Monitoring of effector and target cell stimulation during conjugation by fluorescence polarization

The aim of the present study was to trace early intracellular changes induced in effector and target cells during their conjugation. This was performed by monitoring the intracellular fluorescein fluorescence polarization (IFFP), using the Cellscan apparatus. This apparatus permits the repetitive spectroscopic measurement of individual selected live cells within a population of many cells, while the location of each cell is known and preserved during the various cell manipulations and/or their suspending medium. Both natural killer (NK) and lymphocyte activated killer (LAK) cells were used as effector cells, while NK-sensitive K562 and NK-resistant Daudi cell lines were used as targets. In this study kinetic IFFP measurements were carried out for a period of approximately 4 h following cell attachment. Within minutes following effector-target conjugation, transient reduction of IFFP was observed consecutively, first in the effector and then in the target cells. A continuous reduction of IFFP occurring only in target cells was also found 50 min following conjugation. No reduction in IFFP was observed using NK- and LAK-resistant target cells. Good correlation was found between early stages of conjugation, as assessed by IFFP, and cytolytic efficiency as assessed by ⁵¹chromium release assay. When NK-resistant and LAK-resistant target cells were used, no reduction of IFFP was observed.     

High-resolution 31P-1H two-dimensional Nuclear Magnetic Resonance spectra of unsonicated lipid mixtures spinning at the magic-angle

For multilamellar suspensions of phospholipids, the ¹H and ³¹P Nuclear Magnetic Resonance (NMR) spectra obtained with magic-angle spinning (MAS) exhibit resolution comparable to that of sonicated vesicles. However, specific lipid head groups cannot be recognized in a lipid mixture using one-dimensional NMR spectroscopy. We show here that the combination of MAS and two-dimensional Heteronuclear Overhauser Effect SpectroscopY (HOESY) reveals magnetic interactions between the phosphate and its neighbouring protons and thus allows the distinction in situ of several lipids in a mixture. The ³¹P-¹H HOESY spectra of suspensions of phosphatidylcholine and phosphatidylglycerol or phosphatidylcholine, phosphatidylethanolamine and sphingomyelin are shown as examples. In the course of these experiments, intramolecular spin-diffusion as well as intermolecular interactions between lipids and water were observed. The technique should enable the investigation of lipid-lipid and lipid-protein interactions, lipid hydration as well as lipid asymmetry in membranes without the use of isotopically labeled lipids. Received: 18 April 1996 / Accepted: 26 July 1996     

Design and engineering of photosynthetic light-harvesting and electron transfer using length, time, and energy scales

Decades of research on the physical processes and chemical reaction-pathways in photosynthetic enzymes have resulted in an extensive database of kinetic information. Recently, this database has been augmented by a variety of high and medium resolution crystal structures of key photosynthetic enzymes that now include the two photosystems (PSI and PSII) of oxygenic photosynthetic organisms. Here, we examine the currently available structural and functional information from an engineer's point of view with the long-term goal of reproducing the key features of natural photosystems in de novo designed and custom-built molecular solar energy conversion devices. We find that the basic physics of the transfer processes, namely, the time constraints imposed by the rates of incoming photon flux and the various decay processes allow for a large degree of tolerance in the engineering parameters. Moreover, we find that the requirements to guarantee energy and electron transfer rates that yield high efficiency in natural photosystems are largely met by control of distance between chromophores and redox cofactors. Thus, for projected de novo designed constructions, the control of spatial organization of cofactor molecules within a dense array is initially given priority. Nevertheless, constructions accommodating dense arrays of different cofactors, some well within 1 nm from each other, still presents a significant challenge for protein design.     

Identification of functional modules using network topology and high-throughput data

Background  

With the advent of systems biology, biological knowledge is often represented today by networks. These include regulatory and metabolic networks, protein-protein interaction networks, and many others. At the same time, high-throughput genomics and proteomics techniques generate very large data sets, which require sophisticated computational analysis. Usually, separate and different analysis methodologies are applied to each of the two data types. An integrated investigation of network and high-throughput information together can improve the quality of the analysis by accounting simultaneously for topological network properties alongside intrinsic features of the high-throughput data.     

In vitro enzyme evolution: the screening challenge of isolating the one in a million

The generation of large mutant libraries for in vitro enzyme evolution presents the challenge of effectively screening libraries of 10⁴–10⁷ mutants on the basis of simultaneously assaying their biocatalytic activity. In this review, we highlight the main steps involved in this process, describe the alternative approaches to address this challenge, survey the state-of-the-art technology and assess achievements already made. It is anticipated that, as a result of the expected accomplishment of further improvements in high-throughput screening that will allow routine screening of whole libraries, the number of useful new and improved enzymes derived through in vitro enzyme evolution will expand rapidly in the near future.