Interaction of ochratoxin A with human serum albumin. Binding sites localized by competitive interactions with the native protein and its recombinant fragments

Competitive interactions of ochratoxin A (OTA) and several other acidic compounds were utilized to gain insight into the localization of binding sites and the nature of binding interactions between anionic species and human serum albumin (HSA). Depolarization of OTA fluorescence in the presence of a competing anion was used to quantify ligand-protein interactions. The results obtained were rationalized in terms of OTA displacement from its major binding site. Based on their ability to displace OTA, two distinct groups of the anionic ligands were revealed. The first group contained structurally diverse compounds that shared a common binding site in subdomain IIA (Sudlow Site I). The second group consisted of three non-steroidal anti-inflammatory drugs, which showed much lower affinity to Site I than the OTA dianion. The major site for these drugs was located in domain III. Fluorescence spectroscopy measurements of OTA, warfarin (WAR) and naproxen (NAP) complexes with recombinant proteins corresponding to the domains of HSA (D1-D3) revealed binding to all domains but with different affinities. The binding constants for OTA and WAR decreased in the series D2z.Gt;D3>D1. In contrast, NAP showed the most favorable interaction with D3 and comparable affinities to the two remaining domains. The OTA binding constant for D2, 7.9 x 10(5) M(-1), was smaller than the largest constant for HSA by a factor of approximately 7. The binding constant for OTA with D3, 1.1 x 10(5) M(-1), was very close to that of the secondary binding site for HSA.     

Gliadin T cell epitope selection by tissue transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the transamidation versus deamidation process

Tissue transglutaminase (TG2) can modify proteins by transamidation or deamidation of specific glutamine residues. TG2 has a major role in the pathogenesis of celiac disease as it is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides that are recognized by CD4(+), DQ2-restricted T cells from the celiac lesions. Capillary electrophoresis with fluorescence-labeled gliadin peptides was used to separate and quantify deamidated and transamidated products. In a competition assay, the affinity of TG2 to a set of overlapping gamma-gliadin peptides was measured and compared with their recognition by celiac lesion T cells. Peptides differed considerably in their competition efficiency. Those peptides recognized by intestinal T cell lines showed marked competition indicating them as excellent substrates for TG2. The enzyme fine specificity of TG2 was characterized by synthetic peptide libraries and mass spectrometry. Residues in positions -1, +1, +2, and +3 relative to the targeted glutamine residue influenced the enzyme activity, and proline in position +2 had a particularly positive effect. The characterized sequence specificity of TG2 explained the variation between peptides as TG2 substrates indicating that the enzyme is involved in the selection of gluten T cell epitopes. The enzyme is mainly localized extracellularly in the small intestine where primary amines as substrates for the competing transamidation reaction are present. The deamidation could possibly take place in this compartment as an excess of primary amines did not completely inhibit deamidation of gluten peptides at pH 7.3. However, lowering of the pH decreased the reaction rate of the TG2-catalyzed transamidation, whereas the rate of the deamidation reaction was considerably increased. This suggests that the deamidation of gluten peptides by TG2 more likely takes place in slightly acidic environments.     

Changing the net charge from negative to positive makes ribonuclease Sa cytotoxic

Ribonuclease Sa (pI = 3.5) from Streptomyces aureofaciens and its 3K (D1K, D17K, E41K) (pI = 6.4) and 5K (3K + D25K, E74K) (pI = 10.2) mutants were tested for cytotoxicity. The 5K mutant was cytotoxic to normal and v-ras-transformed NIH3T3 mouse fibroblasts, but RNase Sa and 3K were not. The structure, stability, and activity of the three proteins are comparable, but the net charge at pH 7 increases from -7 for RNase Sa to -1 for 3K and to +3 for 5K. These results suggest that a net positive charge is a key determinant of ribonuclease cytotoxicity. The cytotoxic 5K mutant preferentially attacks v-ras-NIH3T3 fibroblasts, suggesting that mammalian cells expressing the ras-oncogene are potential targets for ribonuclease-based drugs.     

Effect of timing of urea feeding on the yield and quality of embryos in lactating dairy cows

High protein diets, which lead to excess production of nonprotein nitrogen such as ammonia and urea, have been associated with reduced fertility in dairy cows. In this study we test the hypothesis that diets containing high levels of quickly degradable urea nitrogen (QDN) compromise embryo development. Lactating dairy cows were fed mixed silage and concentrates twice daily. At 60 days postpartum, a synchronized estrus was induced and the cows were subsequently superovulated and inseminated using a standard protocol. On Day 7 after insemination, the uteri were flushed and embryos retrieved. At the start of treatment, cows were randomly allocated into three nutritional groups: control (CONT, n = 8), long (L-) QDN (n = 8) and short (S-) QDN (n = 9). The L-QDN cows were fed a supplement of urea from 10 days before insemination, and the S-QDN cows were fed the supplement from insemination until embryo collection. Both L- and S-QDN diets produced significant increases in plasma ammonia and urea 3 h post-feeding. The S-QDN but not the L-QDN diet was associated with a significant reduction in embryo yield. Embryo quality was also significantly reduced in the S-QDN cows. This study indicates that there is no deleterious effect on the yield and quality of embryos recovered 7 days after breeding when QDN feeding is initiated during the previous midluteal phase. However, introduction of a similar diet 10 days later, at the time of insemination, was deleterious. We suggest that QDN is toxic to embryos but cows can adjust within 10 days.     

Distribution of endogenous retinoids,retinoid binding proteins (RBP,CRABPI) and nuclear retinoid X receptor beta (RXRbeta) in the porcine embryo

Retinoids are important signalling molecules in the development of limbs and in the determination of the anterior-posterior orientation of the embryo. The present study examined the content and distribution of retinoic acid, retinol and retinyl esters in porcine embryos during early gestation (gestation days 22-30) macroscopically and microscopically by its autofluorescence and by HPLC. Macroscopically, the yellowish-greenish autofluorescence characteristic of vitamin A was observed in tissues affected by morphogenesis, such as the limbs, in a spatial and temporal manner. Changes in the intensity of autofluorescence in the limbs paralleled changes in the concentration of retinoids in these structures. In the limbs and the body, retinol, retinyl palmitate, and all-trans-retinoic acid but neither the isomers of all-trans retinoic acid nor other retinoid metabolites were detected. In addition, the distribution of specific retinoid-binding proteins was investigated; these are involved in vitamin A transport, metabolism and signal transduction. Immunoreactive retinol-binding protein as well as cellular retinoic acid binding protein type I were only localised in the mesonephros, while the retinoid X receptor beta was widely distributed in most of the tissues and organs of the embryo throughout the time period investigated. The combination of autofluorescence and HPLC analysis allowed for the first time to attribute the yellowish-greenish autofluorescence in specific regions of the embryo to vitamin A, and offers a method to study the local cellular distribution of retinol and/or retinyl esters as well as their concentrations in embryonic tissues.     

Specific visualization of precipitated cerium by energy-filtered transmission electron microscopy for detection of alkaline phosphatase in immunoenzymatic double labeling of tyrosine hydroxylase and serotonin in the rat olfactory bulb

To understand in detail the functional morphology of neuronal circuits it is important to identify at the ultrastructural level the incoming axon, its target neuron, and members of the signaling cascades involved. This, however, represents a formidable task, requiring highly sophisticated electron microscopic multiple-labeling techniques. To extend available double-labeling procedures such as combinations of immunogold and peroxidase methods, an additional, gold- and peroxidase-independent procedure would represent a considerable advantage. The present investigation therefore aimed to use alkaline phosphatase as the immunoenzymatic label at the electron microscopic level via cerium phosphate precipitates. To our surprise we found that available techniques, which are well established for the visualization of endogenous enzymes in sections from various tissues, are not suitable for application to immunocytochemistry. Careful characterization of the individual reaction conditions, however, resulted in an optimized procedure with largely increased sensitivity. The novel technique yields cerium-containing precipitates which are massive enough to allow the detection of the immunoenzymatic reaction product in the electron microscope. Using the rat olfactory bulb as the model system we showed further that our technique allows the combination with the peroxidase/diaminobenzidine system for ultrastructural double labeling. For this purpose, the alkaline phosphatase product is identified by its cerium content via energy-filtered transmission electron microscopy and thereby differentiated from cerium-free peroxidase-derived precipitates. Doing so, we found that ascending serotoninergic fibers do not establish synapses with dopaminergic periglomerular cells in the rat olfactory bulb.     

The sensing of nutritional status and the relationship to filamentous growth in Saccharomyces cerevisiae

Heterotrophic organisms rely on the ingestion of organic molecules or nutrients from the environment to sustain energy and biomass production. Non-motile, unicellular organisms have a limited ability to store nutrients or to take evasive action, and are therefore most directly dependent on the availability of nutrients in their immediate surrounding. Such organisms have evolved numerous developmental options in order to adapt to and to survive the permanently changing nutritional status of the environment. The phenotypical, physiological and molecular nature of nutrient-induced cellular adaptations has been most extensively studied in the yeast Saccharomyces cerevisiae. These studies have revealed a network of sensing mechanisms and of signalling pathways that generate and transmit the information on the nutritional status of the environment to the cellular machinery that implements specific developmental programmes. This review integrates our current knowledge on nutrient sensing and signalling in S. cerevisiae, and suggests how an integrated signalling network may lead to the establishment of a specific developmental programme, namely pseudohyphal differentiation and invasive growth.     

SAR directed design and synthesis of novel beta(1-4)-glucosyltransferase inhibitors and their in vitro inhibition studies

This paper describes SAR directed design and synthesis of novel beta(1-4)-glucosyltransferase (BGT) inhibitors. The designed inhibitors 1-5 provide conformational mimicry of the transition-state in glucosyltransfer reactions. The compounds were tested for in vitro inhibitory activity against (BGT) and the inhibition kinetics were examined. Three of the designed molecules were found to be potential inhibitors of BGT having IC50 values in micromolar (microM) range. Useful structure-activity relationships were established, which provide guidelines for the design of future generations of inhibitors of BGT.     

Substrate activation in acetylcholinesterase induced by low pH or mutation in the pi-cation subsite

Substrate inhibition is considered a defining property of acetylcholinesterase (AChE), whereas substrate activation is characteristic of butyrylcholinesterase (BuChE). To understand the mechanism of substrate inhibition, the pH dependence of acetylthiocholine hydrolysis by AChE was studied between pH 5 and 8. Wild-type human AChE and its mutants Y337G and Y337W, as well as wild-type Bungarus fasciatus AChE and its mutants Y333G, Y333A and Y333W were studied. The pH profile results were unexpected. Instead of substrate inhibition, wild-type AChE and all mutants showed substrate activation at low pH. At high pH, there was substrate inhibition for wild-type AChE and for the mutant with tryptophan in the pi-cation subsite, but substrate activation for mutants containing small residues, glycine or alanine. This is particularly apparent in the B. fasciatus AChE. Thus a single amino acid substitution in the pi-cation site, from the aromatic tyrosine of B. fasciatus AChE to the alanine of BuChE, caused AChE to behave like BuChE. Excess substrate binds to the peripheral anionic site (PAS) of AChE. The finding that AChE is activated by excess substrate supports the idea that binding of a second substrate molecule to the PAS induces a conformational change that reorganizes the active site.