Excited States in Bilayer Graphene Quantum Dots.

We report ground- and excited-state transport through an electrostatically defined few-hole quantum dot in bilayer graphene in both parallel and perpendicular applied magnetic fields. A remarkably clear level scheme for the two-particle spectra is found by analyzing finite bias spectroscopy data within a two-particle model including spin and valley degrees of freedom. We identify the two-hole ground state to be a spin-triplet and valley-singlet state. This spin alignment can be seen as Hund's rule for a valley-degenerate system, which is fundamentally different from quantum dots in carbon nanotubes, where the two-particle ground state is a spin-singlet state. The spin-singlet excited states are found to be valley-triplet states by tilting the magnetic field with respect to the sample plane. We quantify the exchange energy to be 0.35 meV and measure a valley and spin g factor of 36 and 2, respectively.

A cysteine-specific lysosomal transport system provides a major route for the delivery of thiol to human fibroblast lysosomes: possible role in supporting lysosomal proteolysis.

Lysosomes constitute only 4% of the intracellular volume of a normal human fibroblast. When human fibroblasts are incubated for 2-5 min with 20 microM [35S]cystine in Krebs-Ringer phosphate solution at pH 7.4, a minimum of 50-60% of the total radioactivity taken up by the cells is found sequestered into the lysosomal compartment in the form of cysteine. A lysosomal transport system, highly specific for cysteine, appears to facilitate this rapid lysosomal cysteine sequestration. Time courses of [35S]cysteine uptake into isolated, Percoll-purified fibroblast lysosomes at pH 7.0 and 37 degrees C are linear for the first 4-5 min and attain a steady state by 10 min. Lysosomal cysteine uptake displays a Km of 0.05 mM at pH 7.0 and an activation energy of 21 kcal/mol, corresponding to a Q10 of 3.2. The role of this transport system in delivering cysteine into lysosomes is supported by its pH curve showing a slow rate of cysteine transport at the acidic pHs between 5 and 6, but then increasing sevenfold between pH 6 and 7.5 to be maximally active near the cytosolic pH of 7. Carrier mediation by this lysosomal transport route demonstrates a high specificity for cysteine as indicated by the inability of the following amino acids to significantly inhibit at 5 mM the lysosomal uptake of 0.035 mM [35S]L-cysteine: ala, ser, pro, val, gly, homocysteine, D- or L-penicillamine, arg, asp, or leu. Similarly, D-cysteine and beta-mercaptopropionate were poor inhibitors, suggesting that both the L-isomer and alpha-amino group of cysteine appear to be required for recognition by the cysteine-specific transport system. In contrast, cysteamine, which lacks an alpha-carboxyl group, was able to strongly inhibit lysosomal cysteine uptake. The physiological importance of this cysteine-specific lysosomal transport system may be to aid lysosomal proteolysis by delivering cysteine into the lysosomal compartment to (a) maintain the catalytic activity of the thiol-dependent lysosomal enzymes and (b) break protein disulfide bridges at susceptible linkages, thereby allowing proteins to unfold, facilitating their degradation.

Impact of sex and glucose-lowering treatments on hypoglycaemic symptoms in people with type 2 diabetes and chronic kidney disease. The French Chronic Kidney Disease - Renal Epidemiology and Information Network (CKD-REIN) Study.

AIM: To describe current practices of glucose-lowering treatments in people with diabetes and chronic kidney disease (CKD), the associated glucose control and hypoglycaemic symptoms, with an emphasis on sex differences. METHODS: Among the 3033 patients with CKD stages 3-5 recruited into the French CKD-REIN study, 645 men and 288 women had type 2 diabetes and were treated by glucose-lowering drugs. RESULTS: Overall, 31% were treated only with insulin, 28% with combinations of insulin and another drug, 42% with non-insulin glucose-lowering drugs. In CKD stage 3, 40% of patients used metformin, 12% at stages 4&5, similar for men and women; in CKD stage 3, 53% used insulin, similar for men and women, but at stages 4&5, 59% of men and 77% of women used insulin. Patients were reasonably well controlled, with a median HbA1c of 7.1% (54mmol/mol) in men, 7.4% (57mmol/mol) in women (P=0.0003). Hypoglycaemic symptoms were reported by 40% of men and 59% of women; they were not associated with the estimated glomerular filtration rate, nor with albuminuria or with HbA1c in multivariable analyses, but they were more frequent in people treated with insulin, particularly with fast-acting and pre-mixed insulins. CONCLUSION: Glucose-lowering treatment, HbA1c and hypoglycaemic symptoms were sex dependent. Metformin use was similar in men and women, but unexpectedly low in CKD stage 3; its use could be encouraged rather than resorting to insulin. Hypoglycaemic symptoms were frequent and need to be more closely monitored, with appropriate patient-education, especially in women.

[DOPPS estimate of patient life years attributable to modifiable hemodialysis practices in Spain]. / Estimación, según el estudio DOPPS, de los años de vida de pacientes atribuibles a las prácticas de hemodiálisis modificables en España.

The increased mortality risk in hemodialysis (HD) patients unable to meet six targets in different areas of HD practice has been reported previously. Using a prevalent cross-sectional sample of Spanish HD patients (n = 613) from the second stage of the Dialysis Outcomes and Practice Patterns Study to determine the percentage with low dialysis dose, hyperphosphatemia, hypercalcemia, hypoalbuminemia, anemia, and catheter use and based on the mortality hazard ratios and the total HD population in Spain, according to the Spanish Society of Nephrology Report, we estimated the number of patient life years that could potentially be gained in our country. These characteristics of HD practice were selected because each is modifiable through changes in practice, each is associated with mortality, and each has a large number of patients outside the target guidelines. The targets that define "within guidelines" are as follows: dialysis dose (single pool Kt/V >1.2), anemia (hemoglobin >110 g/L), albumin after standardization (>40 g/L), serum phosphorus (1.1-1.5 mmol/L), serum calcium (2.1-2.4 mmol/L), and facility catheter use (<10%). Cox proportional hazards regression models were used to calculate the relative risk of mortality for all patients outside each guideline. In all models, calcium values were adjusted for low serum albumin. A separate Cox survival model adjusted for all six HD practices simultaneously to account for correlation that may exist between some facility practices. All models were adjusted for age, sex, race, time on ESRD, and 14 summary comorbid conditions. Patient years attributable to each of the six practice patterns were estimated and are reported here as the potential patient years gained. Comparison of the estimates by individual guideline shows that, in Spain, increasing patient albumin above 40 g/L in all patients would lead to an estimated gain of 9,269 patient years (a 7.9% increase). Additionally, if all facilities could decrease catheter use to less than 10%, 2,842 patient years could be gained (a 2.4% increase). Though it may be an unrealistic goal, if all Spanish patients currently outside the guidelines achieved all six target levels, an estimated 17,300 life years could be gained over the next five years (a 15% increase). A more achievable goal of bringing 50% of patients who are currently outside targets within targets would result in 9,266 life years gained. In conclusion, this analysis suggests large opportunities to improve HD patient care in Spain.

Evidence for an essential histidine residue located in the binding site of the cysteine-specific lysosomal transport protein.

Previously, we observed that the activity of the cysteine-specific lysosomal transport system increases 7-10-fold between pH 6 and 7.3 to be maximally active in the neutral pH range. To understand what factors contribute to this pH dependence, different chemical modifying agents were used to probe the nature of amino acid residues residing in the transport protein binding site. Diethyl pyrocarbonate (1 mM) and N-ethylmaleimide (5 mM) each strongly inactivated lysosomal cysteine uptake > or = 88%, whereas dicyclohexyl-carbodiimide (2.5 mM), phenylisothiocyanate (2 mM), N-acetylimidazole (33 mM), and phenylglyoxal (2 mM) had a moderate to small effect. Maximal inactivation by DEPC occurs within 12-15 min upon exposure to DEPC concentrations > or = 1 mM. DEPC inactivation is consistent with modification of a histidine residue, displaying no inactivation at pH < 6, half-maximal inactivation at pH 6.6, and maximal inactivation at pH > or = 7.3. The close correspondence of DEPC inactivation to the pH activity curve of cysteine uptake suggests the large increase in lysosomal cysteine transport activity between pH 6 and 7.3 reflects deprotonation of an essential histidine residue. The substrate, L-cysteine (4 mM), fully protects the transport protein from DEPC inactivation suggesting that this histidine residue is located in the carrier's substrate binding site. Finally, part of the pH dependence of the lysosomal cysteine carrier appears to be due to responsiveness to the lysosomal transmembrane proton gradient as indicated by lysosomal membrane vesicles which display a 1.5-fold greater rate of cysteine uptake when pH 7.4out > pH 5.3in than when pH 7.4out = pH 7.4in.

Elevated temperature produces cystine depletion in cystinotic fibroblasts.

Increasing the incubation temperature of cystinotic fibroblasts to 40 or 43 degrees C produces a 70-80% decrease in lysosomal cystine content within 24-48 h. This effect is probably mediated by an altered substrate affinity for another lysosomal transport protein.

Characterization of a transport system for anionic amino acids in human fibroblast lysosomes.

L-Aspartate and L-glutamate are transported into human fibroblast lysosomes by a single, low Km, Na(+)-independent transport system, which has been provisionally named lysosomal system d. This system resembles the Na(+)-dependent plasma membrane system chi-AG, although these differences have been observed: (1) lysosomal system d recognizes the D- as well as the L-isomers of both aspartate and glutamate, whereas only for aspartate is the D-isomer recognized by system chi-AG; (2) the anion L-homocysteate is not accepted by system chi-AG, but is an effective inhibitor of lysosomal system d; (3) N-methyl, alpha-methyl, and omega-hydroxamate derivatives of both aspartate and glutamate inhibit lysosomal system d, but only the aspartate derivatives are accepted by system chi-AG; (4) lysosomal system d shows a preference for the substrate amino group in the alpha-position, a preference not seen for system chi-AG. These points imply differences at the two recognition sites with respect to substrate length, size, and rotation, with the lysosomal site generally being the less restrictive.

Separate and shared lysosomal transport of branched and aromatic dipolar amino acids.

Transport systems analogous to the T and L carriers for aromatic and bulky dipolar amino acids in plasma membranes have been characterized in the membranes of intact lysosomes isolated from human fetal skin fibroblasts. While system L appears ubiquitous in plasma membranes, system T has previously been discriminated only in the plasmalemma of human red blood cells and freshly isolated rat hepatocytes. Our findings with the lysosomal systems, provisionally designated t and l, reveal both shared and dissimilar properties with the plasma membrane systems. These properties include a lack of dependency on extralysosomal Na+, differential sensitivities to the classical system L analog, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), and the system T analog, D-tryptophan, as well as susceptibility to thiol modification at the membrane by reactivity with N-ethylmaleimide. A transport system in lysosomes from the FRTL-5 rat thyroid cell line has been described by Bernar et al. ((1986) J. Biol. Chem. 261, 17107-17112) resembles a composite of both carrier systems reported in this work.

[The Dialysis Outcomes and Practice Patterns Study (DOPPS): results of the Italian cohort]. / Lo studio DOPPS (Dialysis Outcomes and Practice Patterns Study): risultati della coorte italiana.

BACKGROUND: The Dialysis Outcomes and Practice Patterns Study (DOPPS) is an international prospective, longitudinal, observational study examining the relationship between dialysis unit practices and outcomes for hemodialysis (HD) patients in seven developed countries France, Germany, Italy, Spain, United Kingdom, Japan and the United States. Results of the DOPPS in Italy are the subject of this report. METHODS: A national representative sample of 20 dialysis units (21 in Germany) was randomly selected in each of the European DOPPS countries (Euro-DOPPS). In these units, the HD in-center patients were included on a facility census, and their survival rates continuously monitored. A representative sample of incident (269 in Italy, 1553 in the Euro-DOPPS) and prevalent (600 in Italy, 3038 in the Euro-DOPPS) patients was randomly selected from the census for more detailed longitudinal investigation with regard to medical history, laboratory values and hospital admission. RESULTS: Comparing the Italian and Euro-DOPPS cohorts we found comparable mean age for prevalent patients (61.4 vs. 59.5 yrs), but incident patients were older in Italy. Italian prevalent patients had less cardiovascular disease, more satisfactory nutritional status and more frequent use of native vascular access. These data were associated with a comparable mortality (15.7 vs. 16.3 deaths/100 patient yrs), but morbidity was lower in Italy. Kt/V levels were comparable in the two cohorts (1.32 vs. 1.37), but 35% of Italian patients showed a Kt/V below the recommended target. Moreover, hemoglobin levels were below 11 g/dL in 60% of Italian patients. CONCLUSIONS: The DOPPS results bring to light several positive aspects and the opportunity for further possible improvements for Italian patients, but at the same time highlight some critical points that could represent a risk for dialysis quality.

Renoprotective therapy in patients with nondiabetic nephropathies.

End-stage renal failure (ESRF) represents a major health problem. Early diagnosis and effective measures to slow or to stop renal damage are essential goals for nephrologists to prevent or delay progression to ESRF. Identifying mechanisms of progressive parenchymal injury is instrumental in developing renoprotective strategies. Protein traffic through the glomerular barrier is an important determinant of progression in chronic nephropathies and proteinuria is the best predictor of renal outcome. At the moment, ACE inhibition is the most effective treatment in patients with chronic nondiabetic proteinuric nephropathies, reducing protein traffic, urinary protein excretion rate and progression to ESRF more effectively than conventional treatment. Low sodium diet and/or diuretic treatment may help to increase the antiproteinuric effect of ACE inhibitors by maximally activating the renin-angiotensin system. Intensified blood pressure control, whatever treatment is employed, also enhances the antiproteinuric response to ACE inhibitors. However, since this is not always sufficient to normalise urinary proteins and fully prevent renal damage, additional treatments may be needed in patients poorly or not responding to ACE inhibitors. These may include angiotensin II receptor antagonists, non-dihydropyridine calcium antagonists and perhaps low doses of nonsteroidal anti-inflammatory drugs. Preliminary data on multidrug treatments including these additional antiproteinuric agents are encouraging, but additional studies in larger patient numbers are needed to better define the risk/benefit profile of this innovative approach.

Proteinuria predicts end-stage renal failure in non-diabetic chronic nephropathies. The "Gruppo Italiano di Studi Epidemiologici in Nefrologia" (GISEN).

We correlated baseline parameters with glomerular filtration rate (GFR) decline and kidney survival in 274 patients with proteinuric non-diabetic chronic nephropathies (creatinine clearance 20 to 70 ml/min/1.73 m2 and proteinuria > 1 g/24 hr over the last three months) enrolled in the Ramipril Efficacy In Nephropathy (REIN) trial. The GFR, evaluated at baseline, one, three and six months after randomization then every six months, declined linearly by 0.52 +/- 0.83 ml/min/1.73 m2/month (mean +/- SD) over a follow-up (median: range) of 21:3 to 52 months, and kidney survival was 64%. In multivariate analysis, higher baseline proteinuria (P = 0.006), and lower GFR (P = 0.0001) and creatinine clearance (P = 0.0001) correlated with a faster GFR decline. Higher proteinuria was the only baseline predictor of a shorter kidney survival (P = 0.0007) and its predictive value was independent of the underlying renal disease, treatment randomization, and blood pressure control during the followup. Patients in the lowest tertile of baseline proteinuria (< 2.5 g/24 hr) had the slowest rate of GFR decline (-0.25 +/- 0.72 ml/min/1.73 m2/month) and the highest kidney survival (94%), compared with patients in the middle tertile (proteinuria 2.5 to 4.3 g/24 hr; delta GFR, -0.59 +/- 0.82 ml/min/1.73 m2/month, P = 0.008; kidney survival 57%, P = 0.0011) and in the highest tertile (proteinuria > 4.3 g/24 hr; delta GFR, -0.79 +/- 0.87 ml/min/1.73 m2/month, P = 0.0001, kidney survival 44%, P = 0.0001). Kidney survival significantly differed even between the middle and highest tertiles (P < 0.05). Thus, in non-diabetic chronic nephropathies proteinuria is an independent and accurate predictor of disease progression and ESRF.

Drug-induced thrombotic microangiopathy: incidence, prevention and management.

The term thrombotic microangiopathy (TMA) describes syndromes characterised by microangiopathic haemolytic anaemia, thrombocytopenia and variable signs of organ damage due to platelet thrombi in the microcirculation. In children, infections with Shigella dysenteriae type 1 or particular strains of Escherichia coli are the most common cause of TMA; in adults, a variety of underlying causes have been identified, such as bacterial and viral infections, bone marrow and organ transplantation, pregnancy, immune disorders and certain drugs. Although drug-induced TMA is a rare condition, it causes significant morbidity and mortality. Antineoplastic therapy may induce TMA. Most of the cases reported are associated with mitomycin. TMA has also been associated with cyclosporin, tacrolimus, muromonab-CD3 (OKT3) and other drugs such as interferon, anti-aggregating agents (ticlopidine, clopidogrel) and quinine. The early diagnosis of drug-induced TMA may be vital. Strict monitoring of renal function, urine and blood abnormalities, and arterial pressure has to be performed in patients undergoing therapy with potentially toxic drugs. The drug must be discontinued immediately in the case of suspected TMA. Treatment modalities sometimes effective in other forms of TMA have been used empirically. Although plasma exchange therapy seems to be of value, the effectiveness of this approach has yet to be proved in multicentre, randomised clinical studies.

The interaction of platelet proteins with three fibroblast-derived collagenases.

Three collagenases were purified from the culture medium of human skin fibroblasts using ammonium sulfate fractionation, ion-exchange chromatography and gel filtration. The cationic collagenase had a molecular weight of 42,000; two anionic collagenases had molecular weights of 63,000 and 115,000. Preincubation of the individual collagenases with purified human and bovine platelet heparin binding proteins resulted in the inhibition of the two anionic activities, but only by bovine low heparin affinity platelet protein (beta-TG). Such inhibition was dose-dependent at the microgram level, was not antagonized by heparin, and persisted even when the collagenases had been transformed into their 53,000 and 105,000 forms through treatment with p-aminophenylmercuric acetate. Neither human nor bovine high heparin affinity platelet factors (PF-4) nor human low heparin affinity platelet protein (beta-TG) were inhibitory to any of the three collagenases studied. This suggests that the ability of platelet proteins to inhibit collagenase is specifically influenced by the ionic nature of the enzyme and this inhibition is specifically dependent upon the species and type of platelet protein.

How much must blood pressure be reduced in order to obtain the remission of chronic renal disease?

Most chronic nephropathies are characterized by a progressive decline in glomerular filtration rate (GFR) that may lead to renal function replacement by dialysis or transplant. Hypertension has an extremely important role among the various mechanisms contributing to renal function deterioration. High blood pressure levels are associated with increased urinary excretion of proteins and the decrease of systemic and glomerular hypertension reduces urinary excretion of proteins and preserves renal function deterioration. Moreover, recent studies found that an intensified blood pressure control (less than 130/80 mmHg) can slow the progression of diabetic and non diabetic renal disease even more than conventional blood pressure control. The Ramipril Efficacy in Nephropathy (REIN) Study showed that ramipril, an ACE-inhibitor, slowed the rate of GFR decline and halved the combined risk of doubling serum creatinine or end stage renal failure (ESRF) in patients with nephrotic range proteinuria as compared to conventional antihypertensive therapy, at comparable levels of blood pressure control. In these patients, prolonged enough treatment (at least 36 months) with ramipril, lowered the velocity of GFR decline and reduced the risk of dialysis. Thus, both tight blood pressure control and ACE-inhibitors may have a renoprotective effect. It will be interesting to evaluate whether the two combined approaches may have sinergistic effects.

Biosynthesis of proteochondroitin sulfate: substrate requirements for formation of two independent species.

Formation of two species of [14C]proteochondroitin sulfate has previously been demonstrated with UDP-D-[14C]glucuronic acid and UDP-N-acetylgalactosamine as substrates with a microsomal preparation from chick-embryo epiphyseal cartilage. A large species of [14C]proteoglycan that appeared at an earlier stage of synthesis was excluded on Sepharose CL-2B, indicating that it was larger than proteoglycans found in cartilage matrix. Another newly synthesized, smaller [14C]proteoglycan species also formed was retarded on Sepharose CL-2B, and appeared to be at a later stage of synthesis. A 6-h pulse-chase experiment using UDP-[14C]GlcA and UDP-GalNAc followed by cold UDP-GlcA demonstrates that there was no conversion of the large [14C]proteoglycan to the small [14C]proteoglycan. Sulfation of the newly formed large and small [14C]proteoglycans with adenylyl sulfate 3'-phosphate also did not alter their chromatographic patterns, indicating that sulfation did not trigger any post-synthetic size modification. Synthesis with lower concentrations of the sugar nucleotides resulted in a disproportionate diminution in formation of the large proteoglycan. The apparent Km values for UDP-GlcA for the formation of large and small proteoglycans were 0.055 and 0.015 mM, respectively. Concentration requirements for UDP-GalNAc also showed a similar 4-fold difference. These results indicate that, even though the large proteoglycan was at an earlier stage of synthesis, it was not a precursor to the small proteoglycan, and that these proteoglycans represent two separately synthesized species.

Long-term effects of aldosterone blockade in resistant hypertension associated with chronic kidney disease.

Hypertension is a major risk factor for the development and progression of chronic kidney disease (CKD). Mineralocorticoid receptor antagonists (MRAs) are effective in the management of resistant hypertension but are not widely used in CKD because of the risk of hyperkalemia. We retrospectively evaluated the long-term effects and safety of MRAs added to a pre-existing antihypertensive regimen in subjects with resistant hypertension associated with stage 3 CKD. In all, 32 patients were treated with spironolactone and 4 with eplerenone for a median follow-up of 312 days. MRAs induced a significant decrease in systolic blood pressure from 162±22 to 138±14 mm Hg (P<0.0001) and in diastolic blood pressure from 87±17 to 74±12 mm Hg (P<0.0001). Serum potassium increased from 4.0±0.5 to 4.4±0.5 mEq l(-1) (P=0.0001), with the highest value being 5.8 mEq l(-1). The serum creatinine increased from 1.5±0.3 to 1.8±0.5 mg dl(-1) (P=0.0004) and the estimated glomerular filtration rate decreased from 48.6±8.7 to 41.2±11.5 ml min(-1) per 1.73 m(2) (P=0.0002). One case of acute renal failure and three cases of significant hyperkalemia occurred. MRAs significantly reduced blood pressure in subjects with resistant hypertension associated with stage 3 CKD, although close biochemical monitoring is recommended because of an increased risk of hyperkalemia and worsening of renal function.

Dopps estimate of patient life years attributable to modifiable haemodialysis practices in Belgium.

BACKGROUND: Various organizations have published clinical practice guidelines for the care of haemodialysis patients. However, it is unknown to what extent improving or even reaching perfect compliance with guidelines would improve the survival of HD patients in Belgium. METHODS: Using data from the second phase of the Dialysis Outcomes and Practice Patterns Study (DOPPS), the proportion of haemodialysis patients failing to meet six key practice targets (Kt/V > or = 1.2, haemoglobin > or =11 g/dl, phosphate 1.1-1.5 mmol/l, calcium 2.1-2, 4 mmol/l, albumin > or =40 g/l, and facility catheter use < or =10%) was calculated along with the relative risk of mortality associated with being outside these targets. The life years potentially gained from adherence to the six targets, both separately and all six together were then estimated. RESULTS: The percentage of patients outside the targets were as follows: 30.3%, Kt/V; 33.6%, haemoglobin; 56.2%, phosphate; 58.2%, calcium; 67.1%, albumin; and 91.1%, catheter. Estimated patient life years gained with improved compliance with guidelines was highest for albumin (3.670) and catheter use (2.331) but still substantial for the other four targets (ranging from 551 to 1.258). The total of patient years gained if 100% of patients have all six practices brought within target reaches 7.516 years. A conservative estimate of 50% of patients within all targets still yields an improvement of survival of 3.958 patient years. CONCLUSION: This analysis suggests large opportunities to improve HD patient care in Belgium. The avoidance of HD catheters, with the use of AV fistulas whenever possible, should be given a high priority. Admittedly, these calculations assume causality or partial causality that has not been definitively proven. Still, if causality is only partial, the results emphasize that the improvement of patient care through adherence to targets of clinical guidelines might be substantial and all Belgian nephrologists and staff members of dialysis units should carefully pursue every potential effort.