Mitochondrial localization and activity of P-glycoprotein in doxorubicin-resistant K562 cells

It is now well-established that P-glycoprotein 170 (P-gp), an efflux pump involved in multidrug resistance (MDR) is overexpressed at the plasma membrane of doxorubicin-resistant K562 leukemia cells. Nevertheless, several results suggested: (i) that P-gp-mediated drug efflux was not the only mechanism involved in resistance; (ii) that intracellular compartments could accumulate the drug, preventing it from reaching its nuclear targets; (iii) that agents able to reverse multidrug resistance may lead to intracellular drug redistribution. We have studied the localization of P-gp in mitochondria as well as its functional properties in this compartment. Using several monoclonal antibodies (MoAbs) directed against different P-gp epitopes, a protein was detected in the cytoplasm of two doxorubicin-resistant K562 sublines and, by confocal laser scanning microscopy, this protein was shown to co-localize in the Golgi apparatus and in mitochondria, in equivalent proportions. Purified mitochondria were isolated from K562 cell variants; the presence of a protein of about 170 kDa and reacting with several anti-P-gp antibodies was assessed in MDR cells by Western blotting and flow cytometry. Functional assays have shown that mitochondrial P-gp was involved in doxorubicin accumulation inside the organelle but not in its efflux, suggesting an orientation of P-gp in the mitochondrial membrane inverse to that observed in the plasma membrane. A potential role for mitochondrial P-gp in MDR cells would be to protect the nucleus from doxorubicin. This is the first demonstration of the presence and functional activity of P-gp in mitochondria of MDR cells.     

Improved therapeutic efficacy of doxorubicin through conjugation with a novel peptide drug delivery technology (Vectocell)

Improvement in the therapeutic index of doxorubicin, a cytotoxic molecule, has been sought through its chemical conjugation to short (15-23 amino acid) peptide sequences called Vectocell peptides. Vectocell peptides are highly charged drug delivery peptides and display a number of characteristics that make them attractive candidates to minimize many of the limitations observed for a broad range of cytotoxic molecules. The studies reported here characterized the in vitro and in vivo efficacy of a range of Vectocell peptides conjugated to doxorubicin through different linkers. These studies show that the in vivo therapeutic index of doxorubicin can be improved by conjugation with a specific Vectocell peptide (DPV1047) through an ester linker to C14 of doxorubicin, in both colon and breast tumor models. This conjugate was also shown to have significant in vivo antitumoral activity in a model resistant to doxorubicin, suggesting that this conjugate is able to circumvent the multidrug resistance (MDR) phenotype. These experiments therefore provide support for the use of the Vectocell technology with other cytotoxic agents.     

Methicillin-resistant Staphylococcus aureus in veterinary doctors and students, the Netherlands

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the Netherlands, at 1.0%, is among the lowest in Europe. In 2004, a relationship between pig farming and a high risk for MRSA carriage was found. To investigate if those in professional contact with livestock are at higher risk for MRSA carriage, we screened 80 veterinary students and 99 veterinarians and questioned them about animal contacts and known MRSA risk factors. Of these, 27 students who did not have livestock contact were excluded from further analysis. We found 7 carriers of MRSA, a prevalence of 4.6%, which is similar to that found in patients who had previously been treated at foreign hospitals. A correlation of MRSA carriage with a specific animal group could not be established. To preserve the low prevalence of MRSA in the Netherlands, persons involved in the care of livestock should be isolated and screened on admission to the hospital.     

Intrinsic gluconeogenesis is enhanced in renal proximal tubules of Zucker diabetic fatty rats

Recent studies indicate that renal gluconeogenesis is substantially stimulated in patients with type 2 diabetes, but the mechanism that is responsible for such stimulation remains unknown. Therefore, this study tested the hypothesis that renal gluconeogenesis is intrinsically elevated in the Zucker diabetic fatty rat, which is considered to be an excellent model of type 2 diabetes. For this, isolated renal proximal tubules from diabetic rats and from their lean nondiabetic littermates were incubated in the presence of physiologic gluconeogenic precursors. Although there was no increase in substrate removal and despite a reduced cellular ATP level, a marked stimulation of gluconeogenesis was observed in diabetic relative to nondiabetic rats, with near-physiologic concentrations of lactate (38%), glutamine (51%) and glycerol (66%). This stimulation was caused by a change in the fate of the substrate carbon skeletons resulting from an increase in the activities and mRNA levels of the key gluconeogenic enzymes that are common to lactate, glutamine, and glycerol metabolism, i.e., mainly of phosphoenolpyruvate carboxykinase and, to a lesser extent, of glucose-6-phosphatase and fructose-1,6-bisphosphatase. Experimental evidence suggests that glucocorticoids and cAMP were two factors that were responsible for the long-term stimulation of renal gluconeogenesis observed in the diabetic rats. These data provide the first demonstration in an animal model that renal gluconeogenesis is upregulated by a long-term mechanism during type 2 diabetes. Together with the increased renal mass (38%) observed, they lend support to the view so far based only on in vivo studies performed in humans that renal gluconeogenesis may be stimulated by and crucially contribute to the hyperglycemia of type 2 diabetes.     

Science review: carnitine in the treatment of valproic acid-induced toxicity - what is the evidence?

Valproic acid (VPA) is a broad-spectrum antiepileptic drug and is usually well tolerated, but rare serious complications may occur in some patients receiving VPA chronically, including haemorrhagic pancreatitis, bone marrow suppression, VPA-induced hepatotoxicity (VHT) and VPA-induced hyperammonaemic encephalopathy (VHE). Some data suggest that VHT and VHE may be promoted by carnitine deficiency. Acute VPA intoxication also occurs as a consequence of intentional or accidental overdose and its incidence is increasing, because of use of VPA in psychiatric disorders. Although it usually results in mild central nervous system depression, serious toxicity and even fatal cases have been reported. Several studies or isolated clinical observations have suggested the potential value of oral L-carnitine in reversing carnitine deficiency or preventing its development as well as some adverse effects due to VPA. Carnitine supplementation during VPA therapy in high-risk patients is now recommended by some scientific committees and textbooks, especially paediatricians. L-carnitine therapy could also be valuable in those patients who develop VHT or VHE. A few isolated observations also suggest that L-carnitine may be useful in patients with coma or in preventing hepatic dysfunction after acute VPA overdose. However, these issues deserve further investigation in controlled, randomized and probably multicentre trials to evaluate the clinical value and the appropriate dosage of L-carnitine in each of these conditions.     

Effect of ambient temperature on endurance performance while wearing cross-country skiing clothing

This study assessed the effects of exposure to cold (?14 and ?9?°C), cool (?4 and 1?°C) and moderate warm (10 and 20?°C) environments on aerobic endurance performance-related variables: maximal oxygen consumption (VO_2max), running time to exhaustion (TTE), running economy and running speed at lactate threshold (LT). Nine male endurance athletes wearing cross-country ski racing suit performed a standard running test at six ambient temperatures in a climatic chamber with a wind speed of 5?m?s^?1. The exercise protocol consisted of a 10-min warm-up period followed by four submaximal periods of 5?min at increasing intensities between 67 and 91?% of VO_2max and finally a maximal test to exhaustion. During the time course mean skin temperature decreased significantly with reduced ambient temperatures whereas T _re increased during all conditions. T _re was lower at ?14?°C than at ?9 and 20?°C. Running economy was significantly reduced in warm compared to cool environments and was also reduced at 20?°C compared to ?9?°C. Running speed at LT was significantly higher at ?4?°C than at ?9, 10 and 20?°C. TTE was significantly longer at ?4 and 1?°C than at ?14, 10 and 20?°C. No significant differences in VO_2max were found between the various ambient conditions. The optimal aerobic endurance performance wearing a cross-country ski racing suit was found to be ?4 and 1?°C, while performance was reduced under moderate warm (10 and 20?°C) and cold (?14 and ?9?°C) ambient conditions.