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.     

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.     

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.     

Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis

We describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. We measured numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR). We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions.     

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.     

Acyl chain conformational ordering in 1,2 dipalmitoylphosphatidylethanolamine. Integration of FT-IR and 2H NMR results.

The extent of trans-gauche isomerization at the 4 and 4' positions of the acyl chains of fully hydrated 4,4,4',4'-d4 1,2-dipalmitoylphosphatidylethanolamine (4-d4 DPPE) bilayers was quantitatively evaluated from the infrared (IR) intensity of the CD2 rocking modes. About 20% gauche conformers were observed at 72 degrees C (above Tm), while at 23 degrees C, well below Tm, about 4% were noted. The order parameter SC-D was determined from 2H nuclear magnetic resonance (NMR) quadrupolar splittings. SC-D is the product of a segmental order parameter (S gamma), which depends on conformational order, and a chain order parameter (S alpha) which depends on slower motions such as chain wobble. The IR-determined percentage of gauche forms was converted into a segmental order parameter and factored out of the measured value for SC-D to yield an estimate of S alpha = 0.59 for L alpha phase DPPE. A comparison with S alpha for 1,2-dipalmitoylphosphatidylcholine (DPPC) suggests that increased wobble is responsible for enhanced motional averaging of the quadrupolar splittings in the latter at a similar reduced temperature. The extent of conformational disordering [at the 4(4') position] is essentially unchanged between the two molecules. The current study demonstrates the advantage of integrating quantitative IR with 2H NMR data, for elucidation of the contributions of the individual motions that average the NMR quadrupolar splittings.     

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.