Cryoprotectant agent toxicity in porcine articular chondrocytes

Large articular cartilage defects have proven difficult to treat and often result in osteoarthritis of the affected joint. Cryopreservation of articular cartilage can provide an increased supply of tissues for osteochondral allograft but cryoprotective agents are required; however, few studies have been performed on the toxicity of these agents. This study was designed to determine the order of toxicity of five commonly used cryoprotectant agents as well as interactions that occur between them. Isolated porcine articular chondrocytes were exposed to individual cryoprotectant agents and combinations of these agents at 1 M and 3 M concentrations for 5 min and 120 min. Cell viability was determined using membrane integrity dyes and a metabolic activity assay. Subsequently, a regression analysis based study was undertaken to extract the maximum amount of information from this data. Results of this study demonstrated that all 1 M solutions were minimally toxic. The 3 M solutions demonstrated varying toxicity after 120 min. Ethylene glycol and glycerol were less toxic than propylene glycol, dimethyl sulfoxide, and formamide. Combinations of cryoprotectant agents were less toxic than single cryoprotectant agents at the same concentration. This is the most comprehensive study investigating cryoprotectant agent toxicity in articular chondrocytes and has resulted in important information regarding the order of toxicity and interactions that occur between these agents.     

Cryoprotectant agent toxicity in porcine articular chondrocytes

Large articular cartilage defects have proven difficult to treat and often result in osteoarthritis of the affected joint. Cryopreservation of articular cartilage can provide an increased supply of tissues for osteochondral allograft but cryoprotective agents are required; however, few studies have been performed on the toxicity of these agents. This study was designed to determine the order of toxicity of five commonly used cryoprotectant agents as well as interactions that occur between them. Isolated porcine articular chondrocytes were exposed to individual cryoprotectant agents and combinations of these agents at 1 M and 3 M concentrations for 5 min and 120 min. Cell viability was determined using membrane integrity dyes and a metabolic activity assay. Subsequently, a regression analysis based study was undertaken to extract the maximum amount of information from this data. Results of this study demonstrated that all 1 M solutions were minimally toxic. The 3 M solutions demonstrated varying toxicity after 120 min. Ethylene glycol and glycerol were less toxic than propylene glycol, dimethyl sulfoxide, and formamide. Combinations of cryoprotectant agents were less toxic than single cryoprotectant agents at the same concentration. This is the most comprehensive study investigating cryoprotectant agent toxicity in articular chondrocytes and has resulted in important information regarding the order of toxicity and interactions that occur between these agents.     

Recognition sequence design for peptidyl modulators of beta-amyloid aggregation and toxicity

beta-Amyloid (Abeta), the primary protein component of Alzheimer's plaques, is neurotoxic when aggregated into fibrils. We have devised a modular strategy for generating compounds that inhibit Abeta toxicity, based on linking a recognition element for Abeta to a disrupting element designed to interfere with Abeta aggregation. One such compound, with the 15-25 sequence of Abeta as the recognition element and a lysine hexamer as the disrupting element, altered Abeta aggregation kinetics and protected cells from Abeta toxicity [Ghanta et al. (1996) J. Biol. Chem. 271, 29525]. To optimize the recognition element, peptides of 4-8 residues composed of overlapping sequences within the 15-25 domain were synthesized, along with hybrid compounds containing those recognition sequences coupled to a lysine hexamer. None of the recognition peptides altered Abeta aggregation kinetics and only two, KLVFF and KLVF, had any protective effect against Abeta toxicity. The hybrid peptide KLVFF-KKKKKK dramatically altered Abeta aggregation kinetics and aggregate morphology and provided significantly improved protection against Abeta toxicity compared to the recognition peptide alone. In contrast, FAEDVG-KKKKKK possessed only modest inhibitory activity and had no marked effect on Abeta aggregation. The scrambled sequence VLFKF was nearly as effective a recognition domain as KLVFF, suggesting the hydrophobic characteristics of the recognition sequence are critical. None of the cytoprotective peptides prevented Abeta aggregation; rather, they increased aggregate size and altered aggregate morphology. These results suggest that coupling recognition with disrupting elements is an effective generalizable strategy for the creation of Abeta inhibitors. Significantly, prevention of Abeta aggregation may not be required for prevention of toxicity.     

Preparation and analysis of the products of non-enzymatic protein glycosylation and their relationship to cross-linking of proteins

The presence of glycosylated protein-bound lysine residues has led to much speculation regarding changes in structure and function of the modified protein. The synthesis of hexose-lysine adducts and their separation using an amino acid analyser is described. These compounds are also produced during borohydride reduction and subsequent hydrolysis of modified proteins, and misidentification of these may occur depending upon precise chromatographic procedures. The possibility that glucose might participate in a cross-linking reaction between two protein-bound lysine residues was tested but no evidence for such a mechanism was found. The presence of 14C-labelled urinary hexosyllysine indicated that body protein breakdown in addition to ingested dietary hexosyllysine contributes to the excretion of this component.     

Mutational analysis of domain I of Pseudomonas exotoxin. Mutations in domain I of Pseudomonas exotoxin which reduce cell binding and animal toxicity

Pseudomonas exotoxin (PE) is a single polypeptide chain that contains 613 amino acids and is arranged into three structural domains. Domain I is responsible for cell recognition, II for translocation of PE across membranes and III for ADP ribosylation of elongation factor 2. Treatment of PE with reagents that react with lysine residues has been shown to lead to a reduction in cytotoxic activity apparently due to a modification of domain I (Pirker, R., FitzGerald, D. J. P., Hamilton, T. C., Ozols, R. F., Willingham, M. C., and Pastan, S. (1985) Cancer Res. 45, 751-757). To determine which lysine residues are important in cell recognition, all 12 lysines in domain I were converted to glutamates by site-directed mutagenesis. Also, two deletion mutants encompassing almost all of domain I (amino acids 4-252) or most of domain I (amino acids 4-224) were studied. The mutant proteins were produced in Escherichia coli, purified, and tested for their cytotoxic activity against Swiss 3T3 cells and in mice. The data indicate that conversion of lysine 57 to glutamate reduces cytotoxic activity towards 3T3 cells 50-100-fold and in mice about 5-fold. Deletion of amino acids 4-224 causes a similar reduction in toxicity towards cells and mice. Deletion of most of the rest of domain I (amino acids 4-252) causes a further reduction in toxicity toward cells and mice indicating this second region between amino acids 225 and 252 of domain I is also important in the toxicity of PE. Competition assays indicated that the ability of PEGlu57 to bind to 3T3 cells was greatly diminished, accounting for its diminished cytotoxic activity.     

Localized mutagenesis defines regions of the Bacillus thuringiensis delta-endotoxin involved in toxicity and specificity.

Bacillus thuringiensis produces a variety of delta-endotoxins which bind to specific receptors in insect larval midguts. Following insertion into the membrane there is an alteration of ion flux culminating in osmotic lysis. Mutagenic oligonucleotides were used to define regions in one of these toxins involved in specificity and toxicity. One region is highly conserved among all toxins sequenced to date and many mutations resulted in loss of toxicity for three test Lepidoptera. The mutant toxins had lost the capacity to inhibit K(+)-dependent amino acid transport into larval midgut vesicles, but there was no effect on their ability to compete with wild type toxin for binding. The results are consistent with this amphiphilic helical region of the toxin being essential for toxicity. A second mutagenized region overlapped a portion of another potential amphiphilic helix. Mutations of only 2 residues, Ala-92 and Arg-93, resulted in loss of toxicity for two lepidopteran larvae but some activity remained for a third. The A92D mutant toxin competed with the wild type toxin for binding to vesicles prepared from midguts from the sensitive but not from the insensitive larvae. Decreased toxicity was also found when this mutation was transferred to two other related protoxin genes. A number of mutations of each of these residues was analyzed and selective loss of toxicity correlated with the absence of a positive charge. Despite being distal from the presumptive specificity domain, 1 or both of these residues must have an important role in the specific binding of toxins.     

Role of lysine residues of plasma lipoproteins in high affinity binding to cell surface receptors on human fibroblasts.

The low density lipoprotein (LDL) cell surface receptors on human fibroblasts grown in culture bind specific plasma lipoproteins, initiating a series of events which regulate intracellular cholesterol metabolism. Specificity for the interaction with the receptors resides with the protein moieties of the lipoproteins, specifically with the B and E apoproteins of LDL and certain high density lipoproteins (HDLc HDLl), respectively. It was previously established that the amino acid arginine is a functionally significant residue in or near the recognition sites on the B and E apoproteins and that modification of this residue abolishes the ability of these apolipoproteins to bind to the receptor. The present study indicates that lysine residues are also involved in the lipoprotein-receptor interaction. Chemical modification of 15% of the lysine residues of LDL by carbamylation with cyanate or 20% by acetoacetylation with diketene prevents the LDL from competitively displacing unmodified 125I-LDL from the high affinity receptor sites or from binding directly to the receptor. Moreover, quantitative reversal of the aceto-acetylation of the lysine residues of LDL by hydroxylamine treatment regenerates the lysyl residues and reestablishes greater than 90% of the original binding activity of the LDL. The reversibility of this reaction establishes that the loss of binding activity which follows lysine modification is not due to an irreversible alteration of the LDL or HDLc but is probably due to an alteration of a property of the recognition site associated with specific lysine residues. While acetoacetylation and carbamylation neutralize the positive charge on the epsilon-amino group of lysine, reductive methylation selectively modifies lysine residues of LDL and HDLc without altering the positive charge, yet abolishes their ability to bind to the receptor. Preservation of the charge but loss of binding activity following reductive methylation of the lipoproteins suggests that the specificity of the recognition site does not reside simply with the presence of positive charges but depends on other more specific properties of the site determined by the presence of a limited number of the lysine (and arginine) residues. The precise role of lysine remains to be defined, but its function may be to establish and maintain the conformation of the recognition site or the alignment of reactive residues, or both, or to chemically react, through its epsilon-amino group, with the receptor (hydrogen bond formation would be such a possibility).     

Evaluation of alternative cryoprotectants for preserving stallion spermatozoa

Although use of cryopreserved stallion spermatozoa is currently accepted by many breed registries, utilization of this technique remains limited due to poor fertility for some stallions. One reason for these results is osmotic stress that spermatozoa experiences when the cryoprotectant (glycerol) is added to the cells prior to freezing and removal from the cells after thawing. In an effort to minimize osmotic damage, alternative cryoprotectants, having lower molecular weights and greater membrane permeability than glycerol, were evaluated to determine their effectiveness for cryopreserving stallion spermatozoa. In the first experiment, equal molar concentrations of several amides were compared to determine if they could preserve the motility of sperm as well as glycerol. At 0.55 M concentration, addition of glycerol to a skim milk-egg yolk (SMEY) diluent resulted in higher percentages of motile sperm (61%) than methyl formamide (40%) or dimethyl formamide (38%, P<0.05), while formamide, acetamide, and methyl acetamide resulted in recovery of less than 20% motile cells (P<0.05). When methyl formamide or dimethyl formamide were increased to 0.6 or 0.9 M they resulted in percentages of motile cells (48-54%) similar to that achieved with glycerol (52%). Similarly, 0.9 M ethylene glycol also resulted in similar percentages of motile cells (43%). Replacing the glucose and fructose in the SMEY diluent with either raffinose or trehalose did not result in higher percentages of motile sperm (65 and 66%, respectively) than the control SMEY (63%). Similarly, addition of methyl cellulose also did not increase the percentages of motile spermatozoa in the samples, after cryopreservation (P>0.05). In conclusion, both methyl formamide and dimethyl formamide protected stallion spermatozoa from cryodamage as effectively as glycerol. Since these compounds permeate the plasma membrane more effectively than glycerol, they should cause less osmotic damage to stallion spermatozoa than glycerol. Therefore, these compounds may prove very effective in the cryopreservation of stallion spermatozoa, and may be particularly useful for spermatozoa from stallions that produce spermatozoa that have poor post-thaw characteristics when glycerol is used as the cryoprotectant.     

The complete amino acid sequence of rabbit muscle phosphoglucomutase

The complete amino acid sequence of rabbit muscle phosphoglucomutase has been determined by isolating the 11 peptide fragments produced by the cyanogen bromide cleavage reaction and subjecting these to automated sequencing procedures. Products produced by treatment of some of these fragments with hydroxylamine, iodosobenzoic acid, mild acid, cyanogen bromide in formic and heptafluorobutyric acids, Staphylococcus aureus V8 protease, and trypsin (with or without blocking at lysine residues) were used to complete the sequence for each of the cyanogen bromide fragments. The cyanogen bromide fragments were ordered by isolating the four tryptic peptides produced by a limited tryptic digest of the native enzyme in the presence of its substrates and its bivalent metal ion activator, Mg2+, degrading these by means of trypsin, after blocking digestion at lysine residues, and isolating and identifying all fragments thus produced that contained 10 or more residues. The 561-residue sequence thus obtained is one of the longest that has been determined by chemical means. There is excellent agreement between this sequence and published compositions after appropriate normalization. The absorbance of the enzyme is about 7.0 at 278 nm for a 1% solution; this value is 9% lower than that previously used.     

Arginine residues are more effective than lysine residues in eliciting the cellular uptake of onconase

Onconase is an amphibian member of the pancreatic ribonuclease family of enzymes that is in clinical trials for the treatment of cancer. Onconase, which has an abundance of lysine residues, is internalized by cancer cells through endocytosis in a mechanism similar to that of cell-penetrating peptides. Here, we compare the effect of lysine versus arginine residues on the biochemical attributes necessary for Onconase to elicit its cytotoxic activity. In the variant R-Onconase, 10 of the 12 lysine residues in Onconase are replaced with arginine, leaving only the two active-site lysines intact. Cytometric assays quantifying internalization showed a 3-fold increase in the internalization of R-Onconase compared with Onconase. R-Onconase also showed greater affinity for heparin and a 2-fold increase in ribonucleolytic activity. Nonetheless, arginine substitution endowed only a slight increase in toxicity toward human cancer cells. Analysis of denaturation induced with guanidine-HCl showed that R-Onconase has less conformational stability than does the wild-type enzyme; moreover, R-Onconase is more susceptible to proteolytic degradation. These data indicate that arginine residues are more effective than lysine in eliciting cellular internalization but can compromise other aspects of protein structure and function.     

On-Chip Cryopreservation: A Novel Method for Ultra-Rapid Cryoprotectant-Free Cryopreservation of Small Amounts of Human Spermatozoa

Cryopreservation of human spermatozoa free from cryoprotectant can avoid toxicity caused by highly concentrated cryoprotectant and a series of specific carriers have been previously explored, except for PDMS chip. Our study is aimed at exploring a novel device for ultra-rapid cryopreservation of small numbers of spermatozoa without cryoprotectant based on polydimethylsiloxane (PDMS) chips. Spermatozoa from 25 healthy men were involved in this study, comparing on-chip cryopreservation with different micro-channel height (group A: 10 µm height, group B: 50 µm height, group C: 100 µm height) and conventional freezing (group D) in liquid nitrogen for 72 h. The viability, motility, DNA integrity by comet assay and acrosome integrity by fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) staining of frozen-thawed spermatozoa of each group were compared. The motility and viability of post-thawed spermatozoa was significantly decreased than that of pre-freezing spermatozoa. There was no difference of viability and motility of frozen-thawed spermatozoa between group A and D, while viability and motility of group B and C decreased compared to group A. Comet assay showed that no matter for group A or D, there was no difference of CR, TL, TD and OTM between pre-frozen and post-thawed spermatozoa. There was no difference of CR, TL, TD and OTM of post-thawed spermatozoa between group A and group D neither, while spermatozoa DNA damage was more serious in group B and group C with increasing height of micro-channel compared with group A. The proportion of intact acrosome of post-thawed spermatozoa in group A was the highest when compared with group B and group C, though similar to that of group D. In conclusion, PDMS chip with 10 µm height micro-channel is ideal for ultra-rapid cryopreservation of small quantity of spermatozoa without cryoprotectant.     

Pharmacological approaches to counter the toxicity of dopa

Summary Dopa and related catecholamines and their degradation products have been demonstrated to have neurotoxic potential in a number of cellular andin vivo experiments. Several mechanisms have been hypothesized to be involved including generation of prooxidant products that subsequently oxidize membrane lipids and exposed macromolecules. We have utilized a neuronal culture of cerebellar granule cells to study the toxicity of Dopa and the ability of various neuroprotective and antiparkinsonian compounds to offer protection therefrom. This model is apparently based on the ability of Dopa to non-enzymatically induce an oxidative injury to the neuronal cultures. Evidence for this arises from the equal neurotoxic potency of L- and D-Dopa in these cells and the ability of catalase, superoxide dismutase and glutathione to protect the neurons from this toxicity. Further, we found that the neuroprotective antioxidant, PNU-101033 is more effective and potent than vitamin E and deprenyl in this regard. Similarly the D2/D3 agonist, pramipexole is also capable of blocking Dopa toxicity in this model and this effect is independent of dopamine receptor affinity as both enantiomers are equally potent in this assay but disparate in receptor affinity. Also the protection by pramipexole is accompanied by the preservation of reduced glutathione. Thus, this activity seems to be a function of the oxidation potential of pramipexole and it's consequent antioxidant property. Potent antioxidants are effective blockers of Dopa toxicity. If the mechanisms involved in this toxicity have relevance to the progression of Parkinson's pathology in Dopa treated (or untreated) patients, these compounds have the potential to alter the course of the illness.     

E93K charge reversal on actin perturbs steric regulation of thin filaments

Contraction in striated muscles is regulated by Ca2+-dependent movement of tropomyosin-troponin on thin filaments. Interactions of charged amino acid residues between the surfaces of tropomyosin and actin are believed to play an integral role in this steric mechanism by influencing the position of tropomyosin on the filaments. To investigate this possibility further, thin filaments were isolated from troponin-regulated, indirect flight muscles of Drosophila mutants that express actin with an amino acid charge reversal at residue 93 located at the interface between actin subdomains 1 and 2, in which a lysine residue is substituted for a glutamic acid. Electron microscopy and 3D helical reconstruction were employed to evaluate the structural effects of the mutation. In the absence of Ca2+, tropomyosin was in a position that blocked the myosin-binding sites on actin, as previously found with wild-type filaments. However, in the presence of Ca2+, tropomyosin position in the mutant filaments was much more variable than in the wild-type ones. In most cases (approximately 60%), tropomyosin remained in the blocking position despite the presence of Ca2+, failing to undergo a normal Ca2+-induced change in position. Thus, switching of a negative to a positive charge at position 93 on actin may stabilize negatively charged tropomyosin in the Ca2+-free state regardless of Ca2+ levels, an alteration that, in turn, is likely to interfere with steric regulation and consequently muscle activation. These results highlight the importance of actin's surface charges in determining the distribution of tropomyosin positions on thin filaments derived from troponin-regulated striated muscles.     

Central nervous system toxicity of interferons and other cytokines

Prolonged administration of interferons, interleukins and tumor necrosis factor are accompanied by a range of toxic effects of which central nervous system toxicity may be an important dose-limiting factor. While symptoms are widely reported, practically nothing is known about mechanisms of action. This review attempts to distinguish between a direct effect of cytokines upon circumventricular organs and an indirect effect mediated by factors released by endothelial-glial cells of the blood-brain barrier normally impermeable to cytokines. In order to reduce the toxicity of biological response modifiers the definition of the minimum effective dose, the use of the lymphatic route and the observance of the chronobiological rules may help to improve the therapeutic index of these hormone-like compounds. It appears however, that the relationship between cytokine: dose: route: schedule: timing on one side and efficacy: toxicity on the other is complex, and so far no general rule has clearly emerged so that at the moment it appears necessary to find out the optimal therapeutic index for each particular disease.     

Inhibition of bacterial oxidases by formamide and analogs

The enzymatic activity of Paracoccus denitrificans cytochrome c oxidase (COX) and Escherichia coli cytochrome b(o) ubiquinol oxidase (QOX) was determined in the presence of formamide, N,N-dimethyl formamide and N,N-dimethyl acetamide. Formamide was found to inhibit the enzyme activity of the oxidases most significantly, whereas the other two compounds inhibited the activity to a lesser extent. The effects of formamide and analogs on enzyme activity were very similar for COX and QOX, indicating that the mechanism of inhibition might be the same for both of these oxidases. The inhibition kinetics followed a non-competitive mechanism. Studies using proteoliposomes of COX and QOX containing the electron entry site of the enzyme directed towards the outside of the vesicles showed that the effect of inhibition by formamide was higher when the inhibitor was present on the outside of the proteoliposome compared to when it was present only in the aqueous core. This indicates that inhibition of enzyme activity by formamide possibly predominantly involves blocking of the water exit pathway in the oxidases.     

Site-directed mutagenesis and structural studies suggest that the germination protease, GPR, in spores of Bacillus species is an atypical aspartic acid protease

Germination protease (GPR) initiates the degradation of small, acid-soluble spore proteins (SASP) during germination of spores of Bacillus and Clostridium species. The GPR amino acid sequence is not homologous to members of the major protease families, and previous work has not identified residues involved in GPR catalysis. The current work has focused on identifying catalytically essential amino acids by mutagenesis of Bacillus megaterium gpr. A residue was selected for alteration if it (i) was conserved among spore-forming bacteria, (ii) was a potential nucleophile, and (iii) had not been ruled out as inessential for catalysis. GPR variants were overexpressed in Escherichia coli, and the active form (P41) was assayed for activity against SASP and the zymogen form (P46) was assayed for the ability to autoprocess to P41. Variants inactive against SASP and unable to autoprocess were analyzed by circular dichroism spectroscopy and multi-angle laser light scattering to determine whether the variant's inactivity was due to loss of secondary or quaternary structure, respectively. Variation of D127 and D193, but no other residues, resulted in inactive P46 and P41, while variants of each form were well structured and tetrameric, suggesting that D127 and D193 are essential for activity and autoprocessing. Mapping these two aspartate residues and a highly conserved lysine onto the B. megaterium P46 crystal structure revealed a striking similarity to the catalytic residues and propeptide lysine of aspartic acid proteases. These data indicate that GPR is an atypical aspartic acid protease.     

Reduction of cryoprotectant toxicity in cells in suspension by use of a sodium-free vehicle solution

Propane-1,2-diol (PD) possesses physico-chemical properties that make it a useful biological vitrifying agent but, although of relatively low toxicity, it still has substantial damaging effects on cells. This study aimed to identify possible toxic mechanisms using primary cell cultures from vascular tissue: these were exposed to the cryoprotectant at room temperature to avoid any possibility of hypothermic injury. Toxicity was evaluated by measuring the ability of the cells to divide in culture after exposure to the cryoprotectant. A variety of interventions, which addressed either possible consequences of PD exposure, or known mediators of other types of cell injury, were utilized in an attempt to inhibit PD toxicity. Some comparative studies with dimethyl sulphoxide (Me₂SO) exposure were also made.Replacing sodium in the vehicle solution with choline was the only intervention that reduced PD toxicity. It did so both in smooth muscle cells, where the loss of functional capacity was reduced from 56% to 13%, and in endothelial cells. where the reduction was from 40% to18%. Similar observations were also made in smooth muscle cells exposed to Me₂SO. We failed to find evidence for a role of pH regulation, for oxidative injury and/or an involvement of redox-active iron as a mediator of the injury. The results strongly suggest that the influx of sodium into the cell provides one mechanism whereby both PD and Me₂SO exert their toxic effects. We suggest that the use of choline-based vehicle solutions in cryopreservation would be beneficial.     

Pharmacological tuning of heat shock protein 70 modulates polyglutamine toxicity and aggregation

Nine neurodegenerative disorders are caused by the abnormal expansion of polyglutamine (polyQ) regions within distinct proteins. Genetic and biochemical evidence has documented that the molecular chaperone, heat shock protein 70 (Hsp70), modulates polyQ toxicity and aggregation, yet it remains unclear how Hsp70 might be used as a potential therapeutic target in polyQ-related diseases. We have utilized a pair of membrane-permeable compounds that tune the activity of Hsp70 by either stimulating or by inhibiting its ATPase functions. Using these two pharmacological agents in both yeast and PC12 cell models of polyQ aggregation and toxicity, we were surprised to find that stimulating Hsp70 solubilized polyQ conformers and simultaneously exacerbated polyQ-mediated toxicity. By contrast, inhibiting Hsp70 ATPase activity protected against polyQ toxicity and promoted aggregation. These findings clarify the role of Hsp70 as a possible drug target in polyQ disorders and suggest that Hsp70 uses ATP hydrolysis to help partition polyQ proteins into structures with varying levels of proteotoxicity. Our results thus support an emerging concept in which certain kinds of polyQ aggregates may be protective, while more soluble polyQ species are toxic.