Clodronate liposomes: perspectives in research and therapeutics

Liposomes encapsulating the bisphosphonate clodronate can be used for the transient suppression of macrophage functions. Given the important role of macrophages in various disorders, the application of clodronate liposomes has been studied in several models of rheumatoid arthritis, neurological disorders such as experimental allergic encephalomyelitis and spinal cord injury, autoantibody mediated disorders such as immune thrombocytopenic purpura (ITP) and for the improvement of the efficacy of gene transfer and drug targeting.     

Experimental and calculated shift in pK(a) upon binding of phosphotyrosine peptide to the SH2 domain of p56(lck)

The pH dependence of the affinity of a 11-mer phosphotyrosine (pY) peptide (EPQpYEEIPIYL-NH2) for the SH2 domain of the tyrosine kinase p56(lck) was investigated with surface plasmon resonance (SPR). From SPR competition experiments the affinity in solution was obtained. The pH dependence of the affinity in solution can be well described by a proton linkage model with a single pK(a) shift upon binding, from 6.1 to 4.7. This shift is ascribed to the transition from the -2 to the -1 ionisation state of the tyrosine phosphate group. Based on the X-ray structure for the complex with Lck SH2, a pK(a) value of 5.3 for the bound pY peptide was computed, modelling the solvated protein as a system of point charges in a continuum. With the phosphate in the -2 state the binding energy is 1.8 kcal/mol more favourable than for the -1 state, corresponding to a 20-fold higher affinity. A proper charge is relevant in the design of potential therapeutic Lck SH2 ligands with mimics for the metabolically unstable tyrosine phosphate group.     

From time temperature integrator kinetics to time temperature integrator tolerance levels: heat-treated milk

Six milk compounds were studied as potential intrinsic time temperature integrators (TTIs) for the assessment of heat-treated milk. These include the enzymes alkaline phosphatase and lactoperoxidase, the whey protein beta-lactoglobulin and the chemical compounds hydroxymethylfurfural, lactulose and furosine. In previous research the inactivation/denaturation/formation kinetics of these compounds were analyzed under isothermal and nonisothermal conditions and evaluated for variability of the milk composition. The present paper focuses on the implementation of the TTIs. TTIs are validated with respect to microbiological indices and quality attributes, and a quantitative relationship between the denaturation, inactivation or formation of the TTIs and technological processes is established by construction of general time temperature tolerance (TTT) diagrams. In these diagrams temperature time combinations are presented, which lead to the same formation, inactivation or denaturation of TTIs, or result in the same level of microbiological destruction or quality degradation of the product. TTT-diagrams are very informative since they allow visualization of the impact of a thermal process on milk and evaluation of criteria for evaluating milk authenticity (conformity of the product with the terminology applied). Moreover, the optimum combination of temperature and time of heating may be readily deduced from these diagrams.     

Liquid chromatographic method for the quantitative determination of Nepsilon-carboxymethyllysine in human plasma proteins

The modification of the lysine moieties of proteins to Nepsilon-carboxymethyllysine (CML) is supposed to play a major role in the development of long-term complications in patients with diabetes mellitus. This paper presents an analytical method for the quantitative determination of CML in plasma proteins, which could be used for studying the development of diabetic complications. The method is based on isolating proteins from plasma by precipitation with trichloroacetic acid and hydrolysing these under acidic conditions (6M hydrochloric acid at 110 degrees C for 20 h) to the individual amino acids. After hydrolysis, CML is derivatised along with the other amino acids to 9-fluorenylmethoxycarbonyl (FMOC) derivatives, which are subsequently separated by reversed-phase column liquid chromatography using a 150 mm x 4.6 mm C8 column and a mobile phase of 25 mM potassium phosphate buffer (pH 2.0) and acetonitrile (80:20 (v/v)) and detected using fluorescence detection (excitation at 260 nm and emission at 310 nm). Quantification of the protein-bound CML content of a plasma sample is achieved using standard addition. The impact of several aspects of the sample preparation and chromatography on method performance is discussed. Method evaluation results are reported and show that this method is capable of determining CML with good accuracy and precision (below 10%) in the relevant concentration range (1-10 microg/ml), with a limit of detection of 0.2 microg/ml.     

An unusual intraerythrocytic parasite of Parablennius cornutus from South Africa

An intertidal horned blenny, Parablennius cornutus, captured at De Hoop Nature Reserve, South Africa, was found to harbor an unusual blood parasite and the haemogregarine Haemogregarina bigemina. In Giemsa-stained blood films, the enigmatic parasite occurred primarily as intraerythrocytic ringlike stages, with unstained centers and peripheral bands of beaded chromatin, not unlike Haemohormidium spp. Larger forms of the same organism stained pink with Giemsa, with nuclei occurring as 4-8 minute structures around the parasite body or distributed within it. These larger parasites apparently segmented into up to 8 individuals that were rounded or oval with deep-stained, comma-shaped or polar regions surrounding blue cytoplasm. Extracellular, binucleate, sporelike structures in clusters of as many as 16 individuals were also seen in blood films. Praniza larvae of the isopod Gnathia africana were seen in histological sections of gill tissue. Examination of spleen tissue by transmission electron microscopy showed intraerythrocytic organisms with ultrastructural characteristics like those of Haematractidium scombri, namely, a single boundary membrane, sometimes closely apposed nuclei with nucleoli, and profiles of dense material of variable structure. It is concluded that the parasite is probably related to Haemohormidium spp. and H. scombri, but it also shares features with some Microsporida.     

T cell-activated macrophages are capable of both recognition and rejection of pancreatic islet xenografts

Macrophages have been proposed as the major effector cell in T cell-mediated xenograft rejection. To determine their role in this response, NOD-SCID mice were transplanted with fetal pig pancreas (FPP) before reconstitution with CD4(+) T cells from BALB/c mice. Twelve days after CD4(+) T cell reconstitution, purified macrophages (depleted of T cells) were isolated from CD4(+) T cell-reconstituted FPP recipient mice and adoptively transferred to their nonreconstituted counterparts. After adoptive macrophage transfer, FPP recipient mice transferred with macrophages from CD4(+) T cell-reconstituted mice demonstrated xenograft destruction along with massive macrophage infiltration at day 4 and complete graft destruction at day 8 postmacrophage transfer. By contrast, FPP recipients that received macrophages from nonreconstituted mice showed intact FPP xenografts with few infiltrating macrophages at both days 4 and 8 after macrophage transfer. The graft-infiltrating macrophages showed increased expression of their activation markers. Depletion of endogenous macrophages or any remaining CD4(+) T cells did not delay graft rejection in the macrophage-transferred FPP recipients, whereas depletion of transferred macrophages with clodronate liposomes prevented graft rejection. Our results show that macrophages primed by FPP and activated by CD4(+) T cells were attracted from the peripheral circulation and were capable of specific targeting and destruction of FPP xenografts. This suggests that in xenograft rejection, there are macrophage-specific recognition and targeting signals that are independent of those received by T cells.     

Pathways accessory to proteasomal proteolysis are less efficient in major histocompatibility complex class I antigen production

Degradation of cytosolic proteins depends largely on the proteasome, and a fraction of the cleavage products are presented as major histocompatibility complex (MHC) class I-bound ligands at the cell surface of antigen presenting cells. Proteolytic pathways accessory to the proteasome contribute to protein turnover, and their up-regulation may complement the proteasome when proteasomal proteolysis is impaired. Here we show that reduced reliance on proteasomal proteolysis allowed a reduced efficiency of MHC class I ligand production, whereas protein turnover and cellular proliferation were maintained. Using the proteasomal inhibitor adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulphone, we show that covalent inhibition of all three types of proteasomal beta-subunits (beta(1), beta(2), and beta(5)) was compatible with continued growth in cells that up-regulate accessory proteolytic pathways, which include cytosolic proteases as well as deubiquitinating enzymes. However, under these conditions, we observed poor assembly of H-2D(b) molecules and inhibited presentation of endogenous tumor antigens. Thus, the tight link between protein turnover and production of MHC class I ligands can be broken by enforcing the substitution of the proteasome with alternative proteolytic pathways.