Early electrochemical events in lectin-lymphocyte interaction

Concanavalin A, at extremely low concentrations, will produce significant increases in the electrophoretic mobility of murine splenic T lymphocytes. It has been established that the alteration in cellular surface charge is mediated by a factor produced by those lymphocytes that have reacted directly with con A. We originally conjectured that the mobility change might be the consequence of an alteration in the distribution of the charged moieties of membrane glycoproteins. The results of experiments conducted at low temperature raise some questions about this mechanism. Further experiments have been performed to establish the nature of the physicochemical alterations in the peripheral zone of the factor-stimulated lymphocytes that are manifest as changes in cellular surface charge. The results of these studies indicate that, subsequent to the interaction of T lymphocytes with con A, there is a reduction in the number of positively charged amino groups effective at the electrophoretic surface of the cells.     

Isolation of a cDNA clone for the alpha subunit of the human GABA-A receptor

A cDNA clone of an alpha subunit of the human GABA-A receptor has been isolated. The human clone (pCLL800) contains 1055 nucleotides in an open reading frame and 260 nucleotides in the 5' non-coding region. The 351 amino acid sequence of this human alpha subunit shows 97% homology with its bovine counterpart. Hybridization of pCLL800 to Northern blots shows a 3.9/4.3 Kb RNA doublet in human cortex, rat whole brain, cortex, hippocampus, midbrain, olfactory bulb and cerebellum. Developmental studies show that the levels of the rat alpha mRNA increase between one and three weeks of age in a manner similar to the development of the benzodiazepine binding sites.     

Non-ideal mixing and fluid–fluid immiscibility in phosphatidic acid–phosphatidylethanolamine mixed bilayers

Differential scanning calorimetry (DSC) was used to study the miscibility of phosphatidic acids (PAs) with phosphatidylethanolamines (PEs) as a function of chain length (n = 14, 16) and degree of ionization of PAs at pH 4, pH 7, and pH 12. Phase diagrams were constructed using temperature data for onset and end of the phase transition obtained from the direct simulation of the heat-capacity curves. The phase diagrams were analyzed by simulations of the coexistence curves utilizing a four-parameter regular solution model. For PA–PE mixtures, the non-ideality parameters are a function of composition indicating non-symmetric non-ideal mixing behavior. At pH 7, where the PA component is negatively charged, the systems DMPA:DMPE and DPPA:DPPE have positive non-ideality parameters ρ ₁ in both phases, indicating a preferred aggregation of like molecules. In contrast, DMPA:DPPE and DPPA:DMPE mixtures had negative ρ ₁ values. Measurements at pH 4 showed that mixed pair formation is favored when PA is protonated. At pH 12 where PA is doubly charged, highly positive ρ _l1 parameters are obtained for the liquid-crystalline phase except for the system DPPA:DPPE (ρ ₁ < 0). This indicates clustering of like molecules and possibly domain formation in the liquid-crystalline phase. DPPA:DMPE at pH 12 even shows a miscibility gap in the liquid-crystalline phase. Obviously, despite the presence of doubly charged PA a fluid–fluid immiscibility is induced.     

An intelligent bioreactor system for the cultivation of a bioartificial vascular graft

Cardiovascular disease is the most common cause of death, accounting for 31% of deaths worldwide. As purely synthetic grafts implicate concomitant anticoagulation and autologous veins are rare, tissue‐engineered vascular grafts are urgently needed. For successful in vitro cultivation of a bioartificial vascular graft, the suitable bioreactor should provide conditions comparable to vasculogenesis in the body. Such a system has been developed and characterized under continuous and pulsatile flow, and a variety of sensors has been integrated into the bioreactor to control parameters such as temperature, pressure up to 500 mbar, glucose up to 4.5 g/L, lactate, oxygen up to 150 mbar, and flow rate. Wireless data transfer (using the ZigBee specification based on the IEEE 802.15.4 standard) and multiple corresponding sensor signal processing platforms have been implemented as well. Ultrasound is used for touchless monitoring of the growing vascular structure as a quality control before implantation (maximally achieved ultrasound resolution 65 μm at 15 MHz). To withstand the harsh conditions of steam sterilization (120°C for 20 min), all electronics were encapsulated. With such a comprehensive physiologically conditioning, sensing, and imaging bioreactor system, all the requirements for a successful cultivation of vascular grafts are available now.     

Gene expression in Xenopus oocytes

1. Gene expression in Xenopus oocytes is now an integral part of many molecular cloning strategies. 2. For some genes, such as those encoding the ion channels, this system has emerged as the only available means to authenticate and examine the biological activities of the cloned DNA. 3. This review discusses some of the current applications of Xenopus oocytes in modern molecular biology.     

Insights into protein–polysorbate interactions analysed by means of isothermal titration and differential scanning calorimetry

Therapeutic proteins formulated as liquid solutions at high protein concentration are very sensitive to chemical and physical degradation. Especially avoiding the formation of protein aggregates is very crucial for product quality. In order to stabilize the colloidal properties of protein therapeutics various excipient are used. Especially the detergents polysorbate 20 and 80 are common. However, the mechanism upon which the detergents protect the protein from aggregation is not really known. The present study investigates the interaction of polysorbate 20 and 80 with different proteins: lysozyme, bovine serum albumin (BSA) and an immunoglobulin. The interaction and binding of the detergents to the proteins is investigated by isothermal titration calorimetry (ITC). From ITC the thermodynamic parameters (ΔH: change in enthalpy, ΔS: entropy and ΔG: free energy) upon binding are derived as well as the binding constant K _a. The thermal stability of the proteins in the presence of the detergent is assessed by differential scanning calorimetry (DSC). The results show that both detergents bind to BSA with K _a between 8 and 12 × 10³ M⁻¹ with ΔH −50 to −60 kJ/mol (25°C). One to two detergent molecules bind to BSA. The presence of both detergents induces a weak stabilisation of the thermal denaturation properties of BSA. However, the interaction of polysorbate 20 and 80 with lysozyme and the immunoglobulin is quite negligible. The presence of the detergents up to a concentration of 2 mM has no impact on the heat capacity curve neither a destabilisation nor a stabilisation of the native conformation is observed.     

Cadmium,nickel, copper,and zinc influence on microfilament organization in Arabidopsis root cells

The plant cytoskeleton orchestrates such fundamental processes in cells as division, growth and development, polymer cross-linking, membrane anchorage, etc. Here, we describe the influence of Cd²⁺, Ni²⁺, Zn²⁺, and Cu²⁺ on root development and vital organization of actin filaments into different cells of Arabidopsis thaliana line expressing GFP-FABD2. CdSO₄, NiSO₄, CuSO₄, and ZnSO₄ were used in concentrations of 5–20 µM in this study. It was found that Cd, Ni, and Cu cause dose-dependent primary root growth inhibition and alteration of the root morphology, whereas Zn slightly stimulates root growth and does not affect the morphology of Arabidopsis roots. This growth inhibition/stimulation correlated with the various sensitivities of microfilaments to Cd, Ni, Cu, and Zn action. It was established that Cd, Ni, and Cu affected predominantly the actin filaments of meristematic cells. Cells of transition and elongation zones demonstrated strong actin filament sensitivity to Cd and Cu. Microfilaments of elongating root cells were more sensitive to Ni and Cu. Although Cd, Ni, and Cu stimulated root hair growth after long-term treatment, actin filaments were destroyed after 1 h exposure with these metals. Zn did not disrupt native actin filament organization in root cells. Thus, our investigation shows that microfilaments act as sensitive cellular targets for Cd, Ni, and Cu. More data on effects on native actin filaments organization would contribute to a better understanding of plant tolerance mechanisms to the action of these metals.     

Dictyostelium discoideum gene family contains a long internal amino acid repeat

Two different cDNA clones denoted pTO270-6 and pTO270-11 represent two mRNAs that are developmentally regulated during spore germination in Dictyostelium discoideum. The respective mRNAs are found only during early germination and are not present in other stages of growth or multicellular development. Four different genomic clones that hybridize to sequences that are common to both of the 270 cDNA clones were isolated from Dictyostelium libraries and sequenced. Two are the genes for the two cDNAs, and the other two represent genes that do not seem to be transcribed. All four genomic sequences possess a very unusual internal feature in the deduced protein sequences composed of a monotonous repeat of the tetrapeptide threonine-glutamic acid-threonine-proline. The other portions of the proteins have no homology among themselves. The deduced protein corresponding to the 270-6 gene is very similar to avocado (Persea americana) cellulase. Since cellulose in the spore wall has to be digested during spore germination this suggests that this protein may function as an endo-(1,4)-beta-D-glucanase during germination.