Non-immortalized human tenocyte cultures as a vehicle for understanding cellular aspects to tendinopathy.

The biochemical mechanisms underlying tendinopathy are obscure. We briefly describe preliminary observations of human tenocyte behaviour in culture as a vehicle for determining the role of reactive oxygen in tendon pathology.

Phenotypic drift in human tenocyte culture.

Tendon ruptures are increasingly common, repair can be difficult, and healing is poorly understood. Tissue engineering approaches often require expansion of cell numbers to populate a construct, and maintenance of cell phenotype is essential for tissue regeneration. Here, we characterize the phenotype of human Achilles tenocytes and assess how this is affected by passaging. Tenocytes, isolated from tendon samples from 6 patients receiving surgery for rupture of the Achilles tendon, were passaged 8 times. Proliferation rates and cell morphology were recorded at passages 1, 4, and 8. Total collagen, the ratio of collagen types I and III, and decorin were used as indicators of matrix formation, and expression of the integrin beta1 subunit as a marker of cell-matrix interactions. With increasing passage number, cells became more rounded, were more widely spaced at confluence, and confluent cell density declined from 18,700/cm2 to 16,100/cm2 ( p = 0.009). No change to total cell layer collagen was observed but the ratio of type III to type I collagen increased from 0.60 at passage 1 to 0.89 at passage 8 ( p < 0.001). Decorin expression significantly decreased with passage number, from 22.9 +/- 3.1 ng/ng of DNA at passage 1, to 9.1 +/- 1.8 ng/ng of DNA at passage 8 ( p < 0.001). Integrin expression did not change. We conclude that the phenotype of tenocytes in culture rapidly drifts with progressive passage.

The biological activities of new polyamine derivatives as potential therapeutic agents.

A number of polyamine derivatives have demonstrated potential as therapeutic agents. For example, 1,12-bisethylspermine and bisnaphthalimide (elinafide) are currently in phase I clinical trials for the treatment of certain cancers. Here, the biological activities of two new groups of polyamine derivative, namely the oxa-polyamines and the bisnaphthalimides, are presented. The most active compounds in the oxa-polyamine and bisnaphthalimido series possessed IC(50) values of 2.93 and 1.38 microM, respectively, against MCF7 cells after 48 h of exposure. The structure-relationship activities of each group of compounds are discussed. Bisnaphthalimido compounds are DNA-binding agents. Addition of the bisnaphthalimides PK3, PK4, PK5, PK6 and PK7, at a concentration of 10 microM, to the calf thymus DNA duplex increased the T (m) of DNA by 11.55+/-0.56, 14.545+/-1.59, 6.23+/-2.45, 12.56+/-1.84 and 16.45+/-0.39 degrees C respectively. With the exception of PK5, all compounds bind to DNA by intercalation as judged by effect of compounds on DNA mobility. Ethidium bromide displacement assay showed that all the compounds have significant affinity for calf thymus DNA (the drug concentration required to reduce the fluorescence of initially DNA-bound ethidium bromide by 50%, C(50), was 1.21-17.33 microM). The order of DNA-binding strength was PK4 > PK3 > PK7 > PK6 > PK5. In HL-60 promyelocytic leukaemia cells, oxa-polyamine and bisnaphthalimido treatment resulted in a decline in cell proliferation and viability. The assays performed suggested that apoptosis was not the principal cell death mechanism involved in oxa-polyamine cytotoxicity. In contrast, HL-60 cell death induced by the bisnaphthalimido series was characterized by early exposure of phosphatidylserine exclusive from membrane damage, elevated caspase-3 activity, increased DNA instability and, ultimately, DNA fragmentation. Thus the principal cytotoxic members of the bisnaphthalimido series appear to induce apoptosis.

Quercetin modifies reactive oxygen levels but exerts only partial protection against oxidative stress within HL-60 cells.

Quercetin may contribute to the protection afforded by fruit- and vegetable-rich diets against diseases for which excess production of reactive oxygen species (ROS) has been implicated as a causal or contributory factor. We examine the effect of short term (90 min) quercetin (1-100 microM) exposure on the progress of menadione induced oxidative stress within HL-60 cells. 2',7'-dichlorofluorescein and rhodamine-123 fluorescence, resulting from oxidation of the ROS-sensitive dyes dichlorodihydrofluorescein and dihydrorhodamine-123 respectively, were utilised as indicators of general ROS levels. Ethidium fluorescence, resulting from oxidation of dihydroethidium, was used as a potentially more specific indicator of O(2)(-). Exposure to quercetin alone induced a decrease in DCF and rhodamine fluorescence. Conversely, ethidium fluorescence was enhanced by treatment with >or=40 microM quercetin. Incubation with 1-100 microM quercetin reduced the extent of menadione-induced increase in DCF and rhodamine fluorescence but the menadione-induced increase in ethidium fluorescence was further elevated for cells treated with >or=25 microM quercetin. Exposure to >or=10 microM quercetin abrogated menadione-induced DNA single-strand breaks but, paradoxically, quercetin exacerbated membrane damage and failed to enhance the viability of menadione-challenged cells. In conclusion, quercetin exerts only site-specific protection against oxidative stress.

Fatty acid regulation of protein kinase C isoforms in prostate cancer cells.

Polyunsaturated fatty acids influence the aetiology of prostate cancer. Their effects on cellular mechanisms regulating prostate tumorigenesis are unclear. Using prostate cancer cells (LNCaP), we determined effects of n-9-OA, n-6-LA, and n-3-EPA on total PKC and its isoforms in relation to cell proliferation and PSA production. PKC-alpha, delta, gamma, iota, mu, and zeta were present in LNCaP cells; PKC-beta, epsilon, eta, and theta isoforms were not. PKC-alpha was detected only in cytosol; PKC-delta, iota, gamma, and mu were present in cytosol and in membranes. Fatty acids increased cell proliferation, total PKC activity and elicited pro-proliferative effects on specific PKC isoforms (PKC-delta and -iota). EPA and LA increased total PKC activity and reduced membrane-abundance of PKC-delta. OA reduced cytosolic and membrane PKC-delta. Only EPA reduced PKC-gamma membrane abundance. Fatty acids enhanced cytosolic PKC-iota abundance but only EPA and to a lesser extent LA increased its membrane content. Changes in PKC-delta, -iota, and -gamma did not affect PSA production.

Alteration of culture regime modifies antioxidant defenses independent of intracellular reactive oxygen levels and resistance to severe oxidative stress within confluent Caco-2 "intestinal cells".

Reactive oxygen species are implicated in the development of gastrointestinal pathologies. Caco-2 monolayers are routinely used to study intestinal oxidative stress and its potential amelioration by pharmacological agents or dietary micronutrients. Little is known of the plasticity of Caco-2 antioxidant defenses with changes in culture conditions. We examined whether the frequency of culture media renewal alters the antioxidant-prooxidant status and integrity of Caco-2 monolayers. In comparison to monolayers subject to daily media renewal, increasing periods between media exchange resulted in varying degrees of suppression of catalase, glutathione reductase, and glutathione-S-transferase activity. No significant changes to superoxide dismutase activity, total glutathione, or intracellular ROS profiles were observed. Alkaline phosphatase activity, as a marker of differentiation, and mean monolayer cell population size were also unaffected. We suggest that Caco-2 antioxidant enzyme activities are differentially sensitive to changes in culture conditions. Studies employing this cell line for antioxidant-oxidative stress interactions will need to evaluate responses with respect to culture regime.

A loss of resistance to avirulent bacterial pathogens in tobacco is associated with the attenuation of a salicylic acid-potentiated oxidative burst.

The role of salicylic acid (SA) in events occurring before cell death during the hypersensitive reaction (HR) was investigated in leaves of wild-type tobacco Samsun NN and in transgenic lines expressing salicylate hydroxylase (35S-SH-L). Challenge of 35S-SH-L tobacco with avirulent strains of Pseudomonas syringae gave rise to symptoms resembling those normally associated with a compatible response to virulent strains in terms of visible phenotype, kinetics of bacterial multiplication, and escape from the infection site. Compared with responses in wild-type tobacco, both the onset of plant cell death and the induction of an active oxygen species-responsive promoter (AoPR1-GUS) were delayed following challenge of 35S-SH-L plants with avirulent bacteria. The oxidative burst occurring after challenge with avirulent bacteria was visualized histochemically and quantified in situ. H2O2 accumulation at reaction sites was evident within 1 h after inoculation in wild-type tobacco, whereas in 35S-SH-L plants the onset of H2O2 accumulation was delayed by 2-3 h. The delay in H2O2 generation was correlated with a reduction in the transient rise in SA that usually occurred within 1-2 h following inoculation in wild-type plants. Our data indicate that an early transient rise in SA potentiates the oxidative burst, with resultant effects on accumulation of H2O2, plant cell death and also defence-gene induction, factors that together may determine the outcome of plant-pathogen interactions.

Effects of beta-carotene on antioxidant enzyme activity, intracellular reactive oxygen and membrane integrity within post confluent Caco-2 intestinal cells.

As encountered with a plethora of other natural products, the antioxidant activity of beta-carotene has been proposed as one of the mechanisms by which diets rich in this pro-vitamin A active carotenoid apparently afford chemoprevention. Here, we report the ability of beta-carotene to alter endogenous reactive oxygen levels and antioxidant defences within non-stressed 'differentiated' monolayers of an intestinal epithelial cell line (Caco-2) and to subsequently effect resistance to general oxidative insult. The differentiated monolayers efficiently absorbed beta-carotene. Between 3 and 8 days post confluence, cultures exhibited a progressive increase in antioxidant enzyme activity and a corresponding reduction to intracellular ROS levels. The profile for antioxidant enzyme activity was unaffected by sustained daily supplementation with beta-carotene. However, after two daily treatments with 50 microM beta-carotene intracellular ROS levels were significantly reduced and there was a trend towards reduced intracellular ROS within monolayers subject to five daily treatments with 0.5 and 5 microM beta-carotene. Prolonged supplementation with 0.1 and 0.5 microM beta-carotene or short supplementation periods with 5 and 50 microM beta-carotene did not alter susceptibility to H(2)O(2). However, cultures treated daily between 3 and 8 days post confluence with 5 or 50 microM beta-carotene exhibited enhanced LDH release, increased non-adherence and enhanced Trypan blue staining when challenged with 10 mM H(2)O(2). In the absence of H(2)O(2), the beta-carotene treatments were not overtly toxic to the monolayers. These results indicate that beta-carotene does not enhance antioxidant defences within Caco-2 monolayers. The enhancement of H(2)O(2) toxicity by persistent, high doses of beta-carotene may contribute to the failure of this carotenoid to protect high risk individuals from certain degenerative conditions.

Localization of hydrogen peroxide accumulation during the hypersensitive reaction of lettuce cells to Pseudomonas syringae pv phaseolicola.

The active oxygen species hydrogen peroxide (H2O2) was detected cytochemically by its reaction with cerium chloride to produce electron-dense deposits of cerium perhydroxides. In uninoculated lettuce leaves, H2O2 was typically present within the secondary thickened walls of xylem vessels. Inoculation with wild-type cells of Pseudomonas syringae pv phaseolicola caused a rapid hypersensitive reaction (HR) during which highly localized accumulation of H2O2 was found in plant cell walls adjacent to attached bacteria. Quantitative analysis indicated a prolonged burst of H2O2 occurring between 5 to 8 hr after inoculation in cells undergoing the HR during this example of non-host resistance. Cell wall alterations and papilla deposition, which occurred in response to both the wild-type strain and a nonpathogenic hrpD mutant, were not associated with intense staining for H2O2, unless the responding cell was undergoing the HR. Catalase treatment to decompose H2O2 almost entirely eliminated staining, but 3-amino-1,2,4-triazole (catalase inhibitor) did not affect the pattern of distribution of H2O2 detected. H2O2 production was reduced more by the inhibition of plant peroxidases (with potassium cyanide and sodium azide) than by inhibition of neutrophil-like NADPH oxidase (with diphenylene iodonium chloride). Results suggest that CeCl3 reacts with excess H2O2 that is not rapidly metabolized during cross-linking reactions occurring in cell walls; such an excess of H2O2 in the early stages of the plant-bacterium interaction was only produced during the HR. The highly localized accumulation of H2O2 is consistent with its direct role as an antimicrobial agent and as the cause of localized membrane damage at sites of bacterial attachment.

Hrp Mutant of Pseudomonas syringae pv phaseolicola Induces Cell Wall Alterations but Not Membrane Damage Leading to the Hypersensitive Reaction in Lettuce.

Both wild-type (S21-WT) and hrpD- (S21-533) strains of Pseudomonas syringae pv phaseolicola induced the formation of large paramural papillae in lettuce (Lactuca sativa) mesophyll cells adjacent to bacterial colonies. Localized alterations to the plant cell wall included deposition of hydroxyproline-rich glycoproteins, phe-nolics, and callose, and were associated with proliferation of the endoplasmic reticulum and multivesicular bodies. Tissue collapse during the hypersensitive reaction caused by S21-WT was associated with electrolyte leakage and rapid accumulation of the phy-toalexin lettucenin A, both of which followed membrane damage indicated by the failure of mesophyll cells to plasmolyze. A few cells lost the ability to plasmolyze after inoculation with S21-533, and low levels of lettucenin A were recorded, but neither leakage of electrolytes nor tissue collapse were detected. Dysfunction of the plasma membrane in cells adjacent to colonies of S21-WT led to extensive vacuolation of the cytoplasm, organelle disruption, and cytoplasmic collapse[mdash]changes unlike those occurring in cells undergoing apoptosis. Strain S21-533 remained viable within symptomless tissue, whereas cells of S21-WT were killed as a consequence of the hypersensitive reaction. Our observations emphasize the subtle coordination of the plant's response occurring at the subcellular level.

Factors affecting non-response to cervical cytology screening in Norfolk, England.

A geographic information system was used to integrate information on the uptake of cervical cytology screening for general medical practices in Norfolk with other data on the characteristics of the practices and the population they served. Regression analysis was employed to explore the extent to which variations in non-response were associated with explanatory factors. Non-response to the earlier system of opportunistic screening was found to be associated with the size of practice, the presence of a female doctor, the District Health Authority in which the practice was located, the age structure of the practice population, its degree of socio-economic deprivation and levels of rural remoteness. Compared with the earlier system, the new population-based call and recall system was found to be reaching a larger proportion of the population at risk. The former relationships were weaker under the new system, but non-response was still significantly associated with both social and organisational factors.