Comparison of surface proteomes of enterotoxigenic (ETEC) and commensal Escherichia coli strains

Pathogenesis of enterotoxigenic Escherichia coli (ETEC) infections involves colonization of the small intestine mediated by cell-surface fimbriae (CS) or colonization fimbriae antigens (CFA). However, protection against reinfection of ETEC is also conferred by somatic antigens rather than by virulence factors. To discover ETEC specific somatic antigens, the surface proteome of the ETEC H10406 strain was compared with that of non-pathogenic E. coli K12 strains. In this study, we were using stable isotope labelling with amino acids in cell culture (SILAC) technology for the labelling and relative quantification of surface proteins in order to identify polypeptides that are specifically present on ETEC strains. Outer membrane proteins were isolated, separated by gel electrophoresis, and identified by mass spectrometry. Twenty-three differentially expressed cell-surface polypeptides of ETEC were identified and evaluated by bioinformatics for protein vaccine candidates. The combination of being surface-exposed and present differentially makes these polypeptides highly suitable as targets for antibodies and thus for use in passive or active immunisation/vaccination.     

Heme-heme and heme-ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy

Cytochrome bd is a terminal quinol:O₂ oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b₅₅₈, b₅₉₅, and d. The role of heme b₅₉₅ remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b₅₉₅ causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b₅₉₅ and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with τ ∼ 12 μs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with τ ∼ 14 ns, 14 μs, and 280 μs. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-μs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ∼ 4% of the enzyme population. The final, 280-μs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b₅₉₅, and not that of heme b₅₅₈, controls the pathway(s) by which CO migrates between heme d and the medium.     

Different modes of membrane permeabilization by two RTX toxins: HlyA from Escherichia coli and CyaA from Bordetella pertussis

This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: αhemolysin (HlyA) from Escherichia coli and a highly homologous adenylate cyclase toxin (CyaA) from Bordetella pertussis. For this purpose, we employed a fluorescence requenching method using liposomes (extruded through filters of different pore size — 1000 nm, 400 nm or 100 nm) with encapsulated fluorescent dye/quencher pair ANTS/DPX. We showed that both toxins induced a graded leakage of liposome content with different selectivities α for DPX and ANTS. In contrast to HlyA, CyaA exhibited a higher selectivity for cationic quencher DPX, which increased with vesicle diameter. Large unilamellar vesicles (LUV₁₀₀₀) were found to be more suitable for distinguishing between high α values whereas smaller ones (LUV₁₀₀) were more appropriate for discriminating an all-or-none leakage (α = 0) from the graded leakage with low values of α. While disrupting LUV₁₀₀₀, CyaA caused a highly cation-selective leakage (α ~ 15) whereas its mutated form with decreased channel K⁺/Cl⁻ selectivity due to two substitutions in a predicted transmembrane segment (CyaA-E509K + E516K) exhibited much lower selectivity (α ∼ 6). We concluded that the fluorescence requenching method in combination with different size of liposomes is a valuable tool for characterization of pore-forming toxins and their variants.     

Kinetic characterization of the Escherichia coli oligopeptidase A (OpdA) and the role of the Tyr residue

Oligopeptidase A (OpdA) belongs to the M3A subfamily of bacterial peptidases with catalytic and structural properties similar to mammalian thimet-oligopeptidase (TOP) and neurolysin (NEL). The three enzymes have four conserved Tyr residues on a flexible loop in close proximity to the catalytic site. In OpdA, the flexible loop is formed by residues 600-614 (⁶⁰⁰SHIFAGGYAAGYYSY⁶¹⁴). Modeling studies indicated that in OpdA the Tyr⁶⁰⁷ residue might be involved in the recognition of the substrate with a key role in catalysis. Two mutants were constructed replacing Tyr⁶⁰⁷ by Phe (Y607F) or Ala (Y607A) and the influence of the site-directed mutagenesis in the catalytic process was examined. The hydrolysis of Abz-GXSPFRQ-EDDnp derivatives (Abz = ortho-aminobenzoic acid; EDDnp N-[2,4-dinitrophenyl]-ethylenediamine; X = different amino acids) was studied to compare the activities of wild-type OpdA (OpdA WT) and those of Y607F and Y607A mutants The results indicated that OpdA WT cleaved all the peptides only on the X-S bond whereas the Y607F and Y607A mutants were able to hydrolyze both the X-S and the P-F bonds. The kinetic parameters showed the importance of Tyr⁶⁰⁷ in OpdA catalytic activity as its substitution promoted a decrease in the k_cat/K_m value of about 100-fold with Y607F mutant and 1000-fold with Y607A. Both mutations, however, did not affect protein folding as indicated by CD and intrinsic fluorescence analysis. Our results indicate that the OpdA Tyr⁶⁰⁷ residue plays an important role in the enzyme-substrate interaction and in the hydrolytic activity.     

An improved 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay for evaluating the viability of Escherichia coli cells

To reduce the interference in MTT reduction assay, we developed an improved protocol for evaluating the viability of Escherichia coli cells by conducting MTT reduction in 1.5-mL centrifuge tubes and formazan dissolution in test tubes. Our study is helpful in developing protocols for measuring the viability of other gram-negative bacteria.     

Involvement of the AtoS-AtoC signal transduction system in poly-(R)-3-hydroxybutyrate biosynthesis in Escherichia coli

The AtoS–AtoC signal transduction system in E. coli, which induces the atoDAEB operon for the growth of E. coli in short-chain fatty acids, can positively modulate the levels of poly-(R)-3-hydroxybutyrate (cPHB) biosynthesis, a biopolymer with many physiological roles in E. coli. Increased amounts of cPHB were synthesized in E. coli upon exposure of the cells to acetoacetate, the inducer of the AtoS–AtoC two-component system. While E. coli that overproduce both components of the signal transduction system synthesize higher quantities of cPHB (1.5–4.5 fold), those that overproduce either AtoS or AtoC alone do not display such a phenotype. Lack of enhanced cPHB production was also observed in cells overexpressing AtoS and phosphorylation-impaired AtoC mutants. The results were not affected by the nature of the carbon source used, i.e., glucose, acetate or acetoacetate. An E. coli strain with a deletion in the atoS–atoC locus (ΔatoSC) synthesized lower amounts of cPHB compared to wild-type cells. When the ΔatoSC strain was transformed with a plasmid carrying a 6.4-kb fragment encoding the AtoS–AtoC system, cPHB biosynthesis was restored to the level of the atoSC⁺ cells. Introduction of a multicopy plasmid carrying a functional atoDAEB operon, but not one with a promoterless operon, resulted in increased cPHB synthesis only in atoSC⁺ cells in the presence of acetoacetate. These results indicate that the presence of both a functional AtoS–AtoC two-component signal transduction system and a functional atoDAEB operon is critical for the enhanced cPHB biosynthesis in E. coli.     

Excision of the oxidatively formed 5-hydroxyhydantoin and 5-hydroxy-5-methylhydantoin pyrimidine lesions by Escherichia coli and Saccharomyces cerevisiae DNA N-glycosylases

Background

(5R?) and (5S?) diastereomers of 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) are major oxidation products of 2′-deoxycytidine and thymidine respectively. If not repaired, when present in cellular DNA, these base lesions may be processed by DNA polymerases that induce mutagenic and cell lethality processes.

Methods

Synthetic oligonucleotides that contained a unique 5-hydroxyhydantoin (5-OH-Hyd) or 5-hydroxy-5-methylhydantoin (5-OH-5-Me-Hyd) nucleobase were used as probes for repair studies involving several E. coli, yeast and human purified DNA N-glycosylases. Enzymatic reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis after radiolabeling of DNA oligomers or by MALDI-TOF mass spectrometry measurements.

Results

In vitro DNA excision experiments carried out with endo III, endo VIII, Fpg, Ntg1 and Ntg2, show that both base lesions are substrates for these DNA N-glycosylases. The yeast and human Ogg1 proteins (yOgg1 and hOgg1 respectively) and E. coli AlkA were unable to cleave the N-glycosidic bond of the 5-OH-Hyd and 5-OH-5-Me-Hyd lesions. Comparison of the k_cat/K_m ratio reveals that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than 5-OH-Hyd and 5-OH-5-Me-Hyd. The kinetic results obtained with endo III indicate that 5-OH-Hyd and 5-OH-5-Me-Hyd are much better substrates than 5-hydroxycytosine, a well known oxidized pyrimidine substrate for this DNA N-glycosylase.

Conclusions

The present study supports a biological relevance of the base excision repair processes toward the hydantoin lesions, while the removal by the Fpg and endo III proteins are effected at better or comparable rates to that of the removal of 8-oxoGua and 5-OH-Cyt, two established cellular substrates.

General significance

The study provides new insights into the substrate specificity of DNA N-glycosylases involved in the base excision repair of oxidized bases, together with complementary information on the biological role of hydantoin type lesions.     

Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli

Background

F₁F_O ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane. A number of ATP synthases have been characterized to date. The one from the hyperthermophilic bacterium Aquifex aeolicus presents unique features, i.e. a putative heterodimeric stalk. To complement previous work on the native form of this enzyme, we produced it heterologously in Escherichia coli.

Methods

We designed an artificial operon combining the nine genes of A. aeolicus ATP synthase, which are split into four clusters in the A. aeolicus genome. We expressed the genes and purified the enzyme complex by affinity and size-exclusion chromatography. We characterized the complex by native gel electrophoresis, Western blot, and mass spectrometry. We studied its activity by enzymatic assays and we visualized its structure by single-particle electron microscopy.

Results

We show that the heterologously produced complex has the same enzymatic activity and the same structure as the native ATP synthase complex extracted from A. aeolicus cells. We used our expression system to confirm that A. aeolicus ATP synthase possesses a heterodimeric peripheral stalk unique among non-photosynthetic bacterial F₁F_O ATP synthases.

Conclusions

Our system now allows performing previously impossible structural and functional studies on A. aeolicus F₁F_O ATP synthase.

General significance

More broadly, our work provides a valuable platform to characterize many other membrane protein complexes with complicated stoichiometry, i.e. other respiratory complexes, the nuclear pore complex, or transporter systems.     

Low-Level Expression of a Folding-Incompetent Protein in Escherichia coli: Search for the Molecular Determinants of Protein Aggregation In Vivo

Aggregation of peptides and proteins into insoluble amyloid fibrils or related intracellular inclusions is the hallmark of many degenerative diseases, including Alzheimer's disease, Parkinson's disease, and various forms of amyloidosis. In spite of the considerable progress carried out in vitro in elucidating the molecular determinants of the conversion of purified and isolated proteins into amyloid fibrils, very little is known on factors governing this process in the complex environment of living organisms. Taking advantage of increasing evidence that bacterial inclusion bodies consist of amyloid-like aggregates, we have expressed in Escherichia coli both wild type and 21 single-point mutants of the N-terminal domain of the E. coli protein HypF. All variants were expressed as folding-incompetent units in a controlled manner, at low and comparable levels. Their solubilities were measured by quantifying the protein amount contained in the soluble and insoluble fractions by Western blot analysis. A significant negative correlation was found between the solubility of the variants in E. coli and their intrinsic propensity to form amyloid fibrils, predicted using an algorithm previously validated experimentally in vitro on a number of unfolded peptides and proteins, and considering hydrophobicity, β-sheet propensity, and charge as major sequence determinants of the aggregation process. These findings show that the physicochemical parameters previously recognized to govern amyloid formation by fully or partially unfolded proteins are largely applicable in vivo and pave the way for the molecular exploration of a process as complex as protein aggregation in living organisms.     

Leishmania (Viannia) panamensis: An in vitro assay using the expression of GFP for screening of antileishmanial drug

Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known antileishmanial drugs, i.e. amphotericin B and glucantime^®. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus.     

Oligo-(R)-3-hydroxybutyrate modification of sorting signal enables pore formation by Escherichia coli OmpA

The outer membrane protein A (OmpA) of Escherichia coli is a well-known model for protein targeting and protein folding. Wild-type OmpA, isolated either from cytoplasmic inclusion bodies or from outer membranes, forms narrow pores of ∼ 80 pS in planar lipid bilayers at room temperature. The pores are well structured with narrow conductance range when OmpA is isolated using lithium dodecyl sulfate (LDS) or RapiGest surfactant but display irregular conductance when OmpA is isolated with urea or guanidine hydrochloride. Previous studies have shown that serine residues S163 and S167 of the sorting signal of OmpA (residues 163-169), i.e., the essential sequence for outer membrane incorporation, are covalently modified by oligomers of (R)-3-hydroxybutyrate (cOHB). Here we find that single-mutants S163 and S167 of OmpA, which still contain cOHB on one serine of the sorting signal, form narrow pores in planar lipid bilayers at room temperature with lower and more irregular conductance than wild-type OmpA, whereas double mutants S163:S167 and S163:V166 of OmpA, with no cOHB on the sorting signal, are unable to form stable pores in planar lipid bilayers. Our results indicate that modification of serines in the sorting signal of OmpA by cOHB in the cytoplasm enables OmpA to incorporate into lipid bilayers at room temperature as a narrow pore. They further suggest that cOHB modification may be an important factor in protein targeting and protein folding.     

P2X7 receptor antagonists display agonist-like effects on cell signaling proteins

Background

The activation of various P2 receptors (P2R) by extracellular nucleotides promotes diverse cellular events, including the stimulation of cell signaling protein and increases in [Ca²⁺]_i. We report that some agents that can block P2X₇R receptors also promote diverse P2X₇R-independent effects on cell signaling.

Methods

We exposed native rat parotid acinar cells, salivary gland cell lines (Par-C10, HSY, HSG), and PC12 cells to suramin, DIDS (4,4′-diisothiocyano stilbene-2,2′-disulfonic acid), Cibacron Blue 3GA, Brilliant Blue G, and the P2X₇R-selective antagonist A438079, and examined the activation/phosphorylation of ERK1/2, PKCδ, Src, CDCP1, and other signaling proteins.

Results

With the exception of suramin, these agents blocked the phosphorylation of ERK1/2 by BzATP in rat parotid acinar cells; but higher concentrations of suramin blocked ATP-stimulated ⁴⁵Ca²⁺ entry. Aside from A438079, these agents increased the phosphorylation of ERK1/2, Src, PKCδ, and other proteins (including Dok-1) within minutes in an agent- and cell type-specific manner in the absence of a P2X₇R ligand. The stimulatory effect of these compounds on the tyrosine phosphorylation of CDCP1 and its Src-dependent association with PKCδ was blocked by knockdown of CDCP1, which also blocked Src and PKCδ phosphorylation.

Conclusions

Several agents used as P2X₇R blockers promote the activation of various signaling proteins and thereby act more like receptor agonists than antagonists.

General significance

Some compounds used to block P2 receptors have complicated effects that may confound their use in blocking receptor activation and other biological processes for which they are employed, including their use as blockers of various ion transport proteins.     

Purification of the receptor for the N-acetyl-d-glucosamine specific adhesin of Mannheimia haemolytica from bovine neutrophils

The GlcNAc-specific adhesin from Mannheimia haemolytica (MhA) has been shown to participate in pathogenicity of mannheimiosis due to its capacity to adhere to tracheal epithelial cells and activate the oxidative burst of bovine neutrophils. In this work, we purified the MhA receptor from bovine neutrophils (MhAr) by affinity chromatography on MhA-Sepharose. The MhAr, which corresponded to approximately 2% of the protein from cell lysate, is a glycoprotein mainly composed of Glu, Ala, Ser, Gly, and Asp, without cysteine. The glycan portion, which corresponds to 20% by weight, is composed of GalNAc, GlcNAc, Man, Gal, and NeuAc. The receptor is a 165-kDa glycoprotein, as determined by molecular sieve chromatography under native conditions; SDS-PAGE analysis shows a heterodimer of 83 and 80 kDa subunits. This work suggests that the GlcNAc-containing receptor plays a relevant role by activating bovine neutrophils through non-opsonic mechanisms.     

Antioxidant status of Lobiger serradifalci and Oxynoe olivacea (Opisthobranchia, Mollusca)

Invasion of the Mediterranean Sea by the two world-wide famous exotic algae species, Caulerpa taxifolia and Caulerpa racemosa, is still a problem and has adverse effects on the Mediterranean sublittoral ecosystem. Biological control studies revealed that the two native Sacoglossans, Oxynoe olivacea and Lobiger serradifalci, may have an effect on the expansion of invasive Caulerpa spp. in the Mediterranean. In the framework of this study, antioxidant enzyme activities, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), lipid peroxidation (LPO) and oxidized glutathione (GSSG) levels, as oxidative stress markers in L. serradifalci and O. olivacea were determined at two different temperature conditions (20 and 27 °C). In both species, SOD, CAT and GSH-Px activities were found to be positively correlated with temperature. The SOD activities in L. serradifalci were higher than those in O. olivacea at both temperatures, whereas the CAT and GSH-Px activities were significantly (p<0.05) higher in O. olivacea, compared to L. serradifalci. As expected, both species showed decreased LPO levels at 27 °C compared to 20 °C. GSSG level at 27 °C in O.olivacea was significantly (p<0.05) higher than that of 20 °C. On the other hand, no statistical (p>0.05) difference in L.serradifalci existed between GSSG levels at two temperatures. But, despite the variations in the antioxidant enzyme activities, there was no significant difference in LPO levels between the species, suggesting that the oxidative consequences of a given environmental condition may vary among different species. Inasmuch as the GSSG levels were in accordance with antioxidant enzyme activities, GSH might have acted as a cofactor of GSH-Px and an individual antioxidant in these sea slugs.     

Mitochondrial respiration and membrane potential are regulated by the allosteric ATP-inhibition of cytochrome c oxidase

This paper describes the problems of measuring the allosteric ATP-inhibition of cytochrome c oxidase (CcO) in isolated mitochondria. Only by using the ATP-regenerating system phosphoenolpyruvate and pyruvate kinase full ATP-inhibition of CcO could be demonstrated by kinetic measurements. The mechanism was proposed to keep the mitochondrial membrane potential (?Ψ_m) in living cells and tissues at low values (100-140 mV), when the matrix ATP/ADP ratios are high. In contrast, high ?Ψ_m values (180-220 mV) are generally measured in isolated mitochondria. By using a tetraphenyl phosphonium electrode we observed in isolated rat liver mitochondria with glutamate plus malate as substrates a reversible decrease of ?Ψ_m from 233 to 123 mV after addition of phosphoenolpyruvate and pyruvate kinase. The decrease of ?Ψ_m is explained by reversal of the gluconeogenetic enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase yielding ATP and GTP, thus increasing the matrix ATP/ADP ratio. With rat heart mitochondria, which lack these enzymes, no decrease of ?Ψ_m was found. From the data we conclude that high matrix ATP/ADP ratios keep ?Ψ_m at low values by the allosteric ATP-inhibition of CcO, thus preventing the generation of reactive oxygen species which could generate degenerative diseases. It is proposed that respiration in living eukaryotic organisms is normally controlled by the ?Ψ_m-independent “allosteric ATP-inhibition of CcO.” Only when the allosteric ATP-inhibition is switched off under stress, respiration is regulated by “respiratory control,” based on ?Ψ_m according to the Mitchell Theory.     

Vaccenic acid-mediated reduction in cytokine production is independent of c9,t11-CLA in human peripheral blood mononuclear cells

The ruminant trans fatty acid vaccenic acid (tVA) favorably alters markers of inflammation. However, it is not yet clear whether these effects are attributed to its endogenous partial conversion to c9,t11-CLA, which is known to possess anti-inflammatory properties. We compared the cytokine reducing potential of tVA to c9,t11-CLA in human T-helper (Th) cells as a main source of cytokine production during inflammation. Secondly, we assessed whether a bioconversion of tVA to c9,t11-CLA via stearoyl-CoA desaturase (SCD) encoded activity takes place in peripheral blood mononuclear cells (PBMC) in order to relate the outcomes of intracellular cytokine measurement to the degree of conversion. TVA reduced the percentage of both IL-2 and TNF-α expressing Th cells significantly, but to a lesser extent compared to c9,t11-CLA, as determined by flow cytometry after alloreactive stimulation of PBMC. Pre-treatment with the selective PPARγ antagonist T0070907 largely re-established the IL-2 and TNF-α positive Th cell population in both tVA and c9,t11-CLA treated cultures. Interestingly, while the portion of tVA dose-dependently increased within the cellular lipid fraction, the initially marginal amount of c9,t11-CLA remained unaltered. However, SCD mRNA although abundantly expressed in PBMC was not regulated by tVA. Conclusively, these results suggest that the cytokine reducing effect of tVA in human T cells is independent of c9,t11-CLA, since no bioconversion occurred. Moreover, the data provide evidence that tVA mechanistically acts in a manner similar to c9,t11-CLA.     

Modulation of advanced glycation endproduct synthesis by kynurenines in human lens proteins

Human lens proteins (HLP) become chemically modified by kynurenines and advanced glycation end products (AGEs) during aging and cataractogenesis. We investigated the effects of kynurenines on AGE synthesis in HLP. We found that incubation with 5 mM ribose or 5 mM ascorbate produced significant quantities of pentosidine, and this was further enhanced in the presence of two different kynurenines (200–500 µM): N-formylkynurenine (Nfk) and kynurenine (Kyn). Another related compound, 3-hydroxykynurenine (3OH-Kyn), had disparate effects; low concentrations (10–200 µM) promoted pentosidine synthesis, but high concentrations (200–500 µM) inhibited it. 3OH-Kyn showed similar effects on pentosidine synthesis from Amadori-enriched HLP or ribated lysine. Chelex-100 treatment of phosphate buffer reduced pentosidine synthesis from Amadori-enriched HLP by ∼ 90%, but it did not inhibit the stimulating effect of 3OH-Kyn and EDTA. 3OH-Kyn (100–500 μM) spontaneously produced copious amounts of H₂O₂ (10–25 μM), but externally added H₂O₂ had only a mild stimulating effect on pentosidine but had no effect on N^ε-carboxymethyl lysine (CML) synthesis in HLP from ribose and ascorbate. Further, human lens epithelial cells incubated with ribose and 3OH-Kyn showed higher intracellular pentosidine than cells incubated with ribose alone. CML synthesis from glycating agents was inhibited 30 to 50% by 3OH-Kyn at concentrations of 100–500 μM. Argpyrimidine synthesis from 5 mM methylglyoxal was slightly inhibited by all kynurenines at concentrations of 100–500 μM. These results suggest that AGE synthesis in HLP is modulated by kynurenines, and such effects indicate a mode of interplay between kynurenines and carbohydrates important for AGE formation during lens aging and cataract formation.     

Expression of sarco/endoplasmic reticulum Ca ATPase (SERCA) 3 proteins in two major conformational states in native human cell membranes

The SERCA family includes 3 genes (SERCA1-3), each of which giving rise to various isoforms. To date, detailed structural data is only available for the SERCA1a isoform. Here, limited trypsinolysis of either human platelet membranes or recombinant SERCA3a in HEK-293 cells followed by Western blotting using antibodies covering different regions of the SERCA3(a) protein revealed two, kinetically distinct, Early (ETF) and Late (LTF) Tryptic Fragmentations. The ETF uses many tryptic sites while the LTF uses a unique tryptic site. Using site-directed mutagenesis: i) Arg³³⁴, Arg³⁹⁶ and Arg⁶³⁸ were directly assigned to the ETF and ii) Arg¹⁹⁸ was assigned as the only tryptic site to the LTF. Arg⁶⁷¹, Lys⁷¹²/Lys⁷¹³ and Lys⁷²⁸ were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl₂, EGTA or AlF₄⁻ strikingly modified the ETF without any effect on the LTF. Trypsinolysis of the other recombinant SERCA3b-3f isoforms revealed: i) same ETF and LTF as SERCA3a, with variations of the length of the C-terminal fragments; ii) Arg¹⁰⁰² as an additional tryptic site in SERCA3b-3e isoforms. Taken together, the two distinct SERCA3 fragmentation profiles sign the co-expression of SERCA3 proteins in two conformational states in cell membranes.