Osmotic Environment Research Articles

Chimeras Reveal a Single Lipid-Interface Residue that Controls MscL Channel Kinetics as well as Mechanosensitivity

MscL, the highly conserved bacterial mechanosensitive channel of large conductance, serves as an osmotic "emergency release valve," is among the best-studied mechanosensors, and is a paradigm of how a channel senses and responds to membrane tension. Although all homologs tested thus far encode channel activity, many show functional differences. We tested Escherichia coli and Staphylococcus aureus chimeras and found that the periplasmic region of the protein, particularly E.coli I49 and the equivalent S.aureus F47 at the periplasmic lipid-aqueous interface of the first transmembrane domain, drastically influences both the open dwell time and the threshold of channel opening. One mutant shows a severe hysteresis, confirming the importance of this residue in determining the energy barriers for channel gating. We propose that this site acts similarly to a spring for a clasp knife, adjusting the resistance for obtaining and stabilizing an open or closed channel structure.

Proterometra macrostoma(Trematoda: Azygiidae): Location of the Redia and Emergence Path from the Snail,Elimia semicarinata(Gastropoda: Pleuroceridae)

The objectives of this study were to describe (1) the osmotic environment and precise location of the Proterometra macrostoma redia in its snail intermediate host, (2) where retraction of the distome body into the cercarial tail occurs, and (3) the subsequent emergence path of the cercaria out of the snail. Snails, Elimia semicarinata , were collected from North Elkhorn Creek in Scott County, Kentucky and screened daily for patent infections. Live rediae were extracted from infected snails in either artificial pond water (APW) or artificial snail water (ASW) and monitored for changes in morphology and movement every hour over 5 hr at 22 C. Infected and control snails were simultaneously fixed and decalcified in Cal-Ex II, prepared for routine paraffin sectioning, and serial sections subsequently analyzed for rediae and cercariae location. Significantly (χ(2) = 42.45; 1 df; P = 0.0001) more rediae showed movement in ASW than in APW after 5 hr, suggesting a host compartment separate from the mantle cavity. Histological sections clearly showed rediae developing in close association with the snail digestive tract, within the peri-intestinal sinus of the snail, and isolated from the mantle cavity by a mantle membrane. Retraction of the distome body into the cercarial tail follows the emergence of the cercaria from the redia. Cercariae then enter the mantle cavity and emerge into fresh water through a siphon-like structure formed by the mantle collar of the snail.

Molecular Cloning and Evolutionary Analysis of the HOG-Signaling Pathway Genes from Saccharomyces cerevisiae Rice Wine Isolates

The high osmolarity glycerol (HOG) signaling pathway is crucial for yeast to cope with high osmolarity. Here, we showed that Saccharomyces cerevisiae rice wine isolates exhibited higher tolerance to osmotic stress, which was associated with the evolution of HOG pathway genes. Phylogenetic analysis of HOG genes revealed that Chinese rice wine strains were closely related to sake strains, indicating a common origin of rice wine strains. The DNA sequence diversity analysis showed that higher levels of polymorphism tended to accumulate on the osmosensor genes (MSB2 and SLN1), suggesting that most changes in a signaling transduction pathway were concentrated in the receptors. Moreover, the rapid evolution of osmosensors (Sln1/Msb2) and transcription factor (Msn4) might experience positive selection. Our results imply that the evolution of HOG pathway genes in S. cerevisiae rice wine strains is associated with their adaptation to high osmotic environments.

Text-mining network analysis of the response to osmotic stimuli in the intervertebral disc

Intervertebral disc cells experience a broad range of physical stimuli under physiologic conditions, including alterations in their osmotic environment. The purpose of this study was to construct a text-mining network of the genes induced during the response to osmotic stimuli in the intervertebral disc. We obtained a gene expression profile of human intervertebral disc cells from the National Center for Biotechnology Information, after culture under hyper- and hypo-osmotic conditions compared to iso-osmotic conditions, and we identified 65 differentially expressed genes of intervertebral disc cells. We constructed a text-mining network using Biblio-MetReS between the differentially expressed genes and other genes that were included in the same document as the differentially expressed genes. Then, we performed pathway-enrichment analysis to identify the most relevant pathways for the response to osmotic stimuli in intervertebral disc cells. Our data provide a comprehensive bioinformatics analysis of genes and pathways that may be involved in the response to osmotic stimuli in the intervertebral disc.

Nanomechanics measurements of live bacteria reveal a mechanism for bacterial cell protection: the polysaccharide capsule in Klebsiella is a responsive polymer hydrogel that adapts to osmotic stress

A global health problem concerns the evolution of antibiotic-resistant bacteria. A characteristic of these super bug pathogens is their ability to form an exopolysaccharide matrix, the capsule, allowing them to resist antibiotic treatment and other environmental stresses. Here we apply atomic force microscopy (AFM) to interrogate living bacterial cells and provide the first in situ measurements of the biophysical behaviour of the capsule in physiologically relevant conditions. We prepared live Klebsiella pneumoniae, a common pathogen that has evolved super bug strains. We measured how capsule, cell wall and cell cytoplasm of wild type and capsule-deficient bacterial cells respond when indented by an AFM tip, under extreme osmotic conditions. By developing a mechanical model for the indentation of bacteria and fitting the force profiles to this model, we discovered that the organisation and hydration of the capsule alter in response to the osmotic environment, but surprisingly there is no change in cytoplasmic turgor pressure. The capsule deficient mutants however, experience extreme change in turgor pressure under altered osmotic conditions. Our results provide new insight on the biophysical behaviour of the bacterial capsule, demonstrating that it is a polymer hydrogel that acts as an ion sponge to dampen the impact of osmotic stress. Nano-mechanical screening for additives to medical plastics that alter the properties of this ion sponge represents a new way to address the problems associated with antibiotic-resistant bacteria.

Tools to kill: genome of one of the most destructive plant pathogenic fungi Macrophomina phaseolina.

BackgroundMacrophomina phaseolina is one of the most destructive necrotrophic fungal pathogens that infect more than 500 plant species throughout the world. It can grow rapidly in infected plants and subsequently produces a large amount of sclerotia that plugs the vessels, resulting in wilting of the plant.ResultsWe sequenced and assembled ~49 Mb into 15 super-scaffolds covering 92.83% of the M. phaseolina genome. We predict 14,249 open reading frames (ORFs) of which 9,934 are validated by the transcriptome. This phytopathogen has an abundance of secreted oxidases, peroxidases, and hydrolytic enzymes for degrading cell wall polysaccharides and lignocelluloses to penetrate into the host tissue. To overcome the host plant defense response, M. phaseolina encodes a significant number of P450s, MFS type membrane transporters, glycosidases, transposases, and secondary metabolites in comparison to all sequenced ascomycete species. A strikingly distinct set of carbohydrate esterases (CE) are present in M. phaseolina, with the CE9 and CE10 families remarkably higher than any other fungi. The phenotypic microarray data indicates that M. phaseolina can adapt to a wide range of osmotic and pH environments. As a broad host range pathogen, M. phaseolina possesses a large number of pathogen-host interaction genes including those for adhesion, signal transduction, cell wall breakdown, purine biosynthesis, and potent mycotoxin patulin.ConclusionsThe M. phaseolina genome provides a framework of the infection process at the cytological and molecular level which uses a diverse arsenal of enzymatic and toxin tools to destroy the host plants. Further understanding of the M. phaseolina genome-based plant-pathogen interactions will be instrumental in designing rational strategies for disease control, essential to ensuring global agricultural crop production and security.

Engineering of betaine biosynthesis and transport for abiotic stress tolerance in plants

Drought and salinity are the major factors that decrease crop yield. Organisms thriving in osmotic stress environments need adaptive mechanisms for adjusting their intracellular environment to external osmotic stress conditions. One such mechanism, to prevent water loss from the cells is to accumulate large amounts of low molecular weight organic compatible solutes such as proline, betaine and polyols to balance internal osmolarity of the cells. Accumulation of compatible solutes can be achieved by enhanced synthesis and/or reduced catabolism. Certain plants synthesize betaine in chloroplasts via a two-step oxidation of choline and betaine accumulation is associated with enhanced stress tolerance. Many important crop plants have low levels of betaine or none at all. Hence, betaine biosynthetic pathway is a target for metabolic engineering to enhance stress tolerance in crops. Introduction of betaine synthesis pathway into betaine non-accumulating plants has often improved stress tolerance. However, betaine levels of the engineered plants were generally low. To further enhance the betaine accumulation levels, we need to diagnose factors limitng betaine accumulation in engineered plants. Here we discuss recent progress on metabolic engineering of choline precursors for abiotic stress tolerance in plants.

Dissolution Profile of Novel Composite Pellet Cores Based on Different Ratios of Microcrystalline Cellulose and Isomalt

There is a growing interest towards the application of inert cores as starting materials for pharmaceutical pellet manufacturing. They serve as alternatives to develop and adapt a relatively simple manufacturing technology compared with an extrusion/spheronisation process. The major objective of this study was to investigate the effect of the compositions of core materials on the drug release profile. Pure microcrystalline cellulose (MCC), isomalt and different types of novel composite MCC-isomalt cores were layered with model drug (sodium diclofenac) and were coated with acrylic polymer. The effect of the osmolality in the gastrointestinal tract was simulated using glucose as osmotically active agent during in vitro dissolution tests. The results demonstrated the dependence of drug dissolution profile on the ratio of MCC and isomalt in the core and the influence of osmotic properties of the dissolution medium. Isomalt used in the composite core was able to decrease the vulnerability of the dissolution kinetics to the changes in the osmotic environment.

Comparative Analysis on the Key Enzymes of the Glycerol Cycle Metabolic Pathway in Dunaliella salina under Osmotic Stresses

The glycerol metabolic pathway is a special cycle way; glycerol-3-phosphate dehydrogenase (G3pdh), glycerol-3-phosphate phosphatase (G3pp), dihydroxyacetone reductase (Dhar), and dihydroxyacetone kinase (Dhak) are the key enzymes around the pathway. Glycerol is an important osmolyte for Dunaliella salina to resist osmotic stress. In this study, comparative activities of the four enzymes in D. salina and their activity changes under various salt stresses were investigated, from which glycerol metabolic flow direction in the glycerol metabolic pathway was estimated. Results showed that the salinity changes had different effects on the enzymes activities. NaCl could stimulate the activities of all the four enzymes in various degrees when D. salina was grown under continuous salt stress. When treated by hyperosmotic or hypoosmotic shock, only the activity of G3pdh in D. salina was significantly stimulated. It was speculated that, under osmotic stresses, the emergency response of the cycle pathway in D. salina was driven by G3pdh via its response to the osmotic stress. Subsequently, with the changes of salinity, other three enzymes started to respond to osmotic stress. Dhar played a role of balancing the cycle metabolic pathway by its forward and backward reactions. Through synergy, the four enzymes worked together for the effective flow of the cycle metabolic pathways to maintain the glycerol requirements of cells in order to adapt to osmotic stress environments.

Overexpression of mltA in Edwardsiella tarda reduces resistance to antibiotics and enhances lethality in zebra fish

The aim of this study was to investigate the role of membrane-bound lytic murein transglycosylase A (MltA) in a bacterial fish pathogen Edwardsiella tarda. An mltA in-frame deletion mutant (ΔmltA) and an mltA overexpression strain (mltA(+)) of Edw. tarda were constructed through double-crossover allelic exchange and by transformation of a low-copy plasmid carrying the intact mltA into the ΔmltA mutant, respectively. Either inactivation or overexpression of MltA in Edw. tarda resulted in elevated sensitivity to β-lactam antibiotics and lower viability in oligotrophic or high osmotic environment than wild-type strain. Autolysis induced by EDTA was reduced in ΔmltA strain, while mltA(+) strain was virtually flimsy, indicating that MltA is responsible for the lysis effect. Moreover, mltA(+) strain exhibited significant increases in lipopolysaccharide (LPS) biosynthesis and virulence to zebra fish compared with wild-type strain. The results indicated that MltA plays essential roles in β-lactam antibiotics and environmental stresses resistance, autolysis, LPS biosynthesis and pathogenicity of Edw. tarda. This is the first report that MltA has a virulence-related function in Edw. tarda. This study provided useful information for further studies on pathogenesis of Edw. tarda.

Three Routes To Modulate the Pore Size of the MscL Channel/Nanovalve

MscL is a bacterial mechanosensitive channel that protects cells from lysis upon acute decrease in external osmotic environment. It is one of the best characterized mechanosensors known, thus serving as a paradigm of how such molecules sense and respond to stimuli. In addition, the fact that it can be genetically modified, expressed, isolated, and manipulated has led to its proposed use as a triggered nanovalve for various functions including sensors within microelectronic array chips, as well as vesicular-based targeted drug release. X-ray crystallography reveals a homopentameric complex with each subunit containing two transmembrane α-helices (TM1 and TM2) and a single carboxyl terminal α-helix arranging within the complex to form a 5-fold cytoplasmic bundle (CB), whose function and stability remain unclear. In this study, we show three routes that throttle the open channel conductance. When the linker between the TM2 and CB domain is shortened by deletions or constrained by either cross-linking or heavy metal coordination, the conductance of the channel is reduced; in the later two cases, even reversibly. While they have implications for the stability of the CB, these data also provide routes for engineering MscL sensors that are more versatile for potential nanotech devices.

Small molecule-enrichment analysis in response to osmotic stimuli in the intervertebral disc

The intervertebral disc (IVD) is a heterogeneous structure that contributes to load support and flexibility in the spine. IVD cells experience a broad range of physical stimuli under physiological conditions, including alterations in their osmotic environment. To date, the molecular mechanisms regulating the response of IVD to osmotic pressure are still not well understood. We obtained the gene expression profile of human IVD cells from NCBI and looked for potential therapeutic drug candidates. Based on microarray data, we concluded that RAP2A and GNA13 appear to have a role in response to osmotic stimuli in intervertebral discs. Using a computational bioinformatics method, we determined that thioridazine has potential as a therapeutic drug candidate for regulating osmotic pressure changes in IVD cells. We anticipate that our results will be used to generate hypotheses for laboratory, patient, and population-based studies.

Contribution of the Epithelial Sodium Channel to Chondrocyte Regulatory Volume Increase

Chondrocytes are the cells of articular cartilage, responsible for the production and maintenance of the extracellular milieu. They exist in a constantly changing osmotic environment and to survive such osmotic changes they must be able to quickly and effectively regulate their volume. Cells use regulatory volume increase (RVI) to oppose osmotic shrinkage. It has previously been shown that the epithelial sodium channel (ENaC) is important to this process in rat hepatocytes1. Here we investigate the possible contribution of ENaC to RVI mechanisms in canine chondrocytes.Chondrocytes were isolated from cartilage according to standard methods2. Amiloride sensitive single-channel activity reversed at a membrane potential of −1±5mV (n = 5), mean conductance was 9±0.4pS (n = 5) and kinetics were slow. The calculated ENa under these conditions was −6mV, which coupled to the very small conductance would be consistent with this channel being an ENaC. Channel open probability in control conditions was 0.3±0.06 and decreased by 97±2% after application of the ENaC inhibitor, amiloride (10μM; n = 3).Upon exposure to hypertonic solution, cell volume decreased significantly by 35±3% (n = 5; p<0.001). Within 20 minutes of reaching their smallest size, cells under control conditions had returned to 92±4% of their original volume, not significantly different to starting volume (p=0.07). When 100nM benzamil, a specific ENaC inhibitor, was added to the hypertonic solution, cells shrank by 41±3% (n = 5) and were unable to return to their original volume.These data suggest that ENaC contributes to RVI in canine chondrocytes.1. F. Wehner, C. Bohmer, H. Heinzinger et al., Cellular Physiology and Biochemistry 10 (5-6), 335 (2000).2. R. Lewis, K. Asplin, G. Bruce et al., J Cell Physiol 226 (8) (2011).

Effect of periodic fluctuation in the osmotic environment on the adaptation of Salmonella

The growth of Salmonella from different osmotic histories was studied in low water activity conditions. Cell cultures were successively diluted and grown in batch, in minimal medium, without and then with added NaCl, several times and from different inoculum levels. The viable count curves obtained in low water activity conditions showed an initial decline after which the culture either died out or recovered and entered the exponential phase. After the first batch with NaCl added, the subsequent curves at low water activity showed progressively smaller initial decline and shorter lag time as the number of transfers from high to low water activity conditions increased. The observed curves were analyzed by F-tests applying an extension of the model of Baranyi and Roberts (1994). The results suggest that periodic, systematic “training” can improve the adaptation capability of the organism without resulting in higher specific growth rate.

Isolation and characterization of a novel peroxisomal choline monooxygenase in barley

Glycine betaine (GB) is a compatible solute accumulated by many plants under various abiotic stresses. GB is synthesized in two steps, choline→betaine aldehyde→GB, where a functional choline-oxidizing enzyme has only been reported in Amaranthaceae (a chloroplastic ferredoxin-dependent choline monooxygenase) thus far. Here, we have cloned a cDNA encoding a choline monooxygenase (CMO) from barley (Hordeum vulgare) plants, HvCMO. In barley plants under non-stress condition, GB had accumulated in all the determined organs (leaves, internodes, awn and floret proper), mostly in the leaves. The expression of HvCMO protein was abundant in the leaves, whereas the expression of betaine aldehyde dehydrogenase (BADH) protein was abundant in the awn, floret proper and the youngest internode than in the leaves. The accumulation of HvCMO mRNA was increased by high osmotic and low-temperature environments. Also, the expression of HvCMO protein was increased by the presence of high NaCl. Immunofluorescent labeling of HvCMO protein and subcellular fractionation analysis showed that HvCMO protein was localized to peroxisomes. [(14)C]choline was oxidized to betaine aldehyde and GB in spinach (Spinacia oleracea) chloroplasts but not in barley, which indicates that the subcellular localization of choline-oxidizing enzyme is different between two plant species. We investigated the choline-oxidizing reaction using recombinant HvCMO protein expressed in yeast (Saccharomyces cerevisiae). The crude extract of HvCMO-expressing yeast coupled with recombinant BBD2 protein converted [(14)C]choline to GB when NADPH was added as a cofactor. These results suggest that choline oxidation in GB synthesis is mediated by a peroxisomal NADPH-dependent choline monooxygenase in barley plants.

Comparison of osmotic swelling influences on meniscal fibrocartilage and articular cartilage tissue mechanics in compression and shear

Although the contribution of the circumferential collagen bundles to the anisotropic tensile stiffness of meniscal tissue has been well described, the implications of interactions between tissue components for other mechanical properties have not been as widely examined. This study compared the effects of the proteoglycan-associated osmotic swelling stress on meniscal fibrocartilage and articular cartilage (AC) mechanics by manipulating the osmotic environment and tissue compressive offset. Cylindrical samples were obtained from the menisci and AC of bovine stifles, equilibrated in phosphate-buffered saline solutions ranging from 0.1× to 10×, and tested in oscillatory torsional shear and unconfined compression. Biochemical analysis indicated that treatments and testing did not substantially alter tissue composition. Mechanical testing revealed tissue-specific responses to both increasing compressive offset and decreasing bath salinity. Most notably, reduced salinity dramatically increased the shear modulus of both axially and circumferentially oriented meniscal tissue explants to a much greater extent than for cartilage samples. Combined with previous studies, these findings suggest that meniscal proteoglycans have a distinct structural role, stabilizing, and stiffening the matrix surrounding the primary circumferential collagen bundles.

Involvement of redox- and phosphorylation-dependent pathways in osmotic adaptation in sperm cells of euryhaline tilapia

Sperm cells involved in fertilisation must tolerate hypo-osmotic and hyper-osmotic environments. Euryhaline tilapia (Oreochromis mossambicus) can acclimatise to and reproduce in freshwater and seawater because its sperm are able to adapt to these differing osmotic environments. In this study, we found that the dephosphorylation of sperm proteins in O. mossambicus correlated with the activation of flagellar motility when sperm were exposed to hypotonic or hypertonic conditions, and that differences in phosphorylation may reflect adaptations to a given osmotic environment. Of the sperm proteins that were dephosphorylated, the phosphorylation pattern of an 18 kDa protein, identified as the superoxide anion scavenger Cu/Zn superoxide dismutase (Cu/Zn SOD), was different in freshwater- and seawater-acclimatised tilapia sperm. Cu/Zn SOD was distributed from the sperm head to the flagellum. Additionally, differences were observed between freshwater and seawater tilapia in the nitration of tyrosine residues (which might be mediated by SOD) in sperm flagellar proteins in response to osmotic shock. These results demonstrate that reactive-oxygen-species-dependent mechanisms contribute to both osmotic tolerance and the activation of flagellar motility.

Chondroitinase ABC Reduces Brain Tissue SwellingIn Vitro

Increased intracranial pressure (ICP) caused by edema following severe traumatic brain injury (TBI) or stroke contributes to high rates of mortality and morbidity. The search continues for more effective treatments that target the edema that contributes to increased ICP. We previously described the effect of the fixed charge density (FCD) of brain on its swelling behavior according to the Donnan effect. Here we show that reduction of brain tissue FCD is an effective means of reducing brain tissue swelling and edema in rat and porcine cortical brain tissue in vitro. The effect of enzymes directed at digesting candidate contributors to cellular FCD such as chondroitin sulfate proteoglycans (CSPGs), heparin sulfate proteoglycans (HSPGs), and DNA was examined in slices of the adult rat cortex. All enzymes were capable of decreasing FCD in the tissue by ?20%, and reducing tissue swelling over a 24?h period following dissection from ?60% to ?30%. Chondroitinase ABC (ChABC) was most effective at reducing dead brain tissue swelling in response to changes in ionic osmotic environments. ChABC reduced swelling in live slices of tissue even within the first 2?h following dissection. It also significantly reduced the FCD, initial tissue swelling, and volume change in response to hypotonic bathing solution in porcine cortical brain tissue. The use of ChABC to reduce tissue FCD may be an effective method for reducing brain edema and controlling ICP following injury.

Post-transcriptional gene regulation following exposure of osteoarthritic human articular chondrocytes to hyperosmotic conditions

Osmolarity is a major biophysical regulator of chondrocyte function. Modulation of chondrocytic marker gene expression occurs at the post-transcriptional level following exposure of human articular chondrocytes (HAC) to hyperosmotic conditions. This study aims to further characterise the post-transcriptional response of HAC to hyperosmolarity. Gene expression and microRNA (miRNA) levels in freshly isolated HAC after 5h under control or hyperosmotic conditions were measured using microarrays. Regulated genes were checked for the presence of AU rich elements (AREs) in their 3' untranslated regions (3'UTR), whilst gene ontology was examined using Ingenuity Pathway Analysis (IPA). RNA decay rates of candidate ARE-containing genes were determined in HAC using actinomycin D chase experiments and the involvement of the p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathways were investigated using pharmacological inhibitors. Hyperosmolarity led to the regulation of a wide variety of genes. IPA identified enrichment of genes involved with cell stress responses, cell signalling and transforming growth factor β (TGFβ) signalling. Importantly, upregulated genes were over-represented with those containing AREs, and RNA decay analysis demonstrated that many of these were regulated post-transcriptionally by hyperosmolarity in HAC. Analysis of miRNA levels in HAC indicated that they are only modestly regulated by hyperosmotic conditions, whilst inhibitor studies showed that p38 MAPK and ERK1/2 were able to block hyperosmotic induction of many of these genes. Through microarray and bioinformatics analysis we have identified genes which are post-transcriptionally regulated in HAC following exposure to hyperosmotic conditions. These genes have a range of functions, and their regulation involves transduction through the p38 MAPK and ERK1/2 pathways. Interestingly, our results suggest that miRNA regulation is not key to the process. Overall, this work illustrates the range of processes regulated in chondrocytes by changes in their osmotic environment, and underlines the importance of post-transcriptional mRNA regulation to chondrocyte function.

Articular cartilage surface rupture during compression: Investigating the effects of tissue hydration in relation to matrix health

This study aimed at investigating articular cartilage rupture by investigating the response of healthy and degenerate cartilage through altering the osmotic swelling environment of surface-intact, cartilage-on-bone specimens. The osmotic environment in healthy and degenerate bovine cartilage was varied by soaking tissues in either distilled water or 1.5 M NaCl saline to render the tissues into a swollen or dehydrated state (respectively). Creep compression was applied using an 8 mm flat-ended polished indenter that contained a central pore of 450 μm diameter, providing a consistent region for rupture to occur across all specimens. In the first set of experiments, surface rupture of healthy and degenerate specimens required similar levels of nominal compressive stress (8 MPa) when dehydrated than when swollen (7 MPa). In the second set of experiments, the time required for surface rupture to occur (for healthy and degenerate specimens) occurred over similar loading times ( p > 0.05 ). However, the time required for surface rupture for the swollen specimens occurred over a significantly longer time (approximately one order of magnitude) than that required for the dehydrated specimens ( p < 0.05 ). The compressive strains that were measured at rupture in the dehydrated degenerate specimens were significantly lower than those measured in the dehydrated healthy tissues ( p < 0.05 ). Rupture in dehydrated degenerate cartilage suggested a weakened articular surface, and it also suggested that dehydrated cartilage may undergo failure due to stress concentrations as it is unable to redistribute stress away from the site of loading.