Formation of molten globule-like state during acid denaturation of Aspergillus niger glucoamylase

Acid denaturation of Aspergillus niger glucoamylase was studied using different conformational probes. Both far-UV CD spectral signal (MRE_222 nm) and tryptophan fluorescence remained unchanged in the pH range, 7.0–3.0 but decreased significantly below pH 3.0, whereas ANS fluorescence showed a marked increase below pH 1.5. Maximal changes in MRE_222 nm and ANS fluorescence were noticed at pH 1.0. Acid-denatured state of glucoamylase at pH 1.0 retained a significant amount of secondary structure as reflected from far-UV CD spectra but showed a deformed tertiary structure with significant exposure of nonpolar groups as well as tryptophan residues as revealed by increased ANS fluorescence, decreased tryptophan fluorescence and three-dimensional fluorescence spectral signals and increase in K_sv value in acrylamide quenching experiments. Acid-denatured state showed no significant variation in the CD spectral signal throughout the temperature range, 0–100 °C. However, a late cooperative transition was observed upon GdnHCl treatment, compared to the native enzyme. All these results suggested that the acid-denatured state of glucoamylase at pH 1.0 represented the molten globule-like state.     

Molten-globule like partially folded states of human serum albumin induced by fluoro and alkyl alcohols at low pH

Human serum albumin (HSA) exists in a molten-globule like state at low pH (pH 2.0). We studied the effects of trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP) on the acid-denatured state of HSA by far-UV circular dichroism (CD), near-UV CD, tryptophan fluorescence, and 1-anilinonaphthalene-8-sulfonic acid (ANS) binding. At pH 2.0, these alcohols induced the formation of alpha-helical structure as evident from the increase in mean residue ellipticity (MRE) value at 222 nm. On addition of different alcohols, HSA exhibited a transition from the acid-denatured state to the alpha-helical state and loss of ANS-binding sites reflected by the decrease in ANS fluorescence at 480 nm. However, the concentration range required to bring about the transition varied greatly among different alcohols. HFIP was found to have highest potential whereas methanol was least effective in inducing the transition. The order of effectiveness of alcohols was shown to be: HFIP > TFE > 2-chloroethanol > tert-butanol > isopropanol > ethanol > methanol as evident from the Cm values. The near-UV CD spectra and tryptophan fluorescence showed the differential effects of halogenated alcohols with those of alkanols. A comparison of the m values, showing the dependence of Delta GH on alcohol concentration, suggests that the helix stabilizing potential of different alcohols is due to the additive effect of different constituent groups present whereas remarkably higher potential of HFIP involves some other factor in addition to the contribution of constituent groups.     

Molten globule-like state of human serum albumin at low pH.

Human serum albumin (HSA), under conditions of low pH, is known to exist in two isomeric forms, the F form at around pH 4.0 and the E form below 3.0. We studied its conformation in the acid-denatured E form using far-UV and near-UV CD, binding of a hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (ANS), thermal transition by far-UV and near-UV CD, tryptophan fluorescence, quenching of tryptophan fluorescence using a neutral quencher, acrylamide and viscosity measurements. The results show that HSA at pH 2.0 is characterized by a significant amount of secondary structure, as evident from far-UV CD spectra. The near-UV CD spectra showed a profound loss of tertiary structure. A marked increase in ANS fluorescence signified extensive solvent exposure of non-polar clusters. The temperature-dependence of both near-UV and far-UV CD signals did not exhibit a co-operative thermal transition. The intrinsic fluorescence and acrylamide quenching of the lone tryptophan residue, Trp214, showed that, in the acid-denatured state, it is buried in the interior in a non-polar environment. Intrinsic viscosity measurements showed that the acid-denatured state is relatively compact compared with that of the denatured state in 7 M guanidine hydrochloride. These results suggest that HSA at pH 2.0 represents the molten globule state, which has been shown previously for a number of proteins under mild denaturing conditions.     

Protein stabilizing potential of simulated honey sugar cocktail under various denaturation conditions

Protein stabilizing potential of simulated honey sugar cocktail (SHSC) against chemical and thermal denaturations was studied using bovine serum albumin (BSA) as the model protein. The two-step, three-state transition of urea denaturation of BSA became a single-step, two-state transition along with the shift in the whole transition curve towards higher urea concentrations in the presence of increasing SHSC concentrations [8–20% (w/v)] as revealed by far-UV CD, fluorescence and UV difference spectroscopic results. Far-UV and near-UV CD spectra, UV difference spectra, ANS fluorescence and three-dimensional fluorescence results suggested significant retention of native-like conformation in 4.6 M urea-denatured BSA in the presence of 20% (w/v) SHSC. A significant shift was also noticed in thermal and GdnHCl denaturation curves of BSA in the presence of 20% (w/v) SHSC. Taken together, all these results suggested significant stabilization of BSA against urea, GdnHCl and thermal denaturations by SHSC.     

Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase

5-Aminolevulinate synthase (ALAS), a pyridoxal-5′phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in mammals. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0–3.0 and 7.5–10.5) and temperature (20 and 37 °C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH 2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH 10.5 and pH 9.5/37 °C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420 nm to 330 nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphthalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH 1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH 9.5/37 °C), although the k_cat value is significantly decreased. Finally, the binding of 5-aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme.     

Biocatalytic acylation of sugar alcohols by 3-(4-hydroxyphenyl)propionic acid

Enzymatic synthesis of aromatic esters of four different sugar alcohols (xylitol, arabitol, mannitol, and sorbitol) with 3-(4-hydroxyphenyl)propionic acid was performed in organic solvent medium, using immobilized Candida antarctica lipase (Novozyme 435), and molecular sieves for control of the water content. The influence of reaction parameters on the conversion has been investigated, including reaction time, temperature, alcohol/acid molar ratio, and enzyme amount. The highest conversions (94% for xylitol, 98% for arabitol, 80% for mannitol, and 93% for sorbitol) were obtained in pure tert-butanol at 60 °C and 72 h reaction time, 0.3 alcohol/acid molar ratio, and 0.5 g/mol enzyme/substrate ratio. The isolated new sugar alcohols esters were identified by different spectral analyses. MALDI-TOF MS analysis showed the formation of monoesters, diesters, and small quantities of triesters for all investigated sugar alcohols. The catalytic efficiency of the enzyme was higher for the pentitol substrates, decreasing in the following order: arabitol > xylitol > sorbitol > mannitol. These new compounds could have interesting applications in food, pharmaceutical and cosmetic formulations.     

Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein

HET-C2 is a fungal glycolipid transfer protein (GLTP) that uses an evolutionarily-modified GLTP-fold to achieve more focused transfer specificity for simple neutral glycosphingolipids than mammalian GLTPs. Only one of HET-C2's two Trp residues is topologically identical to the three Trp residues of mammalian GLTP. Here, we provide the first assessment of the functional roles of HET-C2 Trp residues in glycolipid binding and membrane interaction. Point mutants HET-C2^W208F, HET-C2^W208A and HET-C2^F149Y all retained > 90% activity and 80–90% intrinsic Trp fluorescence intensity; whereas HET-C2^F149A transfer activity decreased to ~ 55% but displayed ~ 120% intrinsic Trp emission intensity. Thus, neither W208 nor F149 is absolutely essential for activity and most Trp emission intensity (~ 85–90%) originates from Trp109. This conclusion was supported by HET-C2^W109Y/F149Y which displayed ~ 8% intrinsic Trp intensity and was nearly inactive. Incubation of the HET-C2 mutants with 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles containing different monoglycosylceramides or presented by lipid ethanol-injection decreased Trp fluorescence intensity and blue-shifted the Trp λ_max by differing amounts compared to wtHET-C2. With HET-C2 mutants for Trp208, the emission intensity decreases (~ 30–40%) and λ_max blue-shifts (~ 12 nm) were more dramatic than for wtHET-C2 or F149 mutants and closely resembled human GLTP. When Trp109 was mutated, the glycolipid induced changes in HET-C2 emission intensity and λ_max blue-shift were nearly nonexistent. Our findings indicate that the HET-C2 Trp λ_max blue-shift is diagnostic for glycolipid binding; whereas the emission intensity decrease reflects higher environmental polarity encountered upon nonspecific interaction with phosphocholine headgroups comprising the membrane interface and specific interaction with the hydrated glycolipid sugar.     

Spectroscopic characterization of the inclusion complexes of luteolin with native and derivatized β-cyclodextrin

The inclusion complexes of Luteolin (LU) with cyclodextrins (CDs) including β-cyclodextrin (βCD), hydroxypropyl-β-cyclodextrin (HPβCD) and dimethyl-β-cyclodextrin (DMβCD), Scheme 1, have been investigated using the method of steady-state fluorescence. The stoichiometric ratio of the three complexes was found to be 1:1 and the stability constants (K) were estimated from spectrofluorometric titrations, as well as the thermodynamic parameters. Maximum inclusion ability was obtained in the case of HPβCD followed by DMβCD and βCD. Moreover, ¹H NMR and 2D NMR were carried out, revealing that LU has different form of inclusion which is in agreement with molecular modeling studies. These models confirm that when LU–βCD and LU–DMβCD complexes are formed, the B-ring is oriented toward the primary rim; however, for LU–HPβCD complex this ring is oriented toward the secondary rim. The ESR results showed that the antioxidant activity of luteolin was the order LU–HPβCD > LU–DMβCD > LU–βCD > LU, hence the LU-complexes behave are better antioxidants than luteolin free.     

Stop-flow kinetics studies of the interaction of surfactant, sodium dodecyl sulfate, with acid-denatured cytochrome c

Interactions of sodium dodecyl sulfate (SDS) at submicellar and micellar concentration, with the globular protein, horse heart cytochrome c, at low pH have been shown to stabilize two molten globule-like intermediates. These dynamic studies were performed using far-UV, near-UV, and Soret-band circular dichroism (CD) as well as fluorescence methods. Stopped-flow CD and fluorescence studies of acid-denatured cytochrome c refolding with SDS were performed at both submicellar and micellar concentrations. Distinctive refolding mechanisms (from analysis of both CD and fluorescence) were found under these two conditions, and an obvious refolding intermediate was evident in the fluorescence traces. In addition, stopped-flow CD in the Soret region showed multistep kinetics, suggesting that the spectral changes in this region are not only solvent effect related but also connected with the change of secondary structure. A possible folding mechanism is proposed to rationalize the kinetics results.     

A Partially Folded State of Ovalbumin at Low pH Tends to Aggregate

At pH 2, ovalbumin retains native-like secondary structure as seen by far-UV CD and FTIR, but lacks well-defined tertiary structure as seen by the fluorescence and near-UV CD spectra. Addition of 20 mM Trifluoroacetic acid (TFA) or 30 mM Trichloroacetic acid (TCA) on acid-induced state results in protein aggregation. This aggregated state possesses extensive β-sheet structure as revealed by far-UV CD and FTIR spectroscopy. Furthermore, the aggregates exhibit decreased ANS fluorescence and increased thioflavin T fluorescence. The presence of aggregates was confirmed by size exclusion chromatography. Such a formation of β-sheet structure is found in the amyloid of a number of neurological diseases such as Alzheimer’s and scrapie. Ovalbumin at low pH, in the presence of K₂SO₄, exists in partially folded state characterized by native-like secondary structure and tertiary folds.     

Alcohol-induced versus anion-induced states of alpha-chymotrypsinogen A at low pH

Characterization of conformational transition and folding intermediates is central to the study of protein folding. We studied the effect of various alcohols (trifluoroethanol (TFE), butanol, propanol, ethanol and methanol) and salts (K(3)FeCN(6), Na(2)SO(4), KClO(4) and KCl) on the acid-induced state of alpha-chymotrypsinogen A, a predominantly beta-sheet protein, at pH 2.0 by near-UV circular dichroism (CD), far-UV CD and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence measurements. Addition of alcohols led to an increase in ellipticity value at 222 nm indicating the formation of alpha-helical structure. The order of effectiveness of alcohols was shown to be TFE>butanol>propanol>ethanol>methanol. ANS fluorescence data showed a decrease in fluorescence intensity on alcohol addition, suggesting burial of hydrophobic patches. The near-UV CD spectra showed disruption of tertiary structure on alcohol addition. No change in ellipticity was observed on addition of salts at pH 2.0, whereas in the presence of 2 M urea, salts were found to induce a molten globule-like state as evident from the increases in ellipticity at 222 nm and ANS fluorescence indicating exposure of hydrophobic regions of the protein. The effectiveness in inducing the molten globule-like state, i.e. both increase in ellipticity at 222 nm and increase in ANS fluorescence, followed the order K(3)FeCN(6)>Na(2)SO(4)>KClO(4)>KCl. The loss of signal in the near-UV CD spectrum on addition of alcohols indicating disordering of tertiary structure results suggested that the decrease in ANS fluorescence intensity may be attributed to the unfolding of the ANS binding sites. The results imply that the alcohol-induced state had characteristics of an unfolded structure and lies between the molten globule and the unfolded state. Characterization of such partially folded states has important implications for protein folding.     

The role of tryptophans on the cellular uptake and membrane interaction of arginine-rich cell penetrating peptides

Cell-penetrating peptides (CPP) are able to efficiently transport cargos across cell membranes without being cytotoxic to cells, thus present a great potential in drug delivery and diagnosis. While the role of cationic residues in CPPs has been well studied, that of Trp is still not clear. Herein 7 peptide analogs of RW9 (RRWWRRWRR, an efficient CPP) were synthesized in which Trp were systematically replaced by Phe residues. Quantification of cellular uptake reveals that substitution of Trp by Phe strongly reduces the internalization of all peptides despite the fact that they strongly accumulate in the cell membrane. Cellular internalization and biophysical studies show that not only the number of Trp residues but also their positioning in the helix and the size of the hydrophobic face they form are important for their internalization efficacy, the highest uptake occurring for the analog with 3 Trp residues. Using CD and ATR-FTIR spectroscopy we observe that all peptides became structured in contact with lipids, mainly in α-helix. Intrinsic tryptophan fluorescence studies indicate that all peptides partition in the membrane in about the same manner (Kp ~ 10⁵) and that they are located just below the lipid headgroups (~ 10 Å) with slightly different insertion depths for the different analogs. Plasmon Waveguide Resonance studies reveal a direct correlation between the number of Trp residues and the reversibility of the interaction following membrane washing. Thus a more interfacial location of the CPP renders the interaction with the membrane more adjustable and transitory enhancing its internalization ability.     

Position—Specific contribution of interface tryptophans on membrane protein energetics

Interface tryptophans are key residues that facilitate the folding and stability of membrane proteins. Escherichia coli OmpX possesses two unique interface tryptophans, namely Trp76, which is present at the interface and is solvent-exposed, and Trp140, which is relatively more lipid solvated than Trp76 in symmetric lipid membranes. Here, we address the requirement for tryptophan and the consequences of aromatic amino acid substitutions on the folding and stability of OmpX. Using spectroscopic measurements of OmpX-Trp/Tyr/Phe mutants, we show that the specific mutation W76 → Y allows barrel assembly > 1.5-fold faster than native OmpX, and increases stability by ~ 0.4 kcal mol^− 1. In contrast, mutating W140 → F/Y lowers OmpX thermodynamic stability by ~ 0.4 kcal mol^− 1, without affecting the folding kinetics. We conclude that the stabilizing effect of tryptophan at the membrane interface can be position—and local environment—specific. We propose that the thermodynamic contributions for interface residues be interpreted with caution.     

Development of a subunit vaccine for prevention of Clostridium difficile associated diseases: Biophysical characterization of toxoids A and B

Inactivation of bacterial toxins for use in human vaccines traditionally is achieved by treatment with formaldehyde. In contrast, the bivalent experimental vaccine for the prevention of C. difficile infections (CDI) that is currently being evaluated in clinical trials was produced using a different strategy. C. difficile toxins A and B were inactivated using site-directed mutagenesis and treatment with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysulfosuccinimide (EDC/NHS). In the present work we investigate the effect of genetic and chemical modifications on the structure of inactivated toxins (toxoids) A and B. The far-UV circular dichroism (CD) spectra of wild type toxins, mutated toxins, and EDC/NHS-inactivated toxoids reveal that the secondary structure of all proteins is very similar. The near-UV CD spectra show that aromatic residues of all proteins are in a unique asymmetric environment, indicative of well-defined tertiary structure. These results along with the fluorescence emission maxima of 335 nm observed for all proteins suggest that the tertiary structure of toxoids A and B is preserved as well. Analytical ultracentrifugation data demonstrate that all proteins are predominantly monomeric with small fractions of higher molecular weight oligomeric species present in toxoids A and B. Differential scanning calorimetry data reveal that genetic mutations induce thermal destabilization of protein structures. Subsequent treatment with EDC/NHS results either in a minimal (1 °C) increase of apparent thermostability (toxoid B) or no change at all (toxoid A). Therefore, our two-step inactivation strategy is an effective approach for the preparation of non-toxic proteins maintaining native-like structure and conformation.     

Bioluminescent bioreporter Pseudomonas putida TVA8 as a detector of water pollution. Operational conditions and selectivity of free cells sensor

Conditions influencing bioluminescence output from Pseudomonas putida TVA8 harboring chromosomal tod-luxCDABE fusion were followed. In complex media, cell growth was not influenced by the presence of toluene at 53 mg/L. Bioluminescence induction was tested in minimal medium. At 15 °C the highest bioluminescence induced with toluene (1.325 mg/L) was reached after 6 h. At 25 °C the bioluminescence maximum was approximately 20% lower but this was reached after 3.5 h, and at temperatures of 7 °C, 28 °C, 30 °C and 34 °C, bioluminescence peaked at ≤60% of the maximum. Time courses of bioluminescence were dependent on cell concentrations. The heights of bioluminescence maxima were proportional to toluene concentration in the range 0–26 mg/L. Twenty-three organic pollutants (10³× diluted saturated solutions) were tested as bioluminescent inducers. The bioluminescence maximum decreased in the order: ethylbenzene > toluene > phenol > benzene > 4-ethyltoluene > 4-fluorotoluene > cumene > isobutylbenzene > styrene > trichloroethylene > o-, p-xylene > cresol > m-xylene > 2-methylnaphthalene > benzylchloride > naphthalene > salicylic acid > hexachlorobenzene > 2-chloronaphthalene > biphenyl > 2-bromonaphthalene > 1,3,5-triethylbenzene. Bioluminescence was also induced with ethanol and methanol and the presence of these alcohols in concentrations of ≤1% increased bioluminescence of toluene. The induction of bioluminescence from samples of wastewater and groundwater contaminated with BTEX (benzene, toluene, ethylbenzene and xylene) from 0.5 to 120 mg/L was demonstrated.     

Effect of polyols and salts on the acid-induced state of human placental cystatin

Polyols (glycerol and sorbitol) and salts (magnesium sulfate, sodium sulfate, and magnesium chloride) have been used to study the refolding of the acid-induced state of human placental cystatin (HPC), which is a low molecular weight (12,500 daltons) thiol proteinase inhibitor, in terms of CD spectroscopy, binding of hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid (ANS), and intrinsic fluorescence measurements. The helical content of acid-denatured HPC increased with increase in glycerol concentration (0–80%). At 80% glycerol concentration, the secondary structural features observed in the far UV-CD region are similar to those of the native state (pH 6.0). The intrinsic fluorescence and near UV-CD studies showed that this 80% glycerol-induced state has a significant amount of tertiary structure with decreased ANS binding compared to the acid-denatured state. It was found that glycerol is more effective in stabilizing the acid-denatured state of HPC as compared to sorbitol. Among salts the stability effect was more for MgCl₂ (used up to concentration of 3 M) compared to MgSO₄ and Na₂SO₄ (used up to the concentration of 1.5 M due to restricted solubility of HPC at higher sulfate salt concentrations) as determined by CD studies and fluorescence measurements, which showed secondary and tertiary structural resemblance of this MgCl₂-induced state close to native state and showed overall spectral features in between the native state and the acid-denatured state. This MgCl₂ (3 M)-induced state showed decreased ANS fluorescence as compared to the acid-denatured state but more than that of the native state. The results taken together suggest that the acid-denatured state of HPC in the presence of 80% glycerol or 3 M MgCl₂ has a conformation in between that of the native state (pH 6.0) and the acid-induced state at pH 2.0. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 6, pp. 768–777.     

Simultaneous determination of tryptophan and kynurenine in plasma samples of children patients with Kawasaki disease by high-performance liquid chromatography with programmed wavelength ultraviolet detection

A simple, fast, sensitive and specific high-performance liquid chromatography (HPLC) method is developed for simultaneous determination of kynurenine (Kyn) and tryptophan (Trp) with ultraviolet (UV) detection setting programmed wavelength. The separation was carried out on an Agilent Hypersil ODS column (125 mm × 4.0 mm, 5 μm) in less than 6 min and the eluate was monitored by the programmed wavelength detection setting at 360 nm from 0 min to 4 min for Kyn, and at 278 nm from 4 min to 6 min for Trp in a single run with UV detector. The linearities of the method were from 0.20 μmol/L to 21.2 μmol/L for Kyn and 2.25–678.0 μmol/L for Trp, and the detection limits were 0.028 μmol/L for Kyn and 0.053 μmol/L for Trp, respectively. Satisfactory precisions and recoveries were obtained by this method. The assay was employed to analyze plasma samples of children patients with Kawasaki disease (KD). The result showed great difference between Kawasaki disease and control group.     

Effect of the exposure to methyl-β-cyclodextrin prior to chilling or vitrification on the viability of bovine immature oocytes

The present study aimed to evaluate the effect of methyl-β-cyclodextrin (MβCD) as a cholesterol loader to change oocyte plasma membrane and increase its tolerance toward cryopreservation. The first and second experiments were conducted to investigate if MβCD could improve nuclear and cytoplasmic maturation after oocyte exposure to cold stress for 10 or 30 min, respectively. No differences (P > 0.05) in either experiment in the metaphase II (MII) rate of oocytes exposed to MβCD and cold stress; but these oocytes presented lower maturation rates than control groups. In the second experiment, a lower percentage of oocytes showed degenerated chromatin (P < 0.05) after exposure to 2 mg/mL of MβCD compared to the group exposed to 0 mg/mL. However, no differences among treatments were observed in cytoplasmic maturation. Groups exposed to cold stress demonstrated a lower (P < 0.05) capacity for embryonic development compared to the control groups. In the third experiment immature oocytes were exposed to MβCD and then, vitrified (cryotop). After warming, we observed that the ability to reach MII and chromatin degeneration were altered (P < 0.05) by MβCD. The blastocysts rate (P < 0.05) on D7 was higher in the 2 mg/mL MβCD group, but an identical finding was not observed on D8 (P > 0.05). Chromatin degeneration was higher in the vitrification groups. We conclude that MβCD improved nuclear maturation by reducing oocyte degeneration after cold stress or vitrification; however, more studies are required to clarify the usefulness of MβCD use in oocyte cryopreservation.     

Hybrid fluorescent conjugates of COX-2 inhibitors: Search for a COX-2 isozyme imaging cancer biomarker

The observation that the cyclooxygenase-2 (COX-2) isozyme is over-expressed in multiple types of cancer, relative to that in adjacent non-cancerous tissue, prompted this investigation to prepare a group of hybrid fluorescent conjugates wherein the COX inhibitors ibuprofen, (S)-naproxen, acetyl salicylic acid, a chlororofecoxib analog and celecoxib were coupled via a linker group to an acridone, dansyl or rhodamine B fluorophore. Within this group of compounds, the ibuprofen-acridone conjugate (10) showed potent and selective COX-2 inhibition (COX-2 IC₅₀ = 0.67 μM; SI = 110.6), but its fluorescence emission (λ_em = 417, 440 nm) was not suitable for fluorescent imaging of cancer cells that over-express the COX-2 isozyme. In comparison, the celecoxib-dansyl conjugate (25) showed a slightly lower COX-2 potency and selectivity (COX-2 IC₅₀ = 1.1 μM; SI > 90) than the conjugate 10, and it possesses a better fluorescence emission (λ_em = 500 nm). Ultimately, a celecoxib-rhodamine B conjugate (28) that exhibited moderate COX-2 potency and selectivity (COX-2 IC₅₀ = 3.9 μM; SI > 25) having the best fluorescence emission (λ_em = 580 nm) emerged as the most promising biomarker for fluorescence imaging using a colon cancer cell line that over-expresses the COX-2 isozyme.     

Genetic stability of an Escherichia coli strain engineered to produce a novel therapeutic DNA vaccine encoding chicken type II collagen for rheumatoid arthritis

The development of therapeutic DNA vaccines capable of recovering immunological tolerance through the induction of both CD4 + CD25 + FoxP3 + regulatory and CD3 + CD8 + C28-suppressor T cells, and/or inhibition of both autoreactive CD4 + CD28+ type 1 T helper and autoantibody-producing B cells offers a promising new strategy for the treatment of rheumatoid arthritis. Previously, we developed pcDNA-CCOL2A1, a novel therapeutic DNA vaccine, which encodes the full-length chicken type II collagen sequence, and demonstrated that the efficacy of this vaccine for treating rheumatoid arthritis was comparable to that of the current “gold standard” treatment, methotrexate. In this study, we investigated the genetic stability of a strain engineered to produce the vaccine during continuous passage and long-term storage at different temperatures. By screening a panel of 12 strains, we identified a DH5α strain that exhibited high levels (12.30 ± 0.05 mg L⁻¹) of pcDNA-CCOL2A1 production after 15 h cultivation, and subsequently utilized this strain to establish a three-tier cells bank for future studies. Continuous passage of this strain for 100 inoculation times demonstrated that a higher percentage (>95%) of cells maintained the plasmid when cultivated under selective pressure (ampicillin) than under nonselective conditions, suggesting that the presence of antibiotics in the medium prevents the loss of the pcDNA-CCOL2A1 plasmid. Meanwhile, restriction digestion and gene sequencing analyses demonstrated that the pcDNA-CCOL2A1 vector remained stable, and that the plasmid sequence was conserved during this period. Lastly, the DH5α pcDNA-CCOL2A1 strain exhibited a high plasmid preservation (>90%) and high levels of plasmid production (9.05mg L⁻¹) after storage for 60 months at −80 °C. Furthermore the plasmid extracted from the DH5α pcDNA-CCOL2A1 strain after storage for 60 months at −80 °C was transfected to COS-7 cells, it can stably express the target protein chicken type II collagen. Conversely, this strain exhibited a complete loss of capability after 24 and 18 months storage at −20 °C and 4 °C, respectively. These findings will facilitate further pilot-scale testing, and even industrial-scale production, of the novel therapeutic vaccine pcDNA-CCOL2A1.