Anticoagulation in large-volume leukapheresis: comparison between citrate- versus heparin-based anticoagulation on safety and CD34 (+) cell collection efficiency

Background aimsLittle is known of the effect of anticoagulation on peripheral blood progenitor cell (PBPC) harvest during large-volume leukapheresis (LVL). Because of the interaction of heparin with stromal cell-derived factor (SDF)-1α, it has been proposed that a heparin-based anticoagulation may result in an increased PBPC collection efficiency compared with standard citrate-based anticoagulation.MethodsWe conducted a prospective randomized trial to address the effect of both anticoagulation regimes on safety, subjective comfort and CD34 ⁺ collection efficiency in 90 adult patients undergoing standardized LVL. Anticoagulation consisted of either citrate (group C) or a combination of heparin and low-dose citrate (group H).ResultsThe overall incidence of adverse reactions (AR) during LVL was 17%. AR consisted only of citrate-related AR; no bleeding complications were observed. Determination of parameters of the acid–base balance revealed a higher frequency of metabolic alkalosis in group C. Analysis of serum SDF-1α revealed no differences in SDF-1α plasma levels. There were no differences in the CD34 ⁺ cell collection efficiency, resulting in the harvest of equal CD34 ⁺ cell yields independent of the anticoagulation used.ConclusionsOur data show no clinical relevant effect of a heparin containing anticoagulation in terms of an increased overall CD34 ⁺ cell collection during LVL, although this regime shows some benefits in terms of the incidence and subjective tolerance towards AR. Based on our results the decision between a citrate- and heparin-substituted anticoagulation for LVL should be driven by patient-related factors, and should concern potential contraindications of both methods.     

Salicylanilide esters of N-protected amino acids as novel antimicrobial agents

A series of novel, highly antimicrobial salicylanilide esters of N-protected amino acids were synthesized and characterized. Their in vitro antimicrobial activity against eight fungal strains and Mycobacterium tuberculosis was determined. The compounds had the highest level of activity against Aspergillus fumigatus, Absidia corymbifera and Trichophyton mentagrophytes, and these levels were higher than that of the standard drug fluconazole. In addition, three compounds showed interesting antituberculosis activity, with inhibition ranging from 89% to 99%. (S)-4-Chloro-2-(4-trifluoromethylphenylcarbamoyl)-phenyl 2-benzyloxy-carbonylamino-propionate had the highest level of both antifungal and antimycobacterial activity. The structure–activity relationships of the new compounds are discussed.     

New targeting system for antimycotic drugs: beta-glucosidase sensitive amphotericin B-star poly(ethylene glycol) conjugate

A new targeting potentially intravenous conjugate Amphotericin B (AMB)–star poly(ethylene glycol) (sPEG) (M = 25,160) has been synthesized and characterized. It contains a β-d-glucopyranoside molecular switch which is sensitive to β-glucosidases (E.C.3.2.1.21). The β-glucosidase-catalyzed release of AMB from the polymeric carrier was proved in vitro by means of spectrophotometry and HPLC.     

Obesity Triggers Enhanced MDSC Accumulation in Murine Renal Tumors via Elevated Local Production of CCL2

Obesity is one of the leading risk factors for developing renal cell carcinoma, an immunogenic tumor that is treated clinically with immunostimulatory therapies. Currently, however, the mechanisms linking obesity with renal cancer incidence are unclear. Using a model of diet-induced obesity, we found that obese BALB/c mice with orthotopic renal tumors had increased total frequencies of myeloid-derived suppressor cells (MDSC) in renal tumors and spleens by d14 post-tumor challenge, relative to lean counterparts. Renal tumors from obese mice had elevated concentrations of the known myeloid cell chemoattractant CCL2, which was produced locally by increased percentages of dendritic cells, macrophages, B cells, and CD45^- cells in tumors. MDSC expression of the CCL2 receptor, CCR2, was unaltered by obesity but greater percentages of CCR2⁺ MDSCs were present in renal tumors from obese mice. Of note, the intracellular arginase levels and per-cell suppressive capacities of tumor-infiltrating and splenic MDSCs were unchanged in obese mice relative to lean controls. Thus, our findings suggest that obesity promotes renal tumor progression via development of a robust immunosuppressive environment that is characterized by heightened local and systemic MDSC prevalence. Targeted intervention of the CCL2/CCR2 pathway may facilitate immune-mediated renal tumor clearance in the obese.     

Photosynthetic dioxygen formation studied by time-resolved delayed fluorescence measurements--method, rationale, and results on the activation energy of dioxygen formation

The analysis of the time-resolved delayed fluorescence (DF) measurements represents an important tool to study quantitatively light-induced electron transfer as well as associated processes, e.g. proton movements, at the donor side of photosystem II (PSII). This method can provide, inter alia, insights in the functionally important inner-protein proton movements, which are hardly detectable by conventional spectroscopic approaches. The underlying rationale and experimental details of the method are described. The delayed emission of chlorophyll fluorescence of highly active PSII membrane particles was measured in the time domain from 10 mus to 60 ms after each flash of a train of nanosecond laser pulses. Focusing on the oxygen-formation step induced by the third flash, we find that the recently reported formation of an S4-intermediate prior to the onset of O-O bond formation [M. Haumann, P. Liebisch, C. Müller, M. Barra, M. Grabolle, H. Dau, Science 310, 1019-1021, 2006] is a multiphasic process, as anticipated for proton movements from the manganese complex of PSII to the aqueous bulk phase. The S4-formation involves three or more likely sequential steps; a tri-exponential fit yields time constants of 14, 65, and 200 mus (at 20 degrees C, pH 6.4). We determine that S4-formation is characterized by a sizable difference in Gibbs free energy of more than 90 meV (20 degrees C, pH 6.4). In the second part of the study, the temperature dependence (-2.7 to 27.5 degrees C) of the rate constant of dioxygen formation (600/s at 20 degrees C) was investigated by analysis of DF transients. If the activation energy is assumed to be temperature-independent, a value of 230 meV is determined. There are weak indications for a biphasicity in the Arrhenius plot, but clear-cut evidence for a temperature-dependent switch between two activation energies, which would point to the existence of two distinct rate-limiting steps, is not obtained.     

Photosynthetic dioxygen formation studied by time-resolved delayed fluorescence measurements — Method, rationale, and results on the activation energy of dioxygen formation

The analysis of the time-resolved delayed fluorescence (DF) measurements represents an important tool to study quantitatively light-induced electron transfer as well as associated processes, e.g. proton movements, at the donor side of photosystem II (PSII). This method can provide, inter alia, insights in the functionally important inner-protein proton movements, which are hardly detectable by conventional spectroscopic approaches. The underlying rationale and experimental details of the method are described. The delayed emission of chlorophyll fluorescence of highly active PSII membrane particles was measured in the time domain from 10 μs to 60 ms after each flash of a train of nanosecond laser pulses. Focusing on the oxygen-formation step induced by the third flash, we find that the recently reported formation of an S4-intermediate prior to the onset of O-O bond formation [M. Haumann, P. Liebisch, C. Müller, M. Barra, M. Grabolle, H. Dau, Science 310, 1019-1021, 2006] is a multiphasic process, as anticipated for proton movements from the manganese complex of PSII to the aqueous bulk phase. The S4-formation involves three or more likely sequential steps; a tri-exponential fit yields time constants of 14, 65, and 200 μs (at 20 °C, pH 6.4). We determine that S4-formation is characterized by a sizable difference in Gibbs free energy of more than 90 meV (20 °C, pH 6.4). In the second part of the study, the temperature dependence (− 2.7 to 27.5 °C) of the rate constant of dioxygen formation (600/s at 20 °C) was investigated by analysis of DF transients. If the activation energy is assumed to be temperature-independent, a value of 230 meV is determined. There are weak indications for a biphasicity in the Arrhenius plot, but clear-cut evidence for a temperature-dependent switch between two activation energies, which would point to the existence of two distinct rate-limiting steps, is not obtained.     

Introduction of the dansyl group into histidine and tyrosine residues in peptides and proteins

Two dansyl derivatives: 1-(5-dimethylaminonaphthalene) sulfonyl (4-amino)-benzyl amine and 1-(5-dimethylaminonaphthalene) sulfonyl beta(4-aminophenyl) ethylamine, have been recently synthesized. Reaction of these compounds with nitrous acid lead to the corresponding dansyl-bearing diazonium salts. The latter derivatives can couple, under mild basic conditions, to the imidazole moiety of histidine, the phenolic ring of tyrosine and to the epsilon-amino function of lysine. The applicability of the two reagents was tested in the modification of several peptides, including [D-Phe6]LHRH, [D-Gln6]LHRH, Leu-enkephalin and Tyr-tuftsin, and proteins such as calmodulin, bovine serum albumin and nerve growth factor.     

A strategy for obtaining active mammalian enzyme from a fusion protein expressed in bacteria using phospholipase A2 as a model

An active preparation of human phospholipase A2 (PLA2) was made after expression as an insoluble fusion protein in Escherichia coli. The new key elements required for PLA2 isolation were the maintenance of the fusion protein in solution after the initial solubilization and the use of a tryptophan cleavage procedure for regeneration of native PLA2 from the fusion protein. The fusion protein was composed of a beta-galactosidase leader peptide incorporating six consecutive threonine residues to aid in insoluble inclusion body formation, followed by a tryptophan adjacent to the N-terminus of PLA2. The fusion protein was purified from cell lysates, and the leader peptide was cleaved on the C-terminal side of the tryptophan residue with N-chlorosuccinimide. The released PLA2 was refolded and renatured to produce an enzyme with activity comparable to that of other phospholipases A2.     

Structural basis of cyanobacterial photosystem II Inhibition by the herbicide terbutryn

Herbicides that target photosystem II (PSII) compete with the native electron acceptor plastoquinone for binding at the Q_B site in the D1 subunit and thus block the electron transfer from Q_A to Q_B. Here, we present the first crystal structure of PSII with a bound herbicide at a resolution of 3.2 Å. The crystallized PSII core complexes were isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus. The used herbicide terbutryn is found to bind via at least two hydrogen bonds to the Q_B site similar to photosynthetic reaction centers in anoxygenic purple bacteria. Herbicide binding to PSII is also discussed regarding the influence on the redox potential of Q_A, which is known to affect photoinhibition. We further identified a second and novel chloride position close to the water-oxidizing complex and in the vicinity of the chloride ion reported earlier (Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A., and Saenger, W. (2009) Nat. Struct. Mol. Biol. 16, 334–342). This discovery is discussed in the context of proton transfer to the lumen.