Novel zinc(II)-curcumin molecular probes bearing berberine and jatrorrhizine derivatives as potential mitochondria-targeting anti-neoplastic drugs.

Berberine and jatrorrhizine are major bioactive components that are emerging as potential anti-cancer drugs. However, no zinc(II) - berberine/jatrorrhizine - curcumin compounds have been reported in the literature to date. Therefore, the molecular mechanisms associated with their cytotoxicity remain unexplored. To investigate the potential mitochondria-targeting ability, anti-neoplastic activity, and utility in cell imaging of berberine and jatrorrhizine derivates, four novel zinc(II) complexes, [Zn(Ber)(H2O)Cl2] (Zn(Ber)), [Zn(Ber)(Cur)Cl] (Zn(CurBer)), [Zn(Jat)(H2O)Cl2] (Zn(Jat)), and [Zn(Jat)(Cur)Cl] (Zn(CurJat)) bearing the berberine-derived ligand 2,2,2-trifluoroacetate 10-methoxy-9-((9-((2-(pyridin-2-yl)ethyl)amino)nonyl)oxy) -5,6-dihydro- [1,3]dioxolo[4,5-g]isoquinolino [(Torre et al., 2015; de Ruijter et al., 2020) 3,23,2-a]isoquinolin-7-ium (Ber), the jatrorrhizine-derived ligand 2,2,2-trifluoroacetate 2,9,10-trimethoxy-3-((9- ((2-(pyridin-2-yl)ethyl)amino)nonyl)oxy)-5,6-dihydroisoquinolino [(Torre et al., 2015; de Ruijter et al., 2020) 3,23,2-a]isoquinolin-7-ium (Jat), and/or curcumin (H-Cur) were first synthesised in this study. Zn(Ber), Zn(CurBer), Zn(Jat), and Zn(CurJat) showed higher cytotoxicity against human MCF-7 (breast adenocarcinoma) cells than did cisplatin, with IC50 values ranging from 0.21 to 4.45 µM. The anti-neoplastic activities of the zinc(II) - berberine/jatrorrhizine - curcumin complexes were in the following order: Zn(CurBer) > Zn(CurJat) > Zn(Ber) > Zn(Jat) > cisplatin > H-Cur > Ber > Jat > ZnCl2. Among these, Zn(CurBer) displayed the highest cytotoxicity (0.21 ± 0.06 µM). Furthermore, mechanistic investigations revealed that Zn(CurBer) and Zn(CurJat) could accumulate in the mitochondria, exhibit red fluorescence, and trigger mitophagy and apoptosis. In vivo anti-cancer evaluations also suggested that Zn(CurBer) inhibited MCF-7 xenograft tumour growth more effectively than cisplatin and Zn(CurJat). This is the first report describing the synthesis of zinc(II) - berberine/jatrorrhizine - curcumin complexes and their potential use as molecular probes and mitochondria-targeting anti-neoplastic drugs.

Involvement of nitrergic neurons in colonic motility in a rat model of ulcerative colitis.

BACKGROUND: The mechanisms underlying gastrointestinal (GI) dysmotility with ulcerative colitis (UC) have not been fully elucidated. The enteric nervous system (ENS) plays an essential role in the GI motility. As a vital neurotransmitter in the ENS, the gas neurotransmitter nitric oxide (NO) may impact the colonic motility. In this study, dextran sulfate sodium (DSS)-induced UC rat model was used for investigating the effects of NO by examining the effects of rate-limiting enzyme nitric oxide synthase (NOS) changes on the colonic motility as well as the role of the ENS in the colonic motility during UC. AIM: To reveal the relationship between the effects of NOS expression changes in NOS-containing nitrergic neurons and the colonic motility in a rat UC model. METHODS: Male rats (n = 8/each group) were randomly divided into a control (CG), a UC group (EG1), a UC + thrombin derived polypeptide 508 trifluoroacetic acid (TP508TFA; an NOS agonist) group (EG2), and a UC + NG-monomethyl-L-arginine monoacetate (L-NMMA; an NOS inhibitor) group (EG3). UC was induced by administering 5.5% DSS in drinking water without any other treatment (EG1), while the EG2 and EG3 were gavaged with TP508 TFA and L-NMMA, respectively. The disease activity index (DAI) and histological assessment were recorded for each group, whereas the changes in the proportion of colonic nitrergic neurons were counted using immunofluorescence histochemical staining, Western blot, and enzyme linked immunosorbent assay, respectively. In addition, the contractile tension changes in the circular and longitudinal muscles of the rat colon were investigated in vitro using an organ bath system. RESULTS: The proportion of NOS-positive neurons within the colonic myenteric plexus (MP), the relative expression of NOS, and the NOS concentration in serum and colonic tissues were significantly elevated in EG1, EG2, and EG3 compared with CG rats. In UC rats, stimulation with agonists and inhibitors led to variable degrees of increase or decrease for each indicator in the EG2 and EG3. When the rats in EGs developed UC, the mean contraction tension of the colonic smooth muscle detected in vitro was higher in the EG1, EG2, and EG3 than in the CG group. Compared with the EG1, the contraction amplitude and mean contraction tension of the circular and longitudinal muscles of the colon in the EG2 and EG3 were enhanced and attenuated, respectively. Thus, during UC, regulation of the expression of NOS within the MP improved the intestinal motility, thereby favoring the recovery of intestinal functions. CONCLUSION: In UC rats, an increased number of nitrergic neurons in the colonic MP leads to the attenuation of colonic motor function. To intervene NOS activity might modulate the function of nitrergic neurons in the colonic MP and prevent colonic motor dysfunction. These results might provide clues for a novel approach to alleviate diarrhea symptoms of UC patients.

Simultaneous purification and depolymerization of Streptococcus pneumoniae serotype 2 capsular polysaccharides by trifluoroacetic acid.

Development of an effective purification process in order to provide low cost and high-quality vaccine is the necessity of glycoconjugate vaccine manufacturing industries. In the present study, we have attempted to develop a method for simultaneous purification and depolymerization process for capsular polysaccharides (CPS) derived from Streptococcus pneumoniae serotype 2. Trifluoroacetic acid (TFA) was used to precipitate impurities which were then removed by centrifugation. It was observed that the TFA treatment could simultaneously depolymerize the CPS and purify it. The purified and depolymerized CPS was analyzed for its purity, structural identity and conformity, molecular size, antigenicity to meet desired quality specifications. The obtained results showed that the purification and depolymerization of S. pneumoniae serotype 2 CPS did not affect the antigenicity of CPS.

The Effect of Grapefruit Juice on the Pharmacokinetics of Tadalafil in Rats.

We developed and validated a novel, sensitive, selective, and inexpensive high-performance liquid chromatography (HPLC) method for the determination of tadalafil in rats plasma and to investigate the effect of grapefruit juice on the pharmacokinetics of tadalafil in rats. The ZORBAX Eclipse XDB-C18 (4.6 × 150 mm, 5 µm) chromatography column can be used to separate tadalafil and carbamazepine (internal standard, IS). A mixture of acetonitrile-0.2% trifluoroacetic acid-water (48 : 10 : 42, V/V/V) was used as the mobile phase with a flow rate of 1.0 mL/min. The column temperature was set at 35.0°C. The detection wavelength was set at 286 nm. The tadalafil was extracted by ethyl acetate from plasma at the alkaline condition. 12 healthy male Sprague-Dawley (SD) rats were randomly divided into two groups, Group A (experimental group, received grapefruit juice 5 mL/kg for 7 days) and Group B (control group, received normal saline for 7 days). All the rats were given a single dose of tadalafil (5 mg/kg) after the last administration. The main pharmacokinetic parameters were calculated by DAS 2.0 software. Under the conditions of this experiment, the plasma concentrations of tadalafil in the range of 10-2000 ng/ml had a good linear relationship. The intra- and interday precision for tadalafil in plasma were less than 15%, and the relative recovery rate was good at low, medium, and high QC levels. The C max of tadalafil in the control group and the experimental group was (725.89 ± 161.59) ng/mL and (1271.60 ± 179.31) ng/mL, t 1/2 was (9.28 ± 2.07) h and (11.70 ± 1.47) h, AUC (0-t) was (7399.61 ± 696.85) ng·h/mL and (9586.52 ± 2048.81) ng·h/mL, and AUC(0-∞) was (7995.50 ± 707.23) ng·h/mL and (10639.43 ± 2235.94) ng·h/mL, respectively. Results show that the C max of tadalafil in group A was 75.17% higher than that in group B, the Vz/F was also reduced, and the t 1/2 was increased by 2.42 h. The developed HPLC-DAD method for the determination of tadalafil in rats plasma was accurate, reproducible, specific, and it was found to be suitable for the pharmacokinetics of tadalafil and food-drug interactions. Grapefruit juice can inhibit the metabolism of tadalafil and increase the exposure of tadalafil in rats.

Synthesis and in vitro biological evaluation of novel derivatives of Flexicaulin A condensation with amino acid trifluoroacetate.

As our research focus on anticancer drugs, two series of novel derivatives of Flexicaulin A (FA), an ent-kaurene diterpene, condensation with amino acid trifluoroacetate were synthesized, and their anti-proliferative activity against four human cancer cell lines (TE-1, MCF-7, A549 and MGC-803) were evaluated. Compared with FA, the anticancer activity and solubility of most derivatives were significantly improved. Among them, compound 6d had the best activity, and its IC50 value against Esophageal cancer cells (TE-1) was up to 0.75 µM. Subsequent cellular mechanism studies showed that compound 6d could inhibit the proliferation of cancer cells, the formation of cell colonies, and increase the level of ROS on TE-1 cells. In addition, 6d could up-regulate the expressions of SAPK/JNK pathway-associated proteins (p-ASK1, p-MKK4 and p-JNK) and pro-apoptotic proteins (Bak, Bad and Noxa), remarkably increase the ratio of Bax to Bcl-2 and activate Cleaved Caspase-3/9/PARP. These results indicate that compound 6d induces apoptosis through the ROS/JNK/Bcl-2 pathway and holds promising potential as an anti-proliferative agent.

Evaluation of MALDI-TOF mass spectrometry for the identification of bacteria growing as biofilms.

We evaluated MALDI-TOF mass spectrometry to identify bacteria from biofilms. We compared three sample preparation procedures on biofilms grown in vitro. The extended direct transfer method was able to identify 13 isolates out of 18 (72%) at the species level and 15 out of 18 (83%) at the genus level.

Synthesis and Evaluation of the Antioxidant Activity of Lipophilic Phenethyl Trifluoroacetate Esters by In Vitro ABTS, DPPH and in Cell-Culture DCF Assays.

Polyphenols are natural compounds showing a variety of health-promoting effects. Unfortunately, due to low lipid solubility, their applications in the pharmaceutical, food, and cosmetic industries are limited. With the aim of obtaining novel lipophilic derivatives, the present study reports the synthesis of a series of phenethyl trifluoroacetate esters containing up to two hydroxyl groups in the aromatic ring. Experimental logP values confirmed a greater lipophilicity of the novel compounds compared to the parent compounds. The radical scavenging capacity of all phenethyl trifluoroacetate esters was evaluated by in vitro assays (ABTS, DPPH) and in cultured cells (L6 myoblasts and THP-1 leukemic monocytes) using 2',7'-dichlorodihydrofluorescein diacetate. These data revealed that the esters showed a good antioxidant effect that was strictly dependent on the grade of hydroxylation of the phenyl ring. The lack of toxicity, evaluated by the MTT assay and proliferation curves, makes these trifluoroacetates attractive derivatives for pharmaceutical, food, and cosmetic applications.

2D IR Correlation Spectroscopy in the Determination of Aggregation and Stability of KH Domain GXXG Loop Peptide in the Presence and Absence of Trifluoroacetate.

Trifluoroacetate (TFA) is a strong anion byproduct of solid-phase peptide synthesis. Fourier transform infrared (FT-IR) spectroscopy can be used to ascertain the presence of this excipient in peptide samples for quality assessment. TFA absorbs as a strong sharp peak (1675 cm-1) within the amide I' band of the spectral region. A peptide sample and the TFA excipient can be studied simultaneously by FT-IR and 2D IR correlation spectroscopies. In addition, these techniques are able to determine the effect of TFA on the stability of the peptide. Herein, we describe the spectroscopic characterization of the GXXG loop peptide (GXXGlp), which is present in KH domain containing proteins. The sequence of the Homo sapiens Krr1 GXXGlp is evolutionarily conserved (165KRRQRLIGPKGSTLKALELLTNCY189) and has been associated with ssDNA interaction and ribosome biogenesis. Our goal was to determine the structural elements present in this peptide and evaluate whether TFA affects the stability of GXXGlp during thermal stress. We observed differences in the molecular behavior of the synthetic peptide in the presence and absence of TFA at various peptide concentrations. Finally, 2D IR correlation spectroscopy was used for the determination of the unfolding process, mechanism and extent of peptide aggregation, and the effect of TFA on the stability of the peptide. This spectroscopic method can be applied to the characterization of any synthetic peptide.

Comparative proteomic analysis of the influence of gender and acid stimulation on normal human saliva using LC/MS/MS.

PURPOSE: Human saliva is an important source for disease biomarker discovery. This study is to investigate the influence of gender and acid stimulation on the normal human salivary proteome. EXPERIMENTAL DESIGN: Unstimulated and acid-stimulated saliva samples from 5 males and 5 females were labeled with 4-plex iTRAQ and analyzed by 2-DLC MS/MS. By bioinformatics analysis the gender and acid stimulation related proteins were defined. According to protein annotation the important proteins were validated by multiple reaction monitor analysis. RESULTS: A total of 1770 proteins were identified, and 82 proteins in unstimulated saliva were found to be gender-specific, mainly associated with immune function, metabolism and inflammation. However, no gender-specific proteins were found in acid-stimulated saliva. In addition, 182 and 307 differential proteins were found to be acid stimulation-specific in male samples and female samples, respectively, mainly participated in the process of cellular movement, immune function and inflammatory response. Besides, it was found that acid stimulation caused more significant alteration and played a more important role in the human salivary proteome than gender. Gender-specific (IGHG2 and TIMP1) and acid stimulation (PERL, ENOA, ACTB, B4E022 and CALL3) related proteins were validated by MRM analysis. CONCLUSIONS AND CLINICAL RELEVANCE: The results indicate that gender differences exist in the unstimulated salivary proteome, and the influence of acid stimulation on the salivary proteome was more significant than that of gender. The above results may be helpful for salivary proteome research in the future.

Increasing protein charge state when using laser electrospray mass spectrometry.

Femtosecond (fs) laser vaporization is used to transfer cytochrome c, myoglobin, lysozyme, and ubiquitin from the condensed phase into an electrospray (ES) plume consisting of a mixture of a supercharging reagent, m-nitrobenzyl alcohol (m-NBA), and trifluoroacetic acid (TFA), acetic acid (AA), or formic acid (FA). Interaction of acid-sensitive proteins like cytochrome c and myoglobin with the highly charged ES droplets resulted in a shift to higher charge states in comparison with acid-stable proteins like lysozyme and ubiquitin. Laser electrospray mass spectrometry (LEMS) measurements showed an increase in both the average charge states (Zavg) and the charge state with maximum intensity (Zmode) for acid-sensitive proteins compared with conventional electrospray ionization mass spectrometry (ESI-MS) under equivalent solvent conditions. A marked increase in ion abundance of higher charge states was observed for LEMS in comparison with conventional electrospray for cytochrome c (ranging from 19+ to 21+ versus 13+ to 16+) and myoglobin (ranging from 19+ to 26+ versus 18+ to 21+) using an ES solution containing m-NBA and TFA. LEMS measurements as a function of electrospray flow rate yielded increasing charge states with decreasing flow rates for cytochrome c and myoglobin.

Effects of peptide fraction and counter ion on the development of clinical signs in experimental autoimmune encephalomyelitis.

The most commonly used immunogen to induce experimental autoimmune encephalomyelitis is MOG35-55 , a 21-residue peptide derived from myelin oligodendrocyte glycoprotein (MOG). In most studies, mice exhibit a chronic disease; however, in some studies mice show a transient disease. One variable that is not often controlled for is the peptide fraction of the purified MOG material, which can vary from less than 50% to over 90%, with the remainder of mass primarily comprised of the counter ion used for peptide purification. We compared the development of clinical signs in female C57Bl6 mice immunized with two commercially available MOG35-55 peptides of similar purity but different peptide fraction (MOG-A being 45%; MOG-B being 72%). A single immunization with MOG-A induced a chronic disease course with some recovery at later stages, whereas immunization with MOG-B induced a similar course of disease but with significantly lower average clinical scores despite a higher peptide content. The addition of a booster immunization significantly increased clinical severity with both preparations, and significantly reduced the average day of onset using MOG-A. To determine if the counter ion could influence disease, we compared MOG-B-containing trifluoroacetate with MOG-B-containing acetate. Although disease incidence and severity were similar, the average day of disease onset occurred approximately 5 days earlier with the use of MOG-B-containing trifluoroacetate. These results demonstrate that differences in peptide fraction influence the course of encephalomyelitis disease, which may be due in part to the levels of counter ions present in the purified material. These findings underscore the fact that a knowledge of peptide fraction is as critical as knowledge of peptide purity when using peptides from different sources.

Characterization and bacterial adhesion of chitosan-perfluorinated acid films.

We reported herein the study and characterization of films obtained by casting of chitosan solutions in perfluorinated acids, trifluoroacetic (TFA), perfluoropropionic (PFPA), and perfluorooctanoic (PFOA). The films were characterized by FTIR, solid state (13)C NMR, X-ray, AFM, contact angle, thermogravimetric effluent analysis by mass spectrometry, and rheology. The results showed a marked influence of chain length of the perfluorinated acids on the hydrophobic/hydrophilic ratio of the modified chitosan films which was evidenced by the different characteristics observed. The material that showed greater surface stability was chitosan-PFOA. Chitosan film with the addition of PFOA modifier became more hydrophobic, thus water vapor permeability diminished compared to chitosan films alone, this new material also depicted bacterial adhesion which, together with the features already described, proves its potential in applications for bioreactor coating.

Hydration-state change of horse heart cytochrome c corresponding to trifluoroacetic-acid-induced unfolding.

We investigate the hydration state of horse-heart cytochrome c (hh cyt c) in the unfolding process induced by trifluoroacetic acid (TFA). The conformation of hh cyt c changes from the native (N) state (2.9 < pH < 6.0) to the acid-unfolded (U(A)) state (1.7 < pH < 2.0) to the acid-induced molten globule (A) state (pH ∼1.2). Hydration properties of hh cyt c during this process are measured at 20°C by high-resolution dielectric relaxation (DR) spectroscopy, UV-vis absorbance, and circular dichroism spectroscopy. Constrained water of hh cyt c is observed at every pH as an ∼5-GHz Debye component (DC) (DR time, τ(D) ∼30 ps) and its DR amplitude (DRA) is increased by 77% upon N-to-U(A) transition, when pH changes from 6.0 to 2.0. Even in the N state, the DRA of the constrained-water component is found to be increased by 22% with decreasing pH from 6.0 to 2.9, suggesting an increase in the accessible surface area of native hh cyt c. Moreover, hypermobile water around native hh cyt c is detected at pH 6.0 as a 19-GHz DC (τ(D) ∼ 8.4 ps <τ(DW) = 9.4 ps), but is not found at other pH values. The DRA signal of constrained water is found to return to the pH 2.9 (N-state) level upon U(A)-to-A transition. Fast-response water (slightly slower than bulk) around A-state hh cyt c is detected at pH 1.2, and this suggests some accumulation of TFA(-) ions around the peptide chain. Thus, this high-resolution DR spectroscopy study reveals that hh cyt c exhibits significant hydration-state change in the TFA-unfolding process.

Trifluoroacetate is an allosteric modulator with selective actions at the glycine receptor.

Trifluoroacetic acid is a metabolite of the inhaled anesthetics halothane, desflurane and isoflurane as well as a major contaminant in HPLC-purified peptides. Ligand-gated ion channels, including cys-loop receptors such as the glycine receptor, have been the targets of peptide-based drug design and are considered to be likely candidates for mediating the effects of anesthetics in vivo, but the possible secondary contributions of contaminants and metabolites to these effects have not been studied. We used two-electrode voltage-clamp electrophysiology to test glycine, GABA(A) and 5-HT3 receptors expressed in Xenopus oocytes for their sensitivities to sodium trifluoroacetate. Trifluoroacetate (100 µM-3mM) enhanced the currents elicited by low concentrations of glycine applied to α1 homomeric and α1ß heteromeric glycine receptors, but it had no effects when co-applied with a maximally-effective glycine concentration. Trifluoroacetate had no effects on α1ß2γ2S GABA(A) or 5-HT3A receptors at any GABA or serotonin concentration tested. The results demonstrate that trifluoroacetate acts as an allosteric modulator at the glycine receptor with greater specificity than other known modulators. These results have important implications for both the secondary effects of volatile anesthetics and the presence of contaminating trifluoroacetate in HPLC-purified peptides, which is potentially an important source of experimental variability or error that requires control.

Polymorphism of ß2-microglobulin amyloid fibrils manifested by ultrasonication-enhanced fibril formation in trifluoroethanol.

The polymorphic property of amyloid structures has been focused on as a molecular basis of the presence and propagation of different phenotypes of amyloid diseases, although little is known about the molecular mechanism for expressing diverse structures from only one protein sequence. Here, we have found that, in combination with an enhancing effect of ultrasonication on nucleation, ß(2)-microglobulin, a protein responsible for dialysis-related amyloidosis, generates distinct fibril conformations in a concentration-dependent manner in the presence of 2,2,2-trifluoroethanol (TFE). Although the newly formed fibrils all exhibited a similar needle-like morphology with an extensive cross-ß core, as suggested by Fourier transform infrared absorption spectra, they differed in thioflavin T intensity, extension kinetics, and tryptophan fluorescence spectra even in the same solvents, representing polymorphic structures. The hydrophobic residues seemed to be more exposed in the fibrils originating at higher concentrations of TFE, as indicated by the increased binding of 1-anilinonaphthalene-8-sulfonic acid, suggesting that the modulation of hydrophobic interactions is critical to the production of polymorphic amyloid structures. Interestingly, the fibrils formed at higher TFE concentrations showed significantly higher stability against guanidium hydrochloride, the perturbation of ionic strength, and, furthermore, pressurization. The cross-ß structure inside the fibrils seems to have been more idealized, resulting in increased stability when nucleation occurred in the presence of the alcohol, indicating that a weaker contribution of hydrophobic interactions is intrinsically more amenable to the formation of a non-defective amyloid structure.

Evaluation of extractants and precipitants in tear film proteomic analyses.

PURPOSE: To determine the efficiency of several protein extraction or precipitation treatments used in proteomic analyses. METHODS: Tear samples were taken from each eye of 40 normal subjects using glass microcapillaries. Tear volume was measured followed by storage at -86°C. Lotrafilcon B contact lenses were fitted and worn for 14 days, followed by removal and storage at -86°C. Tear samples from each eye within a subject were randomly assigned to either one of four chemical treatments (acetone, trichloroacetic acid, urea, and trifluoroacetic acid/acetonitrile [TFA/ACN]) or no chemical treatment in groups of 10. Contact lens samples were subjected to the same treatments as tear samples for each subject, with a second treatment preceding the first. Protein concentrations were quantified by Bradford assay. RESULTS: For tear samples, a significant reduction in total protein was observed when subjected to any of the four treatments studied compared with those samples left untreated. A positive relationship was noted between protein concentration and tear volume for treated, untreated, and combined tear samples. For contact lens samples, there was a significant reduction in the amount of deposited protein removed when comparing acetone, trichloroacetic acid, and urea with TFA/ACN. A second extraction from contact lenses assigned to the urea and TFA/ACN groups yielded a significant amount of additional protein compared with the amount removed initially. CONCLUSIONS: Tear samples subjected to any of the evaluated chemical treatments provided significantly less protein than untreated samples. For contact lenses, TFA/ACN extraction provided the highest yield of available protein out of the four treatments evaluated.

Characterization of membrane protein non-native states. 1. Extent of unfolding and aggregation of rhodopsin in the presence of chemical denaturants.

Little is known about the general folding mechanisms of helical membrane proteins. Unfolded, i.e., non-native states, in particular, have not yet been characterized in detail. Here, we establish conditions under which denatured states of the mammalian membrane protein rhodopsin, a prototypic G protein coupled receptor with primary function in vision, can be studied. We investigated the effects of the chemical denaturants sodium dodecyl sulfate (SDS), urea, guanidine hydrochloride (GuHCl), and trifluoroacetic acid (TFA) on rhodopsin's secondary structure and propensity for aggregation. Ellipticity at 222 nm decreases in the presence of maximum concentrations of denaturants in the order TFA > GuHCl > urea > SDS + urea > SDS. Interpretation of these changes in ellipticity in terms of helix loss is challenged because the addition of some denaturants leads to aggregation. Through a combination of SDS-PAGE, dependence of ellipticity on protein concentration, and 1D (1)H NMR we show that aggregates form in the presence of GuHCl, TFA, and urea but not in any concentration of SDS, added over a range of 0.05%-30%. Mixed denaturant conditions consisting of 3% SDS and 8 M urea, added in this order, also did not result in aggregation. We conclude that SDS is able to prevent the exposure of large hydrophobic regions present in membrane proteins which otherwise leads to aggregation. Thus, 30% SDS and 3% SDS + 8 M urea are the denaturing conditions of choice to study maximally unfolded rhodopsin without aggregation.

Anti-angiogenic properties of calcium trifluoroacetate.

Endothelial cell proliferation and the formation of new vessels are strictly regulated by angiogenic factors (e.g., VEGF) that induce the activation of signal transduction pathways controlled by calcium dynamics. Using in vitro and in vivo experiments, we investigated the effect of calcium trifluoroacetate (CaTFAc), a complex, poorly dissociated salt that is characterized by its low toxicity, on angiogenesis. In vitro, CaTFAc inhibited VEGF-induced effects on endothelial cell proliferation. In two in vivo models of angiogenesis, a Matrigel plug in mice and a chick embryo chorioallantoic membrane, CaTFAc inhibited the VEGF-induced formation of new vessels. The exact mechanism of action is still under investigation, but in vitro experiments demonstrate that CaTFAc induced a reversible increase in the levels of intracellular calcium under basal conditions and prevented calcium signaling induced by VEGF. These results are the first to suggest that CaTFAc may be useful for the treatment of diseases caused by enhanced angiogenesis.

Hydrolysis of bacterial wall carbohydrates in the microwave using trifluoroacetic acid.

By and large, monosaccharide composition and linkage analyses of bacterial cell-surface carbohydrates are achieved by hydrolysis into the corresponding monomeric constituents, and characterization of these, or their derivatives, by chromatographic and spectrometric methods. Normally, these hydrolyses are carried out conveniently with trifluoroacetic acid (TFA) at high temperatures for long periods of time, for example, in 4M TFA at 100 degrees C for 5h in a heating block. In this study, using a closed-vessel system, we investigated the effectiveness and reliability of microwave-assisted TFA hydrolysis of bacterial lipopolysaccharides, capsule, and teichoic-acid polysaccharides that were variably composed of several glycoses. In all cases, we were able to establish that 5 min of hydrolysis in the microwave at 120 degrees C with 4M TFA (measured pressure of 90 psi) was sufficient time to obtain comparable results to those afforded by conventional hydrolysis. The same observation was made when fully methylated carbohydrates were hydrolyzed. The data obtained with our microwave system (Aurora Instruments MW600) showed that microwave-induced hydrolysis can be used with a high degree of confidence to carry out sugar composition analysis of complex bacterial glycans in markedly shorter periods of time. The results also suggested that non-thermal mechanistic factors must also be involved, at least in part, in accelerating the reaction rate of glycosidic hydrolysis.

MALDI-TOF mass spectrometry compatible inactivation method for highly pathogenic microbial cells and spores.

Identification of microorganisms, specifically of vegetative cells and spores, by intact cell mass spectrometry (ICMS) is an emerging new technology. The technique provides specific biomarker profiles which can be employed for bacterial identification at the genus, species, or even at the subspecies level holding the potential to serve as a rapid and sensitive identification technique in clinical or food microbiology and also for sensitive detection of biosafety level (BSL) 3 microorganisms. However, the development of ICMS as an identification technique for BSL-3 level microorganisms is hampered by the fact that no MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) compatible inactivation procedure for microorganisms, and particularly for bacterial endospores, has been evaluated so far. In this report we describe a new methodology for effective inactivation of microorganisms which is compatible with the analysis of microbial protein patterns by MALDI-TOF mass spectrometry. The main challenge of this work was to define the conditions that ensure microbial inactivation and permit at the same time comprehensive analysis of microbial protein patterns. Among several physical, chemical, and mechanical inactivation procedures, inactivation by trifluoroacetic acid (TFA) proved to be the best method in terms of bactericidal capacity and information content of the mass spectra. Treatment of vegetative cells by 80% TFA alone for 30 min assured complete inactivation of microbial cells under all conditions tested. For spore inactivation, the "TFA inactivation protocol" was developed which is a combination of TFA treatment with basic laboratory routines such as centrifugation and filtering. This MALDI-TOF/ICMS compatible sample preparation protocol is simple and rapid (30 min) and assures reliable inactivation of vegetative cells and spores of highly pathogenic (BSL-3) microorganisms.