sp. JS12 was cloned and expressed as a fusion protein with six histidine residues in Escherichia coli BL21 (DE3) cells, and the protein was primarily found in inclusion bodies. The resulting insoluble proteins were solubilized with 6 M guanidine HCl and refolded using an on-column refolding procedure. Ni-chelation affinity chromatography found the laccase to be a 30 kDa monomeric protein. Spectrophotometry and electron paramagnetic resonance (EPR) analysis indicated LacG as a multi-copper oxidase, with the usual laccase copper sites, Type 1, 2, and 3 Cu(II). The optimum pH for enzymatic activity was 3.0, 6.0, and 6.5 with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), guaiacol and 2,6-dimethoxyphenol (2,6-DMP) as the substrate, respectively. The recombinant protein displayed high thermostability, with a heat inactivation half-life of approximately 2 h at 95 °C, and an optimum temperature of 80 °C with 2,6-DMP. Catalytic efficiency (kcat/Km) showed that guaiacol and 2,6-DMP were highly oxidized by the enzyme. The enzymatic reaction was significantly enhanced by Co2+ and Mn2+, while activity was strongly inhibited in the presence of Fe2+, Zn2+, and thiol compounds. LacG decolorized 43% of Congo red and 14% of Malachite green, and the addition of ABTS as a redox mediator dramatically increased the dye decolorization efficiency.

Characterisation of recombinant thermostable manganese-superoxide dismutase (NeMnSOD) from Nerium oleander.

Superoxide dismutase is one of the key antioxidant enzymes accountable for the eradication of free radicals generated during various metabolic processes. This is first study reporting a thermostable MnSOD obtained from a xerophytic plant, Nerium oleander. The full-length gene identified using Rapid amplification of cDNA ends revealed an open reading frame of 699bp flanked by 5'UTR and 3'UTR of 134bp and 198bp respectively. The corresponding NeMnSOD protein was cloned and expressed in Escherichia coli. The purified protein yields a band of 25.4kDa, which established a specific activity of 2617 units mg-1 of protein and under native condition yield bands of 52kDa and 110kDa, confirming the dimeric and tetrameric state of the protein. The Km and Vmax of 0.078 ± 0.008mM and 1052.3 ± 33.59 units mg-1 of protein, respectively. The purified enzyme demonstrated thermostability by retaining more than 20% activity at a temperature 70 ℃. The enzyme functioned at pH range of 4-9.0 with maximum activity at pH 7.4. Sodium azide, effectively inhibited the activity of enzyme confirming it to be MnSOD. The enzyme activity was least affected on treatment with strong denaturants (Urea, guanidine HCl and SDS) and harsh chemicals (DTT, CHAPS and β-mercapto-ethanol) These experimental data validated with Insilco analysis revealed that NeMnSOD possessed thermo as well as kinetically stable moiety which can be further exploited with its applications in the field of pharmaceutical, food and cosmetic industry, which urge for such thermostable enzyme.

Cystic Fibrosis Sputum Reflections from the Pre‐Modulator Age

Toward the understanding and improvement of cystic fibrosis (CF) airway health, our laboratories have studied the tracheobronchial mucous glycoproteins (mGP) of individuals with and without CF. With the increased mucus viscosity and charged character of the mGP oligosaccharide species, obstructions in the CF gastrointestinal and respiratory tracts readily occur and lead to malnutrition, frequent and persistent pulmonary infections with opportunistic pathogens, and COPD. With new medications (“modulators”) targeting the basic defects, we anticipate marked improvements in CF airway health. With respect for the current airway clearance challenges faced by many with CF, we reflect on our experiences in handling sputum specimens over the years as we processed them toward mGP oligosaccharide structural elucidation. These studies provided opportunities for microscopic, chemical and biochemical observations about CF sputum relative to non‐CF sputum and insights about sputa heterogeneity of an individual. Toward improving therapeutic strategies, here we briefly report these observations. Microscopically, dense CF sputum typically depicted a neutrophil inflammatory response with large numbers of clumped neutrophils, many with degenerating nuclei, and much cellular debris, whereas non‐CF purulent sputa commonly displayed abundant mucous material and scattered inflammatory cells. Average chemical compositions of >1000 sputa (213 non‐purulent, NP; 322 purulent, P; and 468 CF), showed for CF less water content per specimen and greater dry weight (NP, P, CF respectively: water mg/mL 959, 939, 862; macromolecular dry weight [MDW] mg/mL 43, 61, 98 respectively). CF sputa possessed a higher % of MDW as lipid and DNA than non‐CF sputa and surprisingly, decreased total carbohydrate (CHO), (NP, P, CF respectively: lipid 25, 31, 34 % MDW; DNA 0.01, 1.4, 2.7 % MDW, with CF DNA ranging 0.1 – 9.1% MDW; CHO 21, 20, 14 % MDW.) Sputa of 4 CF patients collected over 5 days of hospitalization, showed >35% reduction in sputa DNA content regardless of initial DNA % MDW at admission. Sputa solubilization with guanidinium HCl showed no correlation of lipid content to hours to complete solubilization (hS); a trend to longer times with increased glycoprotein content (GP); and a marked impact of DNA content on solubilization. With CF focus, GP, DNA content, and hS were determined for 77 CF sputa. Rather than linear concentration trends, there were scattered relationships of % MDW of DNA and of GP. 3D graphical analysis revealed longer solubilization times (i.e. 10–20 h) when both mucins and DNA were in significant quantity, than specimens of high levels of DNA with little GP (i.e. 4–8h). Compiling these types of observations, we see CF sputa, over the population, and for a given individual, reflect a heterogenous spectrum of DNA‐mucin relative concentrations. As therapeutic strategies continue to evolve for the mobilization of the viscous obstructive materials, these data suggest that approaches which address both the DNA and mucous glycoprotein components will be the most beneficial in the ever‐changing airway dynamic. As COPDs share many features with CF, these CF sputum reflections may also guide airway clearance strategies in general.Support or Funding InformationUniversity of Missouri Agricultural Experiment Station Chemical Laboratories; Cystic Fibrosis Associations of Missouri and West Plains.

A new method for dentine matrix metalloproteinase extraction

In addition to their involvement in tissue remodelling, matrix metalloproteinases (MMPs), in the oral environment, are linked to leakage in the resin-dentine interface through their involvement in the proteolytic degradation of the resin-dentine hybrid layer.Numerous studies have evaluated dentine MMP activity and the vast majority of those studies have used an MMP extraction protocol to semi-purify the MMPs from dentine, first described around 20 years ago. This is a protocol that requires 32 days for completion. The technique is based on the three-step sequential use of NaCl solution for pulverised dentine washing, then guanidine HCl and EDTA to demineralise the pulverised dentine to extract the MMPs.In this study, a new one-step dentine MMP extraction protocol was adapted to extract dentine MMPs in only four days. This was achieved by eliminating the NaCl washing step and combining the guanidine HCl and EDTA into one extraction solution that also contained proteinase inhibitors.Fifty-two dentine MMP extracts were obtained utilising the two different extraction methods. The amount of total and endogenously active MMP-2 in the specimens was assayed utilising a human MMP-2 activity enzyme-linked immunosorbent assay (ELISA).The study results showed that the new extraction method is as effective as the traditional three-step extraction method in semi-purifying dentine MMP-2.

Thermal and Chemical Unfolding of Lysozyme. Multistate Zimm-Bragg Theory Versus Two-State Model.

Thermal and chemical unfolding of lysozyme in the presence of the guanidine HCl denaturant is a model system to compare the conventional two-state model of protein unfolding with the multistate Zimm-Bragg theory. The two-state model is shown to be the noncooperative limit of the Zimm-Bragg theory. In particular, the Zimm-Bragg theory provides a molecular interpretation of the empirical linear extrapolation method (LEM) of the two-state model. Differential scanning calorimetry (DSC) experiments reported in the literature are analyzed with both methods. Lysozyme unfolding is associated with a large endothermic enthalpy that decreases significantly upon addition of guanidine HCl. In contrast, the Gibbs free energy of unfolding is small, negative, and independent of the guanidine HCl concentration, contradicting, in part, the conclusions of the LEM. The unfolding enthalpy is compensated by an even larger entropy term. The multistate Zimm-Bragg theory predicts a larger conformational enthalpy and a smaller Gibbs free energy than the two-state model. The Zimm-Bragg theory provides the protein cooperativity parameter, the average length of independently folding protein domains, and the Gibbs free energy of unfolding of individual amino acid residues. Guanidine HCl binding to lysozyme is exothermic and counteracts the endothermic unfolding enthalpy. The number of bound denaturant molecules is determined from the decrease in enthalpy and is extrapolated to the guanidine HCl-to-amino acid stoichiometry at complete lysozyme unfolding. Chemical unfolding isotherms measured with circular dichroism (CD) spectroscopy are analyzed with both models. The chemical Zimm-Bragg theory is a cooperative molecular model, yielding the guanidine HCl binding constant and the protein cooperativity parameter. It allows a quantitative comparison between thermal and chemical protein unfolding. The two reactions have almost identical changes in Gibbs free energy. However, thermal unfolding is significantly more cooperative than chemical unfolding. Finally, distinct differences are observed in thermal unfolding between DSC and CD spectroscopy.

Biochemical Characterization of a Novel Monospecific Endo-β-1,4-Glucanase Belonging to GH Family 5 From a Rhizosphere Metagenomic Library.

Cellulases have a broad range of different industrial applications, ranging from food and beverages to pulp and paper and the biofuels area. Here a metagenomics based strategy was used to identify the cellulolytic enzyme CelRH5 from the rhizosphere. CelRH5 is a novel monospecific endo-β-1,4-glucanase belonging to the glycosyl hydrolase family 5 (GH5). Structural based modeling analysis indicated that CelRH5 is related to endo-β-1,4-glucanases derived from thermophilic microorganisms such as Thermotoga maritima, Fervidobacterium nodosum, and Ruminiclostridium thermocellum sharing 30-40% amino acid sequence identity. The molecular weight of the enzyme was determined as 40.5 kDa. Biochemical analyses revealed that the enzyme displayed good activity with soluble forms of cellulose as a substrate such as ostazin brilliant red hydroxyethyl cellulose (OBR-HEC), carboxymethylcellulose (CMC), hydroxyethyl cellulose (HEC), and insoluble azurine cross-linked hydroxyethylcellulose (AZCL-HEC). The enzyme shows highest enzymatic activity at pH 6.5 with high pH tolerance, remaining stable in the pH range 4.5–8.5. Highest activity was observed at 40°C, but CelRH5 is psychrotolerant being active and stable at temperatures below 30°C. The presence of the final products of cellulose hydrolysis (glucose and cellobiose) or metal ions such as Na+, K+, Li+, and Mg2+, as well as ethylenediaminetetraacetic acid (EDTA), urea, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), 2-mercaptoethanol (2-ME) or glycerol, did not have a marked effect on CelRH5 activity. However, the enzyme is quite sensitive to the presence of 10 mM ions Zn2+, Ni2+, Co2+, Fe3+ and reagents such as 1 M guanidine HCl, 0.1% sodium dodecyl sulfate (SDS) and 20% ethanol. Given that it is psychrotolerant and retains activity in the presence of final cellulose degradation products, metal ions and various reagents, which are common in many technological processes; CelRH5 may be potential suitability for a variety of different biotechnological applications.

Novel Pyrimidine Derivatives from 2,5-Dichloro-3-acetylthienyl Chalcones as Antifungal, Antitubercular and Cytotoxic Agents: Design, Synthesis, Biological Activity and Docking Study

Twenty novel pyrimidine derivatives were synthesized from 2,5-dichloro-3-acetylthienyl chalcones by reacting with guanidine HCl in presence of KOH and ethanol under reflux for 6 h. Their structural characterizations were evaluated by ATR-FTIR, 1H NMR, 13C NMR, mass spectroscopy. They were also screened for antifungal, antitubercular and cytotoxicity activities. They were displayed good antifungal activity (MIC = 32-125 μg/mL) against Aspergillus niger and Candida tropicalis fungal species except compound 15 with 4"-pyridinyl moiety (MIC = 8.00 μg/mL) being more potent. Compound 5 with 2",4"-dichlorophenyl moiety was shown with good antitubercular activity (MIC = 6.2 μg/mL) against Mycobacterium tuberculosis H37Rv (MTB) stain. They have also tested for in vitro cytotoxicity activity against DU-145 prostate cancer cell lines. In which the compound 15 with 4"-pyridinyl moiety (IC50 = 2.0 ± 0.1 μg/mL) and compound 17 with 2"-pyrrolyl moiety (IC50 = 6.0 ± 0.1 μg/mL) possess highly potent antiprostate cancer properties. The molecular docking was done with the crystalline structure of mitochondrial 2-enoyl thioester reductase Etr1p/Etr2p heterodimer from Candida tropicalis fungal species with compound 15 (-7.80 kcal/mol) and shown greater binding affinity than fluconazole (-7.60 kcal/mol). Docking was performed with protein crystalline structure (PDB ID: 2WEE) of Mycobacterium tuberculosis H37Rv (MTB) stain and among all, compound 5 was exhibited good binding affinity (-6.90 kcal/mol), compared to pyrazinamide (-4.10 kcal/mol). The protein crystalline structure of a mutant androgen receptor (AR) ligand-binding domain (LBD) (PDB file: 1GS4) was tested with compounds 15 and 17 (-7.60 and -8.20 kcal/mol). They were exhibited good binding properties compared to methotrexate (-5.10 kcal/mol). Hence, these novel pyrimidine compounds are as lead compounds as antifungal, antitubercular and cytotoxic agents.