Existence of Molten Globule State in Homocysteine-Induced Protein Covalent Modifications

Homocysteine thiolactone is a toxic metabolite produced from homocysteine by amino-acyl t-RNA synthetase in error editing reaction. The basic cause of toxicity of homocysteine thiolactone is believed to be due to the adduct formation with lysine residues (known as protein N-homocysteinylation) leading to protein aggregation and loss of enzyme function. There was no data available until now that showed the effect of homocysteine thiolactone on the native state structural changes that led to aggregate formation. In the present study we have investigated the time dependent structural changes due to homocysteine thiolactone induced modifications on three different proteins having different physico-chemical properties (cytochrome-c, lysozyme and alpha lactalbumin). We discovered that N-homocysteinylation leads to the formation of molten globule state—an important protein folding intermediate in the protein folding pathway. We also found that the formation of the molten globule state might be responsible for the appearance of aggregate formation. The study indicates the importance of protein folding intermediate state in eliciting the homocysteine thiolactone toxicity.     

A Functional Loop Spanning Distant Domains of Glutaminyl-tRNA Synthetase Also Stabilizes a Molten Globule State

Molten globule and other disordered states of proteins are now known to play important roles in many cellular processes. From equilibrium unfolding studies of two paralogous proteins and their variants, glutaminyl-tRNA synthetase (GlnRS) and two of its variants [glutamyl-tRNA synthetase (GluRS) and its isolated domains, and a GluRS-GlnRS chimera], we demonstrate that only GlnRS forms a molten globule-like intermediate at low urea concentrations. We demonstrated that a loop in the GlnRS C-terminal anticodon binding domain that promotes communication with the N-terminal domain and indirectly modulates amino acid binding is also responsible for stabilization of the molten globule state. This loop was inserted into GluRS in the eukaryotic branch after the archaea-eukarya split, right around the time when GlnRS evolved. Because of the structural and functional importance of the loop, it is proposed that the insertion of the loop into a putative ancestral GluRS in eukaryotes produced a catalytically active molten globule state. Because of their enhanced dynamic nature, catalytically active molten globules are likely to possess broad substrate specificity. It is further proposed that the putative broader substrate specificity allowed the catalytically active molten globule to accept glutamine in addition to glutamic acid, leading to the evolution of GlnRS.     

Heterogeneity of Equilibrium Molten Globule State of Cytochrome c Induced by Weak Salt Denaturants under Physiological Condition

While many proteins are recognized to undergo folding via intermediate(s), the heterogeneity of equilibrium folding intermediate(s) along the folding pathway is less understood. In our present study, FTIR spectroscopy, far- and near-UV circular dichroism (CD), ANS and tryptophan fluorescence, near IR absorbance spectroscopy and dynamic light scattering (DLS) were used to study the structural and thermodynamic characteristics of the native (N), denatured (D) and intermediate state (X) of goat cytochorme c (cyt-c) induced by weak salt denaturants (LiBr, LiCl and LiClO₄) at pH 6.0 and 25°C. The LiBr-induced denaturation of cyt-c measured by Soret absorption (Δε ₄₀₀) and CD ([θ]₄₀₉), is a three-step process, N ↔ X ↔ D. It is observed that the X state obtained along the denaturation pathway of cyt-c possesses common structural and thermodynamic characteristics of the molten globule (MG) state. The MG state of cyt-c induced by LiBr is compared for its structural and thermodynamic parameters with those found in other solvent conditions such as LiCl, LiClO₄ and acidic pH. Our observations suggest: (1) that the LiBr-induced MG state of cyt-c retains the native Met80-Fe(III) axial bond and Trp59-propionate interactions; (2) that LiBr-induced MG state of cyt-c is more compact retaining the hydrophobic interactions in comparison to the MG states induced by LiCl, LiClO₄ and 0.5 M NaCl at pH 2.0; and (3) that there exists heterogeneity of equilibrium intermediates along the unfolding pathway of cyt-c as highly ordered (X1), classical (X2) and disordered (X3), i.e., D ↔ X3 ↔ X2 ↔ X1 ↔ N.     

1-Anilino-8-Naphthalene Sulfonate (ANS) Is Not a Desirable Probe for Determining the Molten Globule State of Chymopapain

The molten globule (MG) state of proteins is widely detected through binding with 1-anilino-8-naphthalene sulphonate (ANS), a fluorescent dye. This strategy is based upon the assumption that when in molten globule state, the exposed hydrophobic clusters of protein are readily bound by the nonpolar anilino-naphthalene moiety of ANS molecules which then produce brilliant fluorescence. In this work, we explored the acid-induced unfolding pathway of chymopapain, a cysteine proteases from Carica papaya, by monitoring the conformational changes over a pH range 1.0–7.4 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). The spectroscopic measurements showed that although maximum ANS fluorescence intensity was observed at pH 1.0, however protein exhibited ∼80% loss of secondary structure which does not comply with the characteristics of a typical MG-state. In contrast at pH 1.5, chymopapain retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii and nearly 30-fold increase in ANS fluorescence with respect to the native state, indicating that MG-state exists at pH 1.5 and not at pH 1.0. ITC measurements revealed that ANS molecules bound to chymopapain via hydrophobic interaction were more at pH 1.5 than at pH 1.0. However, a large number of ANS molecules were also involved in electrostatic interaction with protein at pH 1.0 which, together with hydrophobically interacted molecules, may be responsible for maximum ANS fluorescence. We conclude that maximum ANS-fluorescence alone may not be the criteria for determining the MG of chymopapain. Hence a comprehensive structural analysis of the intermediate is essentially required.     

pH-Induced Molten Globule State of <Emphasis Type="Italic">Rhizopus niveus</Emphasis> Lipase is More Resistant Against Thermal and Chemical Denaturation Than Its Native State

Here, we have characterized four pH-dependent states: alkaline state, “B” (pH 9.0), native state, “N” (pH 7.4), acid-induced state, “A” (pH 2.2) and molten globule state, “MG” (pH 1.8) of Rhizopus niveus lipase (RNL) by CD, tryptophanyl fluorescence, ANS binding, DLS, and enzyme activity assay. This “MG” state lacks catalytic activity and tertiary structure but it has native-like significant secondary structure. The “R _h” of all the four states of RNL obtained from DLS study suggests that the molecular compactness of the protein increases as the pH of solution decreases. Kinetic analysis of RNL shows that it has maximum catalytic efficiency at state “B” which is 15-fold higher than state “N.” The CD and tryptophanyl fluorescence studies of RNL on GuHCl and temperature-induced unfolding reveal that the “MG” state is more stable than the other states. The DSC endotherms of RNL obtained at pH 9.0, 7.4, and 2.2 were with two transitions, while at pH 1.8 it showed only a single transition.     

Effect of pH on an In Vitro Model of Gastric Microbiota in Enteral Nutrition Patients

Patients with dysphagia due to oropharyngeal disease or cerebrovascular accident require long-term nutritional support via enteral feeding, which often results in microbial overgrowth in the upper gastrointestinal (GI) tract. Gastric acid is the primary innate defense mechanism in the stomach and has been assumed to provide an effective barrier to microbial colonization at pH values of <4. To evaluate the efficacy of gastric acid as a barrier to overgrowth, the microbiota of gastric and duodenal aspirates was assessed by culturing methods. Additionally, a fermentor-based model incorporating enteral nutrition tubing of the gastric microbiota of enteral nutrition (EN) patients was constructed to assess the effect of pH on the microbiota. Results showed that gastric acidity had a relatively small effect on the numbers of microorganisms recovered from intestinal aspirates but did influence microbiota composition. Similarly, at pH 3 in the fermentor, a complex microbiota developed in the planktonic phase and in biofilms. The effect of pH on microbiota composition was similar in aspirates and in the fermentors. Candidas and lactobacilli were aciduric, while recoveries of Escherichia coli and Klebsiella pneumoniae decreased as pH was reduced, although both were still present in significant numbers at pH 3. Only Staphylococcus aureus and Bifidobacterium adolescentis persisted at higher pH values both in vitro and in vivo. Lactate and acetate were the main organic acids detected in both aspirates and fermentors. These data show that the simulator used in this investigation was capable of modeling the effects of environmental influences on the upper GI microbiota of EN patients and that gastric pH of <4 is not sufficient to prevent microbial overgrowth in these individuals.     

Metabolomic Perfusate Analysis during Kidney Machine Perfusion: The Pig Provides an Appropriate Model for Human Studies

Introduction

Hypothermic machine perfusion offers great promise in kidney transplantation and experimental studies are needed to establish the optimal conditions for this to occur. Pig kidneys are considered to be a good model for this purpose and share many properties with human organs. However it is not established whether the metabolism of pig kidneys in such hypothermic hypoxic conditions is comparable to human organs.

Methods

Standard criteria human (n = 12) and porcine (n = 10) kidneys underwent HMP using the LifePort Kidney Transporter 1.0 (Organ Recovery Systems) using KPS-1 solution. Perfusate was sampled at 45 minutes and 4 hours of perfusion and metabolomic analysis performed using 1-D ¹H-NMR spectroscopy.

Results

There was no inter-species difference in the number of metabolites identified. Of the 30 metabolites analysed, 16 (53.3%) were present in comparable concentrations in the pig and human kidney perfusates. The rate of change of concentration for 3-Hydroxybutyrate was greater for human kidneys (p<0.001). For the other 29 metabolites (96.7%), there was no difference in the rate of change of concentration between pig and human samples.

Conclusions

Whilst there are some differences between pig and human kidneys during HMP they appear to be metabolically similar and the pig seems to be a valid model for human studies.     

The Effect of Manipulating Fat Globule Size on the Stability and Rheological Properties of Dairy Creams

Recombined cream (RC, 23 % fat w/w) and standardised commercial cream (CC, 28 % fat w/w) were studied to understand the effects of manipulating fat globule size at the micron-/nano-scale on the stability and rheological properties of cream. All samples were adjusted to a fat: protein ratio of 5:1 and a fat: emulsifier (Tween 80) ratio of 30:1 to stabilize emulsion. For both CC and RC, different emulsions with droplet sizes covering micron- (3.9 μm), sub-micron (0.5 – 0.6 μm) and nano-metric scales (0.13 – 0.29 μm) were obtained using either the homogeniser (7/3 MPa) or the microfluidiser (85 MPa and 42 MPa). Fat globules from both RC and CC had high zeta potential values (-28 to -43 mV) and maintained their reduced size after 1 month of storage at 4 °C, providing evidence of emulsion stability. Droplet size had a significant effect on rheological characteristics of all creams produced. Nano-sized RC tended to have a rigid structure (solid/gel-like form) as compared to micron-sized RC (liquid-like form) as reflected by higher phase angle. Surprisingly, the rheological properties of CC exhibited an opposite tendency to that of RC. This implies that the observed rheological properties of CC and RC could not be fully explained by the discrepancy in droplet size. Differences in interfacial properties between RC and CC might also play a role in the rheological behaviour of the creams. Results indicated the stable high milk fat emulsions could be successfully created by reducing the globule size. These findings would be useful in understanding how micron-/nano-sized emulsions can be utilised in further application or processing of creams.     

The Effect of pH on Staining by Eosins

The effect of pH on staining by 7 dyes of the eosin group was studied by means of specimens fixed 12-18 hr in Gilson's, in Petrunkewitsch's, or in Zenker's fluids, respectively. Sections were soaked in water or alcohol, depending upon the stain solvent, adjusted in 31 equal steps from pH 4.0 to 10.0 before being stained in similarly adjusted dye solutions. The stained sections were rinsed at H-ion concentrations identical to that at which staining took place, until no further color came away, and mounted. Variations in pH elicited different degrees of stain retention; different fixation altered staining affinity even though staining was effected at identical pH values.     

Effect of pH on the Steady State Kinetics of Bovine Heart NADH: Coenzyme Q Oxidoreductase

Complete initial steady state kinetics of NADH-decylubiquinone (DQ) oxidoreductase reaction between pH 6.5 and 9.0 show an ordered sequential mechanism in which the order of substrate bindings and product releases is NADH-DQ–DQH₂-NAD⁺. NADH binding to the free enzyme is accelerated by protonation of an amino acid (possibly a histidine) residue. The NADH release is negligibly slow under the turnover conditions. The rate of DQ binding to the NADH-bound enzyme and the maximal rate at the saturating concentrations of the two substrates, which is determined by the rates of DQH₂ formation in the active site and releases of DQH₂ and NAD⁺ from the enzyme, are insensitive to pH, in contrast to clear pH dependencies of the maximal rates of cytochrome c oxidase and cytochrome bc ₁ complex. Physiological significances of these results are discussed.     

EEG Investigations of Duration Discrimination: The Intermodal Effect Is Induced by an Attentional Bias

Previous studies indicated that empty time intervals are better discriminated in the auditory than in the visual modality, and when delimited by signals delivered from the same (intramodal intervals) rather than from different sensory modalities (intermodal intervals). The present electrophysiological study was conducted to determine the mechanisms which modulated the performances in inter- and intramodal conditions. Participants were asked to categorise as short or long empty intervals marked by auditory (A) and/or visual (V) signals (intramodal intervals: AA, VV; intermodal intervals: AV, VA). Behavioural data revealed that the performances were higher for the AA intervals than for the three other intervals and lower for inter- compared to intramodal intervals. Electrophysiological results indicated that the CNV amplitude recorded at fronto-central electrodes increased significantly until the end of the presentation of the long intervals in the AA conditions, while no significant change in the time course of this component was observed for the other three modalities of presentation. They also indicated that the N1 and P2 amplitudes recorded after the presentation of the signals which delimited the beginning of the intervals were higher for the inter- (AV/VA) compared to the intramodal intervals (AA/VV). The time course of the CNV revealed that the high performances observed with AA intervals would be related to the effectiveness of the neural mechanisms underlying the processing of the ongoing interval. The greater amplitude of the N1 and P2 components during the intermodal intervals suggests that the weak performances observed in these conditions would be caused by an attentional bias induced by the cognitive load and the necessity to switch between modalities.     

pH影响淀粉酶活性的探究

学生在探究pH对淀粉酶活性的影响时．出现了实验结果与预期结果不一致的现象,由此探究pH对淀粉酶活性的影响。     

The Effect of the G 1 - S transition Checkpoint on an Age Structured Cell Cycle Model

Knowledge of how a population of cancerous cells progress through the cell cycle is vital if the population is to be treated effectively, as treatment outcome is dependent on the phase distributions of the population. Estimates on the phase distribution may be obtained experimentally however the errors present in these estimates may effect treatment efficacy and planning. If mathematical models are to be used to make accurate, quantitative predictions concerning treatments, whose efficacy is phase dependent, knowledge of the phase distribution is crucial. In this paper it is shown that two different transition rates at the - checkpoint provide a good fit to a growth curve obtained experimentally. However, the different transition functions predict a different phase distribution for the population, but both lying within the bounds of experimental error. Since treatment outcome is effected by the phase distribution of the population this difference may be critical in treatment planning. Using an age-structured population balance approach the cell cycle is modelled with particular emphasis on the - checkpoint. By considering the probability of cells transitioning at the - checkpoint, different transition functions are obtained. A suitable finite difference scheme for the numerical simulation of the model is derived and shown to be stable. The model is then fitted using the different probability transition functions to experimental data and the effects of the different probability transition functions on the model''s results are discussed.     

The Effect of pH on Copper Toxicity to Blue-Green Algae

The effect of pH on copper toxicity to two planktonic blue-green algae, Aphanizomenon gracile and Oscillatoria redekei, was investigated. Growth rates of the algae without copper treatment decrease with pH, Aphanizomenon is earlier and more affected than Oscillatoria. On the other hand, pH-lowering leads sooner to a toxicity enhancement in Oscillatoria. In the acid range, toxicity retardation occurs in Aphanizomenon. At pH 5.1, shortening of the interval between copper toxicity and copper stimulus is characteristic for both species.     

Effect of calcium on intracellular pH

The effect of intracellular calcium on intracellular pH in the turtle urinary bladder was examined with phosphorus nuclear magnetic resonance. The turtle urinary bladder is capable of acidification in vitro and urinary acidification by this membrane is inhibited by an increase in intracellular calcium. Since calcium is capable of altering intracellular pH, it remains unclear whether the inhibition of urinary acidification is the result of an increase in intracellular pH. In the present study, intracellular calcium was increased by the cholinergic agent, carbachol, the ionophore A23187 and replacement of extracellular Na by sucrose. All agents decreased intracellular pH in the turtle bladder, thus suggesting that inhibition of urinary acidification by these agents is not due to an increase in intracellular pH.     

The Effect of pH on the Covalent Complexes of Thymidylate Synthase

Abstract

The objective of this investigation was to study the effect of pH on the dissociation constants and binding ratios of covalent complexes of thymidylate synthases from Escherichia coli and Lactobacillus casei.     

The Effect of the pH of Meat on the Boiling Test

The effect of the pH on the boiling test was studied in 68 beef and 108 pork muscles. The pH had a significant effect on the sensory scores obtained from the boiling test. The effect was particularly pronounced in the odour of meat. In beef the odour scores remain steady for samples with a pH value under about 6.2 and start to increase rapidly in higher pH values. In pork the increase in scores appears to be linear. The odour observed in high pH meat was described by the judges as abnormal and ammonialike. In the case of meat with a high pH, the results of the boiling test at meat inspection should be interpreted with extreme caution.