Assessing effect of mixing insulins by glucose-clamp technique in subjects with diabetes mellitus

The glucose-clamp technique was used to assess the effect on insulin activity of mixing clinically relevant doses of short- and longer-acting insulins in insulin-treated diabetic subjects. Mixtures of porcine regular and lente insulins resulted in a reduction in activity of the mixture compared with separate injection that was more apparent with premixing for 5 min before injecting than with premixing for 2 min. These findings were not explained by differences in volume of the injected insulin preparations. Mixing of porcine regular and bovine ultralente insulins for 2 min before injecting resulted in a reduced activity compared with separate injection. No difference in activity was obvious for porcine regular and NPH insulins given separately or premixed for 5 min. A reduction of activity during the 1st h after injection was observed when separate injection of one brand of these insulins (Velosulin or Insulatard) was compared with the manufactured mixture (Mixtard). These results indicate that premixing regular and NPH insulins in a 1-to-2 ratio does not alter the biological activity compared with separate administration of the insulins. However, the mixing of regular and insulin-zinc suspensions results in a loss of insulin activity, the magnitude of which depends on the time between mixing and injecting the insulin. The clinical significance of the effect of mixing on the efficacy of subcutaneous insulin therapy should be considered in the context that this is only one of many factors that may affect the activity of subcutaneously injected insulin.     

Cell proliferation depends on mitochondrial Ca2+ uptake: inhibition by salicylate

Store-operated Ca²⁺ entry (SOCE) is a ubiquitous Ca²⁺ influx pathway involved in control of multiple cellular and physiological processes including cell proliferation. Recent evidence has shown that SOCE depends critically on mitochondrial sinking of entering Ca²⁺ to avoid Ca²⁺-dependent inactivation. Thus, a role of mitochondria in control of cell proliferation could be anticipated. We show here that activation of SOCE induces cytosolic high [Ca²⁺] domains that are large enough to be sensed and avidly taken up by a pool of nearby mitochondria. Prevention of mitochondrial clearance of the entering Ca²⁺ inhibited both SOCE and cell proliferation in several cell types including Jurkat and human colon cancer cells. In addition, we find that therapeutic concentrations of salicylate, the major metabolite of aspirin, depolarize partially mitochondria and inhibit mitochondrial Ca²⁺ uptake, as revealed by mitochondrial Ca²⁺ measurements with targeted aequorins. This salicylate-induced inhibition of mitochondrial Ca²⁺ sinking prevented SOCE and impaired cell growth of Jurkat and human colon cancer cells. Finally, direct blockade of SOCE by the pyrazole derivative BTP-2 was sufficient to arrest cell growth. Taken together, our results reveal that cell proliferation depends critically on mitochondrial Ca²⁺ uptake and suggest that inhibition of tumour cell proliferation by salicylate may be due to interference with mitochondrial Ca²⁺ uptake, which is essential for sustaining SOCE. This novel mechanism may contribute to explaining the reported anti-proliferative and anti-tumoral actions of aspirin and dietary salicylates.     

Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.     

Effect of S-adenosyl-L-methionine on rat brain oxidative stress damage in a combined model of permanent focal ischemia and global ischemia-reperfusion

We analyzed the effects of S-adenosyl-L-methionine (SAM) on tissue oxidative status in a combined model of permanent focal ischemia and global reperfusion in the rat brain. The production of thiobarbituric acid reactive substances (TBARS) was measured under basal conditions and after induction with ferrous salt as an indicator of brain lipid peroxidation. Total, oxidized and reduced glutathione were measured as indicators of the antioxidant defense capacity of brain tissue. Mitochondrial reduction of tetraphenyl tetrazolium (TPT) was quantified morphometrically. Results obtained in vitro showed that incubation with SAM reduced lipid peroxidation, with a maximum inhibition of 65.12+/-5.99% after incubation with 1 mmol/l; glutathione production was not significantly modified. In the brain ischemia-reperfusion model, TBARS production increased and glutathione content decreased, and mitochondrial reduction of TPT decreased significantly after ischemia-reperfusion in areas dependent on carotid circulation. The administration of 50 mg/kg SAM per day for 3 days led to the inhibition of brain lipid peroxidation and increased total glutathione production. These changes were accompanied by an increase in mitochondrial capacity to reduce TPT. We conclude that SAM reduces oxidative damage in the rat brain in an experimental model of ischemia-reperfusion.     

A longitudinal communication approach in advanced lung cancer: A qualitative study of patients’, relatives’ and staff's perspectives

Communication and the care of patients with advanced cancer are a dynamic, interactive and challenging process, often characterised in every day practice by discontinuity and lack of coordination. The objective of this study was to explore the patients’ and family‐caregivers’ needs and preferences regarding communication, quality of life and care over the trajectory of disease. The second aim was to assess health professionals’ views on a longitudinally structured, forward‐thinking communication approach based on defined milestones. A qualitative approach was chosen incorporating semi‐structured interviews with nine patients with metastatic lung cancer and nine relatives, and focus groups with 15 healthcare providers from different professions involved in the care of these patients. Patients and relatives described a situation of shock and coping deficits with moments of insufficient communication and lack of continuity in care. Healthcare providers reported the strong need for improvement in communication within the team and between patients and professionals and welcomed the implementation of a longitudinal communication approach. Requirements for the implementation of a longitudinal communication approach include specific communication training with focus on the process that patients and relatives are involved in. Team‐building measures and the necessary flexibility to respect individuality in life should be incorporated.     

Ocular Redness Measured with the Keratograph 5M in Patients Using Anti-Glaucoma Eye Drops

Purpose: To examine correlations between ocular redness measured with the new topographer Keratograph 5M and the use of topical anti-glaucoma medication. Methods: A total of 211 eyes of 211 patients with open-angle glaucoma or ocular hypertension on topical medication and 51 eyes of 51 healthy volunteers were recruited over 10 months. Outcome variables were keratograph redness scores (RS): overall, bulbar temporal (BT), bulbar nasal (BN), limbar temporal (LT), and limbar nasal (LN). In each subject, we also recorded the intraocular pressure-lowering eye drops used, daily doses and daily and cumulative preservative concentrations, fluorescein corneal staining score (OXFORD), lower tear meniscus height (Fourier-domain OCT), non-invasive tear film breakup time (Keratograph 5M), and ocular surface disease questionnaire index (OSDI). Results: Higher RS were recorded in the medication than control group (P < 0.01 all scores). Within the medication group, older patients returned greater RS (P < 0.05 all scores). Prostaglandin was a strong predictor of higher scores, except LN RS. A higher OSDI was associated with a higher LN RS (β = 0.007; P < 0.05), while the use of β-blockers was linked to a lower LN RS (β = ?0.225; P < 0.05). The use of ≥3 daily eye drops with preservative gave rise to a higher BN RS and ≥3 daily eye drops to a higher LN RS (β = 0.366, P < 0.01; β = 0.296, P < 0.05, respectively). Conclusion: Keratograph 5M can objectively detect the hyperaemia induced by glaucoma medication. The factors contributing to ocular redness were advanced age, more daily eye drops (nasal sectors), a higher OSDI, and prostaglandin as the medication used.