Increased production of 27-hydroxycholesterol in human colorectal cancer advanced stage: Possible contribution to cancer cell survival and infiltration.

So far, the investigation in cancer cell lines of the modulation of cancer growth and progression by oxysterols, in particular 27-hydroxycholesterol (27HC), has yielded controversial results. The primary aim of this study was the quantitative evaluation of possible changes in 27HC levels during the different steps of colorectal cancer (CRC) progression in humans. A consistent increase in this oxysterol in CRC mass compared to the tumor-adjacent tissue was indeed observed, but only in advanced stages of progression (TNM stage III), a phase in which cancer has spread to nearby sites. To investigate possible pro-tumor properties of 27HC, its effects were studied in vitro in differentiated CaCo-2 cells. Relatively high concentrations of this oxysterol markedly increased the release of pro-inflammatory interleukins 6 and 8, monocyte chemoattractant protein-1, vascular endothelial growth factor, as well as matrix metalloproteinases 2 and 9. The up-regulation of all these molecules, which are potentially able to favor cancer progression, appeared to be dependent upon a net stimulation of Akt signaling exerted by supra-physiological amounts of 27HC.

Exploring oxidative modifications of tyrosine: an update on mechanisms of formation, advances in analysis and biological consequences.

Protein oxidation is increasingly recognised as an important modulator of biochemical pathways controlling both physiological and pathological processes. While much attention has focused on cysteine modifications in reversible redox signalling, there is increasing evidence that other protein residues are oxidised in vivo with impact on cellular homeostasis and redox signalling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor the formation of oxidised derivatives, which depends on a variety of analytical techniques. While antibody-dependent techniques such as ELISAs are commonly used, these have limitations, and more specific assays based on spectroscopic or spectrometric techniques are required to provide information on the exact residues modified and the nature of the modification. These approaches have helped understanding of the consequences of tyrosine oxidation in biological systems, especially its effects on cell signalling and cell dysfunction, linking to roles in disease. There is mounting evidence that tyrosine oxidation processes are important in vivo and can contribute to cellular pathology.

Ophthalmic and molecular genetic findings in Kniest dysplasia.

PURPOSE: To study the variability of the ophthalmic phenotype in Kniest dysplasia. Kniest dysplasia is an inherited disorder associated with defects in type II collagen and characterised by short-trunked dwarfism, kyphoscoliosis, and enlarged joints with restricted mobility. Other features include marked hand arthropathy, cleft palate, hearing loss, and ocular abnormalities (myopia, abnormal vitreous, and high risk of developing retinal detachment). METHODS: Data from eight unrelated individuals with a clinical and molecular diagnosis of Kniest dysplasia are reported. Clinical assessment included an audiogram and ophthalmological examination in all but one patient who died in the immediate postnatal period. Sanger sequencing of the COL2A1 gene was performed. RESULTS: Six of the seven patients tested were high myopes with one patient being an emmetrope. Bilateral quandratic cataracts and subluxed lenses were noted in one subject. Variable but abnormal vitreous architecture was observed in all seven individuals tested. Six of the seven patients had significant hearing impairment and five of the seven patients exhibited clefting abnormalities. One patient had bilateral retinal detachments in his twenties. Six dominant disease-causing COL2A1 variants were detected. In three cases, testing of parental samples revealed that the disease-causing variant was not present in either parent. CONCLUSION: The ophthalmic features in Kniest dysplasia are very similar to those in other disorders of type II collagen such as Stickler syndrome. It is likely that different type II collagenopathies have a similar level of ocular morbidity and regular ophthalmologic examination is recommended. Kniest dysplasia is associated with heterozygous COL2A1 mutations that are frequently de novo.

Impairment of calcium ATPases by high glucose and potential pharmacological protection.

The review deals with impairment of Ca(2+)-ATPases by high glucose or its derivatives in vitro, as well as in human diabetes and experimental animal models. Acute increases in glucose level strongly correlate with oxidative stress. Dysfunction of Ca(2+)-ATPases in diabetic and in some cases even in nondiabetic conditions may result in nitration of and in irreversible modification of cysteine-674. Nonenyzmatic protein glycation might lead to alteration of Ca(2+)-ATPase structure and function contributing to Ca(2+) imbalance and thus may be involved in development of chronic complications of diabetes. The susceptibility to glycation is probably due to the relatively high percentage of lysine and arginine residues at the ATP binding and phosphorylation domains. Reversible glycation may develop into irreversible modifications (advanced glycation end products, AGEs). Sites of SERCA AGEs are depicted in this review. Finally, several mechanisms of prevention of Ca(2+)-pump glycation, and their advantages and disadvantages are discussed.

The in vivo susceptibility of Leishmania donovani to sodium stibogluconate is drug specific and can be reversed by inhibiting glutathione biosynthesis.

Resistance to pentavalent antimonial (Sb(v)) agents such as sodium stibogluconate (SSG) is creating a major problem in the treatment of visceral leishmaniasis. In the present study the in vivo susceptibilities of Leishmania donovani strains, typed as SSG resistant (strain 200011) or SSG sensitive (strain 200016) on the basis of their responses to a single SSG dose of 300 mg of Sb(v)/kg of body weight, to other antileishmanial drugs were determined. In addition, the role of glutathione in SSG resistance was investigated by determining the influence on SSG treatment of concomitant treatment with a nonionic surfactant vesicle formulation of buthionine sulfoximine (BSO), a specific inhibitor of the enzyme gamma-glutamylcysteine synthetase which is involved in glutathione biosynthesis, and SSG, on the efficacy of SSG treatment. L. donovani strains that were SSG resistant (strain 200011) and SSG sensitive (strain 200016) were equally susceptible to in vivo treatment with miltefosine, paromomycin and amphotericin B (Fungizone and AmBisome) formulations. Combined treatment with SSG and vesicular BSO significantly increased the in vivo efficacy of SSG against both the 200011 and the 200016 L. donovani strains. However, joint treatment that included high SSG doses was unexpectedly associated with toxicity. Measurement of glutathione levels in the spleens and livers of treated mice showed that the ability of the combined therapy to inhibit glutathione levels was also dependent on the SSG dose used and that the combined treatment exhibited organ-dependent effects. The SSG resistance exhibited by the L. donovani strains was not associated with cross-resistance to other classes of compounds and could be reversed by treatment with an inhibitor of glutathione biosynthesis, indicating that clinical resistance to antimonial drugs should not affect the antileishmanial efficacies of alternative drugs. In addition, it should be possible to identify a treatment regimen that could reverse antimony resistance.

Erythrocyte glutathione balance and membrane stability during preeclampsia.

This study aimed to determine whether oxidative damage to the erythrocyte occurs in preeclampsia, and relates to disease severity. The oxidative status of intact erythrocytes from preeclamptic patients and normal pregnant women was determined using spin echo 1H-NMR, which measures both the concentration and redox state of intracellular glutathione. Previous studies of preeclampsia have only measured total glutathione levels. Membrane fragility was determined from the degree of lysis caused by incubation in hypotonic saline. Erythrocytes from moderate-severe preeclamptic patients underwent more lysis than erythrocytes from control pregnant women (p < .05) or mild preeclamptic patients. It is suggested that increased lysis results from oxidative damage to the erythrocyte membrane, causing a decrease in membrane fluidity and reducing its ability to withstand osmotic changes. Intracellular glutathione was more oxidized in erythrocytes from pregnant women compared to nonpregnant controls (p < .05), and there was a less significant trend toward more oxidized glutathione with increasing severity of preeclampsia. The moderate-severe group showed a clear division in glutathione redox status: some patients had very oxidized glutathione while others had a normal redox balance. This novel finding suggests that some patients may be unusually susceptible to erythrocyte glutathione oxidation, possibly leading to general cellular damage, in particular HELLP Syndrome.

Oxidation of erythrocyte glutathione by monocytes stimulated with interleukin-6. Analysis by 1H spin echo NMR.

1H spin echo NMR was used to follow the release of reactive oxygen species (ROS) from human monocytes by monitoring erythrocyte glutathione status, which is sensitive to applied oxidative stress. This allowed the ability of the cytokine interleukin-6 (IL-6) to stimulate release of ROS from monocytes to be assessed in terms of oxidative damage to other cells, providing an estimation of its importance in vivo. It was found that incubation of monocytes with erythrocytes in the presence of IL-6 resulted in oxidation of the erythrocyte glutathione pool, indicating that oxidants are released in sufficient amounts to cause oxidative stress. High levels of IL-6 occurring in plasma of women with severe pre-eclampsia could therefore be responsible for depleted plasma antioxidants and haemolysis. The oxidation of erythrocyte glutathione was inhibited by the presence of the cyclooxygenase inhibitor indomethacin, suggesting that this may be of value in the treatment of oxidative pathologies.

Alteration of the erythrocyte glutathione redox balance by N-acetylcysteine, captopril and exogenous glutathione.

The effect of the thiol containing compounds N-acetylcysteine and captopril on glutathione metabolism in human erythrocytes has been investigated non-invasively using 1H spin echo NMR. N-Acetylcysteine was found to increase the reduced form of glutathione while captopril increased the oxidized form, but no changes in the total glutathione concentration were observed. Incubation of the cells with buthionine sulphoximine to inhibit de novo glutathione synthesis did not affect the response. The results show that these compounds act by altering the redox balance of glutathione rather than by stimulating its synthesis, and that their mechanisms of action are different, and not simply due to the presence of the thiol group.

Metabolic studies of a fructose-intolerant yeast by in vivo 31P-nuclear magnetic resonance spectroscopy.

The metabolic effects of the administration of fructose to a yeast expressing the cDNA for rat liver ketohexokinase have been investigated by 31P-nuclear magnetic resonance spectroscopy. Cessation of growth suffered by the yeast on exposure to 5 and 25 mM-fructose was accompanied by a large accumulation of fructose 1-phosphate at the expense of cytoplasmic orthophosphate and nucleoside triphosphate. Shifts in resonances were consistent with a drop in cytoplasmic pH. Arresting growth with 1 mM-fructose, however, did not result in these changes, although a large accumulation of fructose 1-phosphate occurred which may have been supported by the mobilization of polyphosphates.