High Ionic Strength Conditions Research Articles

Assessment of Net Charge and Protein–Protein Interactions of Different Monoclonal Antibodies

The purpose of this work was to compare biophysical properties of different monoclonal antibodies (mAbs). mAbs' theoretical isoelectric point (IEP) and theoretical net charge were compared with experimentally assessed values. IEP was determined by isoelectric focusing capillary electrophoresis, determination of zero electrophoretic mobility, or the minimum mutual diffusion coefficient during pH titration. Net charge was determined using electrophoretic mobility and self-diffusion coefficient. It was found that antibodies differ substantially in their biophysical properties, that is, in IEP, net charge, and zeta potential. Also, the importance of these properties was studied with respect to protein-protein interactions. This was achieved by assessing the second virial coefficient (A(2)) determined by static light scattering (SLS) and dynamic light scattering (DLS). It was found that at low ionic strength formulation conditions [20 mM histidine (His)/His-HCl buffer, pH 6.0] proteins' charge is the main driver for overall repulsive protein interactions. At high ionic strength conditions (20 mM His/His-HCl buffer, pH 6.0, + 150 mM NaCl), where counterions are shielding ionic interactions, proteins' repulsive forces were weakened, but to a different extent. Furthermore, a DLS method was developed allowing fast and easy assessment of A(2) by minimum need of material.

Elongation factor 4 (EF4/LepA) accelerates protein synthesis at increased Mg2+concentrations

Elongation factor 4 (EF4) is one of the most conserved proteins present in bacteria as well as in mitochondria and chloroplasts of eukaryotes. Although EF4 has the unique ability to catalyze the back-translocation reaction on posttranslocation state ribosomes, the physiological role of EF4 remains unclear. Here we demonstrate that EF4 is stored at the membrane of Escherichia coli cells and released into the cytoplasm upon conditions of high ionic strength or low temperature. Under such conditions, wild-type E. coli cells overgrow mutant cells lacking the EF4 gene within 5-10 generations. Elevated intracellular Mg(2+) concentrations or low temperature retard bacterial growth and inhibit protein synthesis, probably because of formation of aberrant elongating ribosomal states. We suggest that EF4 binds to these stuck ribosomes and remobilizes them, consistent with the EF4-dependent enhancement (fivefold) in protein synthesis observed under these unfavorable conditions. The strong selective advantage conferred by the presence of EF4 at high intracellular ionic strength or low temperatures explains the ubiquitous distribution and high conservation of EF4.

Discovery of Biphasic Thermal Unfolding of OmpC with Implications for Surface Loop Stability

Escherichia coli outer membrane protein C (osmoporin) is a close homologue of OmpF or matrix porin, expressed under conditions of high osmolarity or ionic strength. Despite the fact that the proteins display very similar structures (rmsd = 0.78 Å), the channel activities (gating or selectivity) of the two proteins are markedly different, and compared to OmpF, there is much less published information about the stability and folding of OmpC. In this paper, we report a structural study of nine OmpC mutations that affect channel size and voltage gating. The secondary and tertiary structural analysis by circular dichroism (CD) indicated that the single-amino acid substitutions have little impact on the protein fold. However, a thermal denaturation study using CD and differential scanning calorimetry shows that different mutations lead to varied levels of destabilization, with the largest showing a 15 °C lower T(m) than the wild type and a 40% reduction in ΔH(cal). CD thermal denaturation measurements revealed that OmpC unfolds in a biphasic process, in which only the second phase is affected by the known mutations. The first stage of unfolding was shown to be reversible and separate from the main unfolding and loss of trimeric structure occurring in the second phase, leaving the flexible extracellular loops as the likely site of unfolding. The first phase is abolished as OmpC becomes more stable at lower pH.

Colorimetric Assay for S-Adenosylhomocysteine Hydrolase Activity and Inhibition Using Fluorosurfactant-Capped Gold Nanoparticles

This study reports a simple colorimetric method for the sensitive detection of S-adenosylhomocysteine hydrolase (SAHH) activity and inhibition using fluorosurfactant-capped gold nanoparticles (FSN-AuNPs). FSN stabilizes the AuNPs against conditions of high ionic strength, and FSN-AuNPs are merely aggregated in the presence of homocysteine (HCys) and cysteine. Because of this feature, FSN-AuNPs were found to be dispersed in the presence of S-adenosylhomocysteine (SAH) that lacks a free thiol group. After SAHH catalyzed the hydrolysis of SAH, the produced HCys molecules were bound to the surface of AuNPs through the formation of Au-S bonds. As a result, the nanoparticle (NP) aggregation occurred through electrostatic attraction between each HCys-attached AuNP. This approach had a minimum detectable concentration of 100 units/L (~6 nM). Additionally, because adenosine analogs are capable of inhibiting SAHH activity, the addition of adenosine analogs to a solution containing SAH and SAHH resulted in the suppression of hydrolyzed SAH-induced NP aggregation. Adenosine analogs exhibited the following trend in the half-maximal inhibitory concentrations: adenosine > adenosine monophosphate > adenosine diphosphate ~ adenosine triphosphate. We have demonstrated that the combination of SAHH inhibition and FSN-AuNPs can be utilized for the selective detection of adenosine.

Colorimetric Sensing of Silver(I) and Mercury(II) Ions Based on an Assembly of Tween 20-Stabilized Gold Nanoparticles

We have developed a rapid and homogeneous method for the highly selective detection of Hg(2+) and Ag(+) using Tween 20-modified gold nanoparticles (AuNPs). Citrate ions were found to still be adsorbed on the Au surface when citrate-capped AuNPs were modified with Tween 20, which stabilizes the citrate-capped AuNPs against conditions of high ionic strength. When citrate ions had reduced Hg(2+) and Ag(+) to form Hg-Au alloys and Ag on the surface of the AuNPs, Tween 20 was removed from the NP surface. As a result, the AuNPs were unstable under a high-ionic-strength solution, resulting in NP aggregation. The formation of Hg-Au alloys or Ag on the surface of the AuNPs was demonstrated by means of inductively coupled plasma mass spectroscopy and energy-dispersive X-ray spectroscopy. Tween 20-AuNPs could selectively detect Hg(2+) and Ag(+) at concentrations as low as 0.1 and 0.1 microM in the presence of NaCl and EDTA, respectively. Moreover, the probe enables the analysis of AgNPs with a minimum detectable concentration that corresponds to 1 pM. This probe was successfully applied to detect Hg(2+) in drinking water and seawater, Ag(+) in drinking water, and AgNPs in drinking water.

Uptake of Ozone to Deliquesced KI and Mixed KI/NaCl Aerosol Particles

The kinetics of uptake of ozone to deliquesced potassium iodide (KI) aerosol particles has been investigated in an aerosol flow tube at 72-75% relative humidity, room temperature, and atmospheric pressure. The observed loss of ozone was further analyzed in terms of a numeric model to explicitly track the iodide concentration in the particles. This allowed retrieving a value alpha(b) = 0.6 +/- (0.5)(0.4) for the bulk accommodation coefficient (alpha(b)). The second order rate constant in the bulk phase agreed with available literature (k(b) = (1.0 +/- 0.3) x 10(9) M(-1) s(-1)) even for the high ionic strength conditions of the present experiments. As long as iodide remained in excess, the average uptake coefficient was gamma = (1.10 +/- 0.20) x 10(-2). Different experiments were performed where the iodide to chloride ratio, the ozone concentration, and the surface to volume ratio of particles were varied. In combination, the results obtained indicate that uptake was driven by fast bulk accommodation and reaction in the bulk for all conditions investigated. The results further suggest that ozone uptake is not limited by the bulk accommodation coefficient alpha(b) under atmospheric conditions.

Ferredoxin:NADP+ Oxidoreductase Association with Phycocyanin Modulates Its Properties

In photosynthetic organisms, ferredoxin:NADP(+) oxidoreductase (FNR) is known to provide NADPH for CO(2) assimilation, but it also utilizes NADPH to provide reduced ferredoxin. The cyanobacterium Synechocystis sp. strain PCC6803 produces two FNR isoforms, a small one (FNR(S)) similar to the one found in plant plastids and a large one (FNR(L)) that is associated with the phycobilisome, a light-harvesting complex. Here we show that a mutant lacking FNR(L) exhibits a higher NADP(+)/NADPH ratio. We also purified to homogeneity a phycobilisome subcomplex comprising FNR(L,) named FNR(L)-PC. The enzymatic activities of FNR(L)-PC were compared with those of FNR(S). During NADPH oxidation, FNR(L)-PC exhibits a 30% decrease in the Michaelis constant K(m)((NADPH)), and a 70% increase in K(m)((ferredoxin)), which is in agreement with its predicted lower activity of ferredoxin reduction. During NADP(+) reduction, the FNR(L)-PC shows a 29/43% decrease in the rate of single electron transfer from reduced ferredoxin in the presence/absence of NADP(+). The increase in K(m)((ferredoxin)) and the rate decrease of single reduction are attributed to steric hindrance by the phycocyanin moiety of FNR(L)-PC. Both isoforms are capable of catalyzing the NADP(+) reduction under multiple turnover conditions. Furthermore, we obtained evidence that, under high ionic strength conditions, electron transfer from reduced ferredoxin is rate limiting during this process. The differences that we observe might not fully explain the in vivo properties of the Synechocystis mutants expressing only one of the isoforms. Therefore, we advocate that FNR localization and/or substrates availability are essential in vivo.

MFI–UF constant pressure at high ionic strength conditions

It has been clearly proven that particles much smaller than 0.45 μm are responsible for colloidal fouling of the surface of RO and NF membranes. As a consequence the predictive value of SDI and MFI (0.45 µm) in particulate fouling is doubtful. To overcome this deficiency the MFI–UF has been developed, making use of ultrafiltration membrane with different pore sizes and measured at constant pressure or constant flux. The MFI–UF has not yet been tested and evaluated for brackish/estuarine water and seawater. The objective of this work is to investigate the effect of salinity on the MFI–UF constant pressure for higher ionic strength conditions present in brackish/estuarine waters and seawater prior to application to reverse osmosis. For this, specific objectives are to ascertain the effect of pressure and ionic strength on various membrane filters. Membrane filters consisting of three membrane materials were tested – PES, RC, and PVDF – with different MWCOs (100, 30 and 10 kDa and 0.1 µm for PDVF). Ultra pure water was used to prepared standard solutions having different salinities, together with Delft canal water and Delft tap water. One membrane filter showed significant compaction after testing with ultra pure water namely 100 kDa RC membrane at pressures above 1.5 bar. “Salt compaction” measured at 1 bar and by increasing the salt concentration in 5 g/L steps was higher for PES than for RC membranes. PVDF membranes only showed apparent/salt compaction starting at 20 g/L. For PES membranes,the observed flux decline due to sodium chloride was faster and higher than for RC and PVDF membranes. The calculated “blank” or apparent MFI–UF values due to salinity were low compared to MFI values usually observed in surface and seawaters. Pre- conditioning of the membranes by soaking in sodium chloride solutions might eliminate this blank previous testing. Addition of different amounts of sodium chloride to tap water and to canal water resulted in higher values of MFI for MF membranes, while for UF membranes the effect was not significant (7% deviation from 0 to 35 g/L).

Colorimetric Sensing of Adenosine Based on Aptamer Binding Inducing Gold Nanoparticle Aggregation

AbstractA simple and rapid colorimetric approach for the determination of adenosine has been developed via target inducing aptamer structure switching, thus leading to Au colloidal solution aggregation. In the absence of the analytes, the aptamer/gold nanoparticle (Au NP) solution remained well dispersed under a given high ionic strength condition in that the random‐coil aptamer was readily wrapped on the surface of the Au NPs, which resulted in the enhancement of the repulsive force between the nanoparticles due to the high negative charge density of DNA molecules. While in the presence of adenosine, target‐aptamer complexes were formed and the conformation of the aptamer was changed to a folded structure which disfavored its adsorption on the Au NP surface, thus leading to the reduction of the negative charge density on each Au NP and then the reduced degree of electrostatic repulsion between Au nanoparticles. As a result, the aggregation of the Au colloidal solution occurred. The changes of the absorption spectrum could be easily monitored by a UV‐Vis spectrophotometer. A linear correlation exists between the ratio of the absorbance of the system at 522 to 700 nm (A522 nm/A700 nm) and the concentration of adenosine between 100 nmol·L−1 and 10 µmol·L−1, with a detection limit of 51.5 nmol·L−1.

Restructuring of small flocs of polystyrene latex with polyelectrolyte

The structure of small flocs formed of negatively charged polystyrene latex particles with cationic polyelectrolyte was investigated to clarify the characteristics of the restructuring process of colloidal flocs. Experiments were designed in order to elucidate the effect of the different kinetical patterns in the adsorption of polyelectrolytes at the surface of colloidal particles on the restructuring process of flocs. In the experiments, flocs were formed in a standard mixing apparatus and their morphology was monitored by using a microscope. The relation between the maximum distance in the projection of a formed floc and the number of primary particles comprising the floc was used as the evaluation index of the floc structure. It was found that the restructuring of flocs takes place facilely under the condition of high ionic strength. On the other hand, under the condition of low ionic strength, the degree of restructuring varied with the molecular weight. Flocs with bulky structure were formed when polyelectrolytes with high molecular weight were used. This result of the restructuring of floc as functions of the ionic strength of solution and the molecular weight of polyelectrolyte is consistent with the rearrangement behavior of adsorbing polyelectrolytes reported previously [K. Aoki, Y. Adachi, J. Colloid Interface Sci. 300 (2006) 69–77].

Protamine-induced biosilica as efficient enzyme immobilization carrier with high loading and improved stability

β-glucuronidase (GUS) was encapsulated inside mesoporous biosilica prepared via a biomimetic silicification approach. Protamine, a natural cationic polypeptide, was used to induce the condensation of sodium silicate and the enzyme GUS was thus encapsulated during the precipitation process. The enzyme loading efficiency was found to be as high as 100% within its solubility limit in aqueous solution. Furthermore, all the enzyme molecules were tightly entrapped in the biosilica particles without any leaching even under a high ionic strength condition. The biosilica particles displayed a dramatic enrichment effect toward the substrate, baicalin, through nonspecific adsorption. The catalytic activity of the encapsulated GUS was evaluated by converting baicalin into baicalein, two important effective components of herbal medicines. It was found that the thermal and pH tolerance as well as the storage and recycling stability of the encapsulated GUS were significantly enhanced compared with those of the free enzyme. Under the optimum reaction conditions (37 °C, pH 7.0), a high productivity of baicalein of 75% was obtained, only 5% lower than the case for free enzyme counterpart. No appreciable loss in activity was observed after 30-day storage, and 80% of the initial activity could be retained after 4 repeated cycles.

Balance between Coulombic Interactions and Physical Confinement in Silica Hydrogel Encapsulation

We examined the behavior of various entrapped guest molecules within silica hydrogel and evaluated the effect of Coulombic interactions and physical confinement on molecular mobility. Although rhodamine 6G (R6G) and fluorescein (FL) share similar size and molecular structure, their behavior in silica hydrogel was found to be dramatically different. A good majority of R6G was immobilized with little to no exchangeable molecules, whereas FL displayed a considerable amount of mobility in silica hydrogel. Moreover, silica hydrogel encapsulated R6G failed to gain mobility even under low pH or high ionic strength conditions to minimize Coulombic interactions, implying that encapsulated R6G molecules were inaccessible and likely trapped deep inside the silica matrix of a hydrogel. On the contrary, FL was relatively free to rotate and translate inside a silica hydrogel, implying that FL remained solvated in the solvent phase and was able to maintain its mobility throughout the hydrogel formation process. Fluorescence recovery after photobleaching measurements put the diffusion coefficient of FL in silica hydrogel at ca. 2.1 x 10(-6) cm(2) s(-1), about a factor of 3 slower than that in solution. The substantial difference in mobility between cationic R6G and anionic FL led us to conclude that the effect of Coulombic interactions on mobility is more dominating in hydrogel than in alcogel. Our results also suggest that Coulombic interactions are strong enough to influence the eventual placement of a guest molecule in a silica hydrogel, causing R6G and FL to reside in different microenvironments. This has a profound implication on the use of molecular probes to study silica hydrogel since a slight difference in physical attribute may result in very diverse observations even from identically prepared silica hydrogel samples. As demonstrated, the repulsion between FL and silica renders FL liquid-bound, making FL more suitable for monitoring the change in viscosity and physical confinement during hydrogel formation, whereas other researchers have shown that silica-bound R6G is more suitably used as a reliable probe for monitoring the growth of silica colloids because of its strong attraction toward silica.

Copper modulates the heat-induced sulfhydryl/disulfide interchange reactions of β-Lactoglobulin

This study describes the effect of copper on the heat-denaturation/aggregation of β-Lactoglobulin AB at neutral pH. The kinetics of disappearance of native β-Lactoglobulin under different ionic strength and Cu2+/β-Lactoglobulin molar ratio conditions were followed and the type of interactions (covalent or non-covalent) shared between non-native structures during the heating process were examined. On heating, the rate of disappearance of native β-Lactoglobulin was accelerated by increasing the Cu2+/β-Lactoglobulin molar ratio. Copper induces oxidation of the free sulfhydryl group of β-Lactoglobulin resulting mainly in the formation of covalent dimers, which were further associated into large non-covalent aggregates under high ionic strength conditions. Characterisation of the β-Lactoglobulin dimers reveals the existence of three different molecular species arising randomly (dimers A–A, A–B and B–B), in which tertiary structure was completely lost. The quantity of added copper constitutes a powerful way to control the heat-denaturation/aggregation process of β-Lactoglobulin in particular regarding the relative proportion of covalent and non-covalent interactions into formed aggregates.

Regulation of the nucleic acid chaperone activity of HTLV-1 Nucleocapsid Protein

Nucleocapsid proteins (NC) of retroviruses are nucleic acid chaperones that facilitate nucleic acid remodeling. This property of NC proteins is critical for their role in viral genome dimerization, maturation and reverse transcription. In contrast to all other NC proteins studied to date, the human T-cell leukemia virus type 1 (HTLV-1) NC protein was shown to be an extremely poor chaperone. In this work, we demonstrate that the anionic C-terminal domain (CTD) of this protein is responsible for its poor chaperone function. Single molecule DNA stretching studies suggest that HTLV-1 NC dissociates very slowly from single-stranded DNA, which may be a primary reason for its poor chaperone activity. In contrast, a truncation mutant that lacks the CTD is a more effective annealing agent and displays faster off-rate kinetics. Under conditions of high ionic strength, the properties of the WT and CTD-deletion variant are much more similar to each other. Taken together, our data suggest that an electrostatic attraction between the anionic CTD and cationic N-terminal domain of HTLV-1 NC leads to polymerization onto ssDNA resulting in a poor ability to aggregate nucleic acids or to promote their annealing. This property of HTLV-1 NC makes it similar to typical SSB proteins, and may be related to this NC's role in excluding the viral restriction factor APOBEC3G from HTLV particles.

Synchronous Gene Expression of theYersinia enterocoliticaYsa Type III Secretion System and Its Effectors

Type III secretion systems (T3SSs) are complex units that consist of many proteins. Often the proteins are encoded as a cohesive unit on virulence plasmids, but several systems have their various components dispersed around the chromosome. The Yersinia enterocolitica Ysa T3SS is such a system, where the apparatus genes, some regulatory genes, and four genes encoding secreted proteins (ysp genes) are contained in a single locus. The remaining ysp genes and at least one additional regulator are found elsewhere on the chromosome. Expression of ysa genes requires conditions of high ionic strength, neutral/basic pH, and low temperatures (26 degrees C) and is stimulated by exposure to solid surfaces. The AraC-like regulator YsaE and the dual-function chaperone/regulator SycB are required to stimulate the sycB promoter, which transcribes sycB and probably yspBCDA as well. The putative phosphorelay proteins YsrRS (located at the distal end of the ysa locus) and RcsB, the response regulator of the RcsBCD phosphorelay system, are required to initiate transcription at the ysaE promoter, which drives transcription of many apparatus genes. In this work, we sought to determine which ysp genes were coordinately regulated with the genes within the ysa locus. We found that six unlinked ysp genes responded to NaCl and required YsaE/SycB, YsrRS, and RcsB for expression. Three ysp genes had unique patterns, one of which was unaffected by all elements tested except NaCl. Thus, while the ysp genes were likely to have been acquired independently, most have acquired a synchronous regulatory pattern.

One-Step Label-Free Optical Genosensing System for Sequence-Specific DNA Related to the Human Immunodeficiency Virus Based on the Measurements of Light Scattering Signals of Gold Nanorods

A one-step label-free optical genosensing method has been developed in this contribution by taking short DNA target with its sequence related to the human immunodeficiency virus type 1 (HIV-1) as an example. By employing anisotropic nonspherical and positively charged gold nanorods (Au-NRs) as the recognition platform, which show high stability against aggregation under high ionic strength conditions without any additional stable reagent, we found that the addition of target DNA to the mixture of nonmodified Au-NRs suspension and label-free probe DNA in high ionic strength buffer leads to a color change from red to light purple in less than 5 min, displaying strong plasmon resonance light scattering (PRLS) signals. Mechanism investigations showed that the strong PRLS signals should be ascribed to the aggregation of Au-NRs induced by the formed double-stranded oligonucleotides (dsDNA) from the hybridization of target DNA with probe DNA. With the PRLS signals, we monitored the hybridization process of a 21-mer single-stranded oligonucleotide (ssDNA) from the HIV-1 U5 long terminal repeat (LTR) sequence with its complementary oligonucleotide and detected the effect of single-base-pair mismatches. Two polymerase chain reaction (PCR) amplicon artificial samples derived from Mycobacterium tuberculosis glmS and genes encoding for Bacillus glucanase and an HIV-1 LTR sample isolated from HIV-1-positive blood were detected with satisfactory results, showing that the present method has simplicity, sensitivity, specificity, and reliability for sequence-specific DNA detection related to the HIV gene.

A New Optical Parametric Amplifier Based on Lithium Thioindate Used for Sum Frequency Generation Vibrational Spectroscopic Studies of the Amide I Mode of an Interfacial Model Peptide

We describe a new optical parametric amplifier (OPA) that employs lithium thioindate, LiInS2 (LIS), to create tunable infrared light between 1500 cm(-1) and 2000 cm(-1). The OPA based on LIS described within provides intense infrared light with a good beam profile relative to similar OPAs built on silver gallium sulfide, AgGaS2 (AGS), or silver gallium selenide, AgGaSe2 (AGSe). We have used the new LIS OPA to perform surface-specific sum frequency generation (SFG) vibrational spectroscopy of the amide I vibrational mode of a model peptide at the hydrophobic deuterated polystyrene (d8-PS)-phosphate buffered saline interface. This model polypeptide (which is known to be an alpha-helix in the bulk solution under the high ionic strength conditions employed here) contains hydrophobic leucyl (L) residues and hydrophilic lysyl (K) residues, with sequence Ac-LKKLLKLLKKLLKL-NH2. The amide I mode at the d8-PS-buffer interface was found to be centered around 1655 cm(-1). This can be interpreted as the peptide having maintained its alpha-helical structure when adsorbed on the hydrophobic surface, although other interpretations are discussed.

Temperature dependence of myosin-II tail fragment assembly

Dictyostelium myosin-II bipolar thick filament (BTF) assembly is heavily dependent on ionic strength and temperature and is reversible by the phosphorylation of just three threonines. Truncated tail fragments of Dictyostelium myosin-II are commonly used as models for BTF assembly, as they self-assemble into regular paracrystals that recapitulate the ionic strength and phosphorylation dependence of full-length Dictyostelium myosin-II BTF assembly. Here we show that Dictyostelium myosin-II tail fragment assembly is highly temperature dependent, similar to full-length Dictyostelium myosin-II. Assembly of paracrystals was far more robust at 4 degrees C than at higher temperatures. Pre-assembled paracrystals disassembled completely when shifted to 37 degrees C, indicating that assembly does not greatly improve the thermostability of these tail fragments. The melting temperatures of individual Dictyostelium myosin-II tail coiled-coils under both low and high ionic strength conditions that prohibit paracrystal assembly are extremely low, 21 degrees C and 28 degrees C, respectively. These data are consistent with reversible thermal denaturation of the coiled-coil as the most likely explanation for assembly incompetence under either very low ionic strength or high temperature conditions. Assembled paracrystals of a structurally similar fragment of nonmuscle myosin-IIA were far more thermodynamically stable than their Dictyostelium counterparts at the temperatures examined here.

Cardiolipin deficiency leads to decreased cardiolipin peroxidation and increased resistance of cells to apoptosis

Cardiolipin (CL), a unique mitochondrial phospholipid synthesized by CL synthase (CLS), plays important, yet not fully understood, roles in mitochondria-dependent apoptosis. We manipulated CL levels in HeLa cells by knocking down CLS using RNA interference and selected a clone of CL-deficient cells with ~ 45% of its normal content. ESI–MS analysis showed that the CL molecular species were the same in CL-deficient and CL-sufficient cells. CL deficiency did not change mitochondrial functions (membrane potential, reactive oxygen species generation, cellular ATP levels) but conferred resistance to apoptosis induced by actinomycin D (ActD), rotenone, or γ-irradiation. During ActD-induced apoptosis, decreased CL peroxidation along with suppressed cytochrome (cyt) c release was observed in CL-deficient cells, whereas Bax translocation to mitochondria remained similar to that in CL-sufficient HeLa cells. The amounts of loosely bound cyt c (releasable under high ionic strength conditions) were the same in CL-deficient and CL-sufficient cells. Given that CL peroxidation during apoptosis is catalyzed by CL/cyt c complexes and CL oxidation products are essential for cyt c release from mitochondria, our results suggest that CL deficiency prevents adequate assembly of productive CL/cyt c complexes and CL peroxidation, resulting in increased resistance to apoptosis.

Length of exposure to high post-rigor temperatures affects the tenderisation of the beef M. longissmus dorsi

This experiment examined the effect of length of exposure of bovine M. longissmus dorsi to high temperatures (37°C) on proteolytic activity during post-mortem aging and subsequent meat tenderness. To avoid confounding between pH decline and incubation treatments, the experiment was conducted on post-rigor meat (pH < 5.6), which had entered rigor at 15°C. This meat was first incubated at 15°C until rigor (pH < 5.6), whereupon samples were then reheated and maintained at 37°C for 0, 1 or 3 h. Following incubation, samples were aged at 1°C for 1–21 days. Control groups were incubated at either 15 or 37°C until pH < 5.6, followed by aging at 1°C. High rigor temperatures accelerated post-mortem proteolysis early in the post-mortem period, as indicated by a rapid decline in shear force (P < 0.01), but post-mortem aging ceased at about day 3 post-mortem and the high rigor temperature treatment was ultimately 30% tougher at day 21 post-mortem (P < 0.01). The process of reheating samples from 15 to 37°C had minimal effect on tenderness levels, but was associated with a slight increase in proteolysis, identified by myofibril length, and was associated with an increase in cook loss percentage (P < 0.05). Shear force in the length of exposure experiment was affected by an incubation time × post-mortem aging interaction (P < 0.01). This indicated an initial acceleration of post-mortem aging with increased length of exposure, but also a reduction in the ultimate tenderness of product after extended post-mortem aging. This was presumably due to the loss of proteolytic enzyme activity caused by the instability of proteolytic enzymes at high ionic strength conditions such as those present in post-mortem muscle.