Impairment of anterograde and retrograde neurofilament transport after anti-kinesin and anti-dynein antibody microinjection in chicken dorsal root ganglia

The purpose of the present study was to investigate the participation of the motor proteins kinesin and dynein in axonal transport of neurofilaments (NF) in cultured dorsal root ganglia neurons. Therefore, we performed live-recording studies of the green fluorescent protein-tagged neurofilament M (GFP-NF-M) to assay transport processes in neurons. Co-localization studies revealed that GFP-NF-M was capable to build a functional NF network with other NF subunits, including phosphorylated heavy neurofilaments (NF-H-PH). Time-lapse recordings using confocal laser scanning microscopy exhibited fast transport of NF dots in anterograde and retrograde direction through a photobleached gap. Following microinjection of anti-kinesin antibodies or colchicine treatment an impairment of anterograde as well as retrograde NF transport was observed during live-recording experiments. In contrast, microinjection of anti-dynein antibodies only impaired retrograde transport of NF whereas the anterograde movement of GFP-NF-M was unaffected. Treatment of the cells with unspecific antibodies had no effect.     

Reversible permeabilization using high-intensity femtosecond laser pulses: applications to biopreservation

Non-invasive manipulation of live cells is important for cell-based therapeutics. Herein we report on the uniqueness of using high-intensity femtosecond laser pulses for reversibly permeabilizing mammalian cells for biopreservation applications. When mammalian cells were suspended in a impermeable hyperosmotic cryoprotectant sucrose solution, femtosecond laser pulses were used to transiently permeabilize cells for cytoplasmic solute uptake. The kinetics of cells exposed to 0.2, 0.3, 0.4, and 0.5 M sucrose, following permeabilization, were measured using video microscopy, and post-permeabilization survival was determined by a dual fluorescence membrane integrity assay. Using appropriate laser parameters, we observed the highest cell survival for 0.2 M sucrose solution (>90%), with a progressive decline in cell survival towards higher concentrations. Using diffusion equations describing the transport of solutes, the intracellular osmolarity at the inner surface of the membrane (x = 10 nm) and to a diffusive length of x = 10 microm was estimated, and a high loading efficiency (>98% for x = 10 nm and >70% for x = 10 microm) was calculated for cells suspended in 0.2 M sucrose. This is the first report of using femtosecond laser pulses for permeabilizing cells in the presence of cryoprotectants for biopreservation applications.     

Entamoeba histolytica: analysis of the trophozoite proteome by two-dimensional polyacrylamide gel electrophoresis

The Entamoeba histolytica genome project carried out at TIGR and the Sanger Institute has produced a near-complete set of deduced open reading frame sequences. These data provide strong support for the identification of signals from proteomic analyses such as two-dimensional electrophoresis by protein sequencing and/or mass spectrometric methods. To carry out an initial investigation of the E. histolytica proteome, appropriate sample preparation and silver staining protocols were adapted. After preparation of protein extracts from E. histolytica HM-1:IMSS trophozoites, solubilized proteins were separated in the first dimension in IPG (immobilized pH gradient) strips depending on their pI and subsequently in the second dimension according to their molecular weight by SDS-polyacrylamide gel electrophoresis. Of the more than 1500 protein spots visualized, several landmark spots were isolated from the gels and identified by either tryptic cleavage and subsequent MALDI-TOF mass spectrometry or by protein sequencing.     

Direct interaction between the reductase domain of endothelial nitric oxide synthase and the ryanodine receptor

We have performed the recombinant expression and purification of the reductase domain of endothelial nitric oxide synthase (eNOS) and used it as a bait in search for interacting proteins present in endothelial cells. Using mass spectrometry of the bound proteins run in a PAGE-SDS gel, we were able to identify the ryanodine receptor (RyR) as a novel eNOS-binding partner. This interaction was confirmed through immunoprecipitation of both RyR and eNOS from endothelial cells and cardiac myocytes. Immunofluorescence data indicated that a subpopulation of eNOS associates with RyR in perinuclear regions of the cell, where eNOS might be responsible for the known nitrosylation of RyR.     

Binding of JAB1/CSN5 to MIF is mediated by the MPN domain but is independent of the JAMM motif

Macrophage migration inhibitory factor (MIF) binds to c-Jun activation domain binding protein-1 (JAB1)/subunit 5 of COP9 signalosome (CSN5) and modulates cell signaling and the cell cycle through JAB1. The binding domain of JAB1 responsible for binding to MIF is unknown. We hypothesized that the conserved Mpr1p Pad1p N-terminal (MPN) domain of JAB1 may mediate binding to MIF. In fact, yeast two hybrid (YTH) and in vitro translation/coimmunoprecipitation (CoIP) analysis showed that a core MPN domain, which did not cover the functional JAB1/MPN/Mov34 metalloenzyme (JAMM) deneddylase sequence, binds to MIF comparable to full-length JAB1. YTH and pull-down analysis in conjunction with nanobead affinity matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry demonstrated that MIF(50-65) and MPN are sufficient to mediate MIF-JAB1 interaction, respectively. Finally, endogenous CoIP of MIF-CSN6 complexes from mammalian cells demonstrated that MPN is responsible for MIF-JAB1 binding in vivo, and, as CSN6 does not contain a functional JAMM motif, confirmed that the interaction does not require JAMM.     

Cyanobacterial Photosystem I lacks specificity in its interaction with cytochrome c<Subscript>6</Subscript> electron donors

In cyanobacteria, plastocyanin and cytochrome c ₆, the alternate donor proteins to Photosystem I, can be acidic, neutral or basic; the role of electrostatics in their interaction with photosystem I varies accordingly. In order to elucidate whether these changes in the electron donors’ properties correlate with complementary changes in the docking site of the corresponding photosystem, we have investigated the kinetics of reactions between three cytochrome c ₆ with isoelectric points of 5.6, 7.0 and 9.0, with Photosystem I particles from the same three genera of cyanobacteria which provided the cytochromes. The model systems compared here thus sample the full range of charge properties observed in cytochromes c ₆: acidic, basic and neutral. The rate constants and dependence on ionic strength for photosystem I reduction were distinctive for each cytochrome c ₆, but independent of Photosystem I. We conclude that the specific structural features of each cytochrome c ₆ dictate their different kinetic behaviours, whereas the three photosystems are relatively indiscriminate in docking with the electron donors.     

A four-subunit cytochrome bc1 complex complements the respiratory chain of Thermus thermophilus

Several components of the respiratory chain of the eubacterium Thermus thermophilus have previously been characterized to various extent, while no conclusive evidence for a cytochrome bc₁ complex has been obtained. Here, we show that four consecutive genes encoding cytochrome bc₁ subunits are organized in an operon-like structure termed fbcCXFB. The four gene products are identified as genuine subunits of a cytochrome bc₁ complex isolated from membranes of T. thermophilus. While both the cytochrome b and the FeS subunit show typical features of canonical subunits of this respiratory complex, a further membrane-integral component (FbcX) of so far unknown function copurifies as a subunit of this complex. The cytochrome c₁ carries an extensive N-terminal hydrophilic domain, followed by a hydrophobic, presumably membrane-embedded helical region and a typical heme c binding domain. This latter sequence has been expressed in Escherichia coli, and in vitro shown to be a kinetically competent electron donor to cytochrome c₅₅₂, mediating electron transfer to the ba₃ oxidase. Identification of this cytochrome bc₁ complex bridges the gap between the previously reported NADH oxidation activities and terminal oxidases, thus, defining all components of a minimal, mitochondrial-type electron transfer chain in this evolutionary ancient thermophile.     

Characterization of the temperature- and pressure-induced inverse and reentrant transition of the minimum elastin-like polypeptide GVG(VPGVG) by DSC, PPC, CD, and FT-IR spectroscopy

We investigated the temperature- and pressure-dependent structure and phase behavior of a solvated oligopeptide, GVG(VPGVG), which serves as a minimalistic elastin-like model system, over a large region of the thermodynamic phase field, ranging from 2 to 120°C and from ambient pressure up to ~10 kbar, applying various spectroscopic (CD, FT-IR) and thermodynamic (DSC, PPC) measurements. We find that this octapeptide behaves as a two-state system which undergoes the well-known inverse-temperature folding transition occurring at T ≈ 36°C, and, in addition, a slow trend reversal at higher temperatures, finally leading to a reentrant unfolding close to the boiling point of water. Furthermore, the pressure-dependence of the folding/unfolding transition was studied to yield a more complete picture of the p, T-stability diagram of the system. A molecular-level picture of these processes, in particular on the role of water for the folding and unfolding events of the peptide, presented with the help of molecular-dynamics simulations, is presented in a companion article in this issue.     

Membrane topography of HLA I, HLA II, and ICAM-1 is affected by IFN-gamma in lipid rafts of uveal melanomas

The lateral distribution and colocalization of HLA I, HLA-DR, and ICAM-1 proteins was studied for the first time in the plasma membrane of two human uveal melanoma cell lines, OCM-1 and OCM-3. Our fluorescence resonance energy transfer and confocal laser scanning microscopic experiments revealed that these molecules are mostly confined to the same membrane regions, where they form similar protein patterns (homo- and hetero-associates) to those found previously on other cell types of lymphoid as well as colorectal carcinoma origin. Confocal microscopic colocalization experiments with GM(1) gangliosides and the GPI-anchored CD59 molecules showed enrichment of HLA I, HLA-DR, and ICAM-1 molecules in specific membrane domains (lipid rafts) excluding the transferrin receptor. IFN-gamma remarkably increased the expression levels of these molecules and rearranged their association patterns, which can affect the adoptive immune response of effector cells.     

Capillary electrophoretic characterization of PEGylated human parathyroid hormone with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A capillary electrophoretic method (CE) for characterizing PEGylated human parathyroid hormone 1-34 (PTH) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is described. CE was used to optimize the PEGylation of PTH through control of the reaction pH and the molar ratio of reactants with the advantages of minimal sample consumption and high separation capacity. The mono-PEGylated PTH (mono-PEG-PTH) was isolated and then digested with endoproteinase Lys-C. Resistance to Lys-C digestion on the PEGylation sites in the mono-PEG-PTH resulted in patterns of CE electropherograms different from that of the native PTH, and the PEGylation sites were assigned accordingly. The extent of positional isomers present in the mono-PEG-PTH was also determined by quantifying PEGylated fragments in the same CE electropherogram. In conclusion, the CE analysis of the Lys-C-digested sample allowed for simultaneous analysis of the PEGylation site and the extent of positional isomers in the mono-PEG-PTH. The results were confirmed by MALDI-TOF MS. This method will be applicable for characterizing PEGylation of other therapeutic peptides.