Resonant energy transfer from proteins to pyridine nucleotides in mitochondria

BACKGROUND: Anaesthetic agents inhibit certain functions of human neutrophils. The respiratory burst (RB) enzyme in the plasma membrane of neutrophils leads to the production of superoxide anion. The oxygen radicals are responsible for killing phagocytised micro-organisms. We investigated the in vitro influence of remifentanil, fentanyl, and alfentanil on the respiratory burst of human neutrophils. METHODS: For the flow-cytometric evaluation, leukocytes were obtained as supernatant following sedimentation and were incubated with the tested drugs. The concentrations in vitro were adjusted to conform to the plasma concentrations reported for anaesthesia and also to 10-fold higher concentrations. The RB was measured by intracellular oxidation of dihydrorhodamine to fluorescent rhodamine after induction of phorbol-myristate-acetate (PMA), Escherichia coli (E. coli) or priming by tumour necrosis factor alpha followed by stimulation of n-formyl-methionyl-leucyl-phenylalanine (TNF-alpha/FMLP). In order to exclude prestimulation of the neutrophil granulocytes, negative controls were carried out. Propidium iodide (PI) was added for viability discrimination immediately prior to flow cytometry measurement. RESULTS: Regardless of the triggering agents chosen (PMA, E. coli, TNF-alpha/FMLP), remifentanil, fentanyl, and alfentanil had no significant effect on the neutrophils' respiratory burst even in concentrations which were higher than those encountered during in vivo conditions. CONCLUSION: With respect to peri- and postoperative risk of infection, anaesthetics and analgetics with no inhibiting effect on neutrophil function should be used. These results show that remifentanil, fentanyl, and alfentanil do not influence the neutrophils' respiratory burst in vitro.     

On the Formation of a Diffusion Bond from Cold-Spray Coatings

To understand the development of diffusion bonding, which can increase the bonding strength, three different cold-sprayed coating/substrate systems were investigated, Ni/Cu, Cu/Cu, and Al/Mg, by annealing at increased temperatures for various times. The formation of intermetallic compounds in the Al/Mg system reduced the bonding strength dramatically. In Cu/Cu and Ni-Cu, diffusion bonds developed at lower temperatures as Ni-Cu forms an isomorphous system, which increased the bonding strength effectively. However, higher temperature annealing reduced bonding strength ultimately because of the Kirkendall pores.     

Mammalian glycophosphatidylinositol anchor transfer to proteins and posttransfer deacylation

The glycophosphatidylinositol (GPI) anchors of proteins expressed on human erythrocytes and nucleated cells differ with respect to acylation of an inositol hydroxyl group, a structural feature that modulates their cleavability by PI-specific phospholipase C (PI-PLC). To determine how this GPI anchor modification is regulated, the precursor and protein-associated GPIs in two K562 cell transfectants (ATCC and .48) exhibiting alternatively PI-PLC-sensitive and resistant surface proteins were analyzed and the temporal relationship between GPI protein transfer and acquisition of PI-PLC sensitivity was determined. Nondenaturing PAGE analyses demonstrated that, whereas in .48 transfectants the GPI anchors in decay accelerating factor (DAF) and placental alkaline phosphatase (PLAP) were >95% acylated, in ATCC transfectants, they were 60 and 33% unsubstituted, respectively. In contrast, TLC analyses revealed that putative GPI donors in the two lines were identical and were >/=95% acylated. Studies of de novo DAF biosynthesis in HeLa cells bearing proteins with >90% unacylated anchors showed that within 5 min at 37 degreesC (or at 18 degreesC, which does not permit endoplasmic reticilum exit), >50% of the anchor in nascent 44-kDa proDAF protein exhibited PI-PLC sensitivity. In vitro analyses of the microsomal processing of miniPLAP, a truncated PLAP reporter protein, demonstrated that the anchor donor initially transferred to prominiPLAP was acylated and then progressively was deacylated. These findings indicate that (i) the anchor moiety that initially transfers to nascent proteins is acylated, (ii) inositol acylation in mature surface proteins is regulated via posttransfer deacylation, which in general is cell-specific but also can be protein-dependent, and (iii) deacylation occurs in the endoplasmic reticulum immediately after GPI transfer.     

Myoglobin-induced oxidative damage: evidence for radical transfer from oxidized myoglobin to other proteins and antioxidants

Reaction of equine Fe(III) myoglobin with H2O2 gives rise to an Fe(IV)-oxo species at the heme center and protein (globin)-derived radicals. Studies have shown that there are two (or more) sites for the protein-derived radical: at tyrosine (Tyr-103) or tryptophan (Trp-14). The latter radical reacts rapidly with oxygen to give a Trp-derived peroxyl radical. The formation of both the tyrosine phenoxyl radical and the tryptophan-derived peroxyl species have been confirmed in the present study; the latter appears to be the major initial radical, with the phenoxyl radical appearing at longer reaction times, possibly via secondary reactions. We have investigated, by EPR spectroscopy, the reactivity of the Trp-14 peroxyl radical with amino acids, peptides, proteins, and antioxidants, with the aim of determining whether this species can damage other targets, i.e., whether intermolecular protein-to-protein radical transfer and hence chain-oxidation occurs, and the factors that control these reactions. Three amino acids show significant reactivity: Tyr, Trp, and Cys, with Cys the least efficient. Evidence has also been obtained for (inefficient) hydrogen abstraction at peptide alpha-carbon sites; this may result in backbone cleavage in the presence of oxygen. The myoglobin Trp-14 peroxyl radical has been shown to react rapidly with a wide range of proteins to give long-lived secondary radicals on the target protein. These reactions appear to mainly involve Tyr residues on the target protein, although evidence for reaction at Trp has also been obtained. Antioxidants (GSH, ascorbate, Trolox C, vitamin E, and urate) react with the myoglobin-derived peroxyl radical; in some cases antioxidant-derived radicals are detected. These reactions are only efficient at high antioxidant concentrations, suggesting that protein-to-protein damage transfer and protein chain-oxidation may occur readily in biological systems.     

Intraparticle mass transfer in high-speed chromatography of proteins

Styrene 7,8-oxide and ethylene oxide are widely used genotoxic bulk chemicals, which have been associated with potential carcinogenic hazard for occupationally exposed workers. Both epoxides alkylate DNA preferentially at the N-7 position of guanine and consequently produce single-strand breaks and alkali labile sites in the DNA of exposed cells. In order to study the role of human microsomal epoxide hydrolase (hmEH) in protecting cells against genotoxicity of styrene 7,8-oxide and ethylene oxide, we expressed the cDNA of hmEH in V79 Chinese hamster cells. We obtained a number of cell clones that expressed functionally active epoxide hydrolase. Among these, the clone 92hmEH-V79 revealed an especially high enzymatic mEH activity toward styrene 7,8-oxide (10 nmol converted per mg of protein per min, measured in the 9,000 x g supernatant of the cell homogenate), that was 100 times higher than that determined in mock-transfected cells and within the range of mEH activity in human liver. Styrene 7,8-oxide-induced DNA single-strand breaks/alkali labile sites (dose range 10 microM to 1 mM styrene 7,8-oxide) measured by the alkaline elution technique were significantly lower in the 92hmEH-V79 cells as compared to the mock-transfected cells. The protection against styrene 7,8-oxide genotoxicity in 92hmEH-V79 cells could be abolished by addition of valpromide, a selective inhibitor of microsomal epoxide hydrolase. These results clearly show that the metabolism of styrene 7,8-oxide by hmEH in 92hmEH-V79 cells was responsible for the protection against styrene 7,8-oxide genotoxicity. On the other hand, no protective effect of epoxide hydrolase expression could be observed on ethylene oxide-induced DNA damage with the recombinant cell line over a dose range of 0.5-2.5 mM ethylene oxide. This selectivity of the protective effect on epoxide genotoxicity thus appears to be an important factor that must be taken into account for the prediction of the genotoxic risk of epoxides themselves or compounds that can be metabolically activated to epoxides.     

Lessons from a monoclonal antibody to double-stranded DNA

In this graduate school of Tokyo Medical and Dental University, I was given a somewhat vague but farsighted theme by a professor: "protein-DNA interaction." Within these simple words, I was given a free hand. Being curious about the etiology of connective tissue diseases, I began to study the biochemistry and pathophysiology of autoimmunity, especially the nature of anti-DNA antibodies that are the principal anti-nuclear antibodies observed in patients with systemic lupus erythematosus (SLE). My thesis was on the characterization of serum anti-DNA antibodies purified by a novel method of affinity column chromatography. Thereafter, I remained involved in this fascinating field. In spite of the rapid progress in molecular immunology, the etiology of any particular systemic autoimmune disorder remains elusive at this point. Here, works on a monoclonal anti-DNA antibody performed in the laboratories of Dr. B. D. Stollar (Tufts University, Boston), Dr. Y. Kanai (University of Tokyo), and in our laboratory will be reviewed along with related articles.     

The role of structure in antibody cross-reactivity between peptides and folded proteins

Peptides have the potential for targeting vaccines against pre-specified epitopes on folded proteins. When polyclonal antibodies against native proteins are used to screen peptide libraries, most of the peptides isolated align to linear epitopes on the proteins. The mechanism of cross-reactivity is unclear; both structural mimicry by the peptide and induced fit of the epitope may occur. The most effective peptide mimics of protein epitopes are likely to be those that best mimic both the chemistry and the structure of epitopes. Our goal in this work has been to establish a strategy for characterizing epitopes on a folded protein that are candidates for structural mimicry by peptides. We investigated the chemical and structural bases of peptide-protein cross-reactivity using phage-displayed peptide libraries in combination with computational structural analysis. Polyclonal antibodies against the well-characterized antigens, hen eggwhite lysozyme and worm myohemerythrin, were used to screen a panel of phage-displayed peptide libraries. Most of the selected peptide sequences aligned to linear epitopes on the corresponding protein; the critical binding sequence of each epitope was revealed from these alignments. The structures of the critical sequences as they occur in other non-homologous proteins were analyzed using the Sequery and Superpositional Structural Assignment computer programs. These allowed us to evaluate the extent of conformational preference inherent in each sequence independent of its protein context, and thus to predict the peptides most likely to have structural preferences that match their protein epitopes. Evidence for sequences having a clear structural bias emerged for several epitopes, and synthetic peptides representing three of these epitopes bound antibody with sub-micromolar affinities. The strong preference for a type II beta-turn predicted for one peptide was confirmed by NMR and circular dichroism analyses. Our strategy for identifying conformationally biased epitope sequences provides a new approach to the design of epitope-targeted, peptide-based vaccines.     

Preliminary immunohistochemical characterization of a monoclonal antibody (PRO:4-216) prepared from human prostate cancer nuclear matrix proteins

OBJECTIVES: A nuclear matrix protein (PC-1) was previously identified and reported to be present only in human prostate cancer but absent in tissue from the same prostate containing either benign prostatic hyperplasia (BPH) or normal prostate tissue. The PC-1 protein was identified by high resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and exhibited a molecular mass of 56 kDa and an isoelectric point of 6.58. This work investigates the immunohistochemical characterization of PRO:4-216, a monoclonal antibody to PC-1. METHODS: Areas of the 2D-PAGE gels containing the human prostate cancer nuclear matrix proteins near PC-1 were isolated, eluted, and injected into mice to develop monoclonal antibodies. Antibodies were screened by immunofluorescence for nuclear reactivity to a human prostate cancer cell line (LnCaP) and by 1D and 2D Western blots for reactivity with prostate cancer nuclear matrix proteins. Monoclonal antibodies from the selected clones were affinity purified. The monoclonal antibody PRO:4-216 was used to analyze frozen tissue from 20 cancerous, 22 BPH, and 22 normal regions from fresh human prostate specimens. Tissue sections were analyzed for their immunohistochemical (IHC) (horseradish peroxidase) staining. RESULTS: Using a reference value for positive staining at an IHC score of greater than 50, 85% (17 of 20) of the cancerous, 5% (1 of 22) of the BPH, and 9% (2 of 22) of the normal prostate tissues stained positive. The one BPH and two normal tissues that stained positive were taken from prostates in which the adjacent cancerous tissue also demonstrated high IHC scores (greater than 225). CONCLUSIONS: These data demonstrate nuclear reactivity on fresh frozen human prostate cancer tissue for the monoclonal antibody PRO:4-216. PRO:4-216 may aid in distinguishing normal prostate and BPH from cancerous tissue.     

Cell-to-cell transfer of glycosylphosphatidylinositol-anchored membrane proteins during sperm maturation

The majority of gene therapy protocols have used or plan to use retroviral vectors based upon murine leukaemia virus. These vectors are able to infect many different cell types, and the retroviral promoter, which is often used to control the expression of a therapeutic gene, is active in a wide range of different cell types. Safe and efficient gene transfer systems, whether based upon retroviruses or other agents, should deliver beneficial genes only to cells that require their therapeutic action, and these genes ideally should be expressed exclusively in such cells. In this paper, strategies for redirecting the infection spectrum of retroviral vectors in order to obtain cell-targeted gene delivery are discussed. These strategies include the engineering of the retroviral envelope protein, which, together with the availability of its cognate receptor, determines infectivity, and the use of proteins from other enveloped viruses of both retroviral and nonretroviral origin in the cell lines used to produce retroviral vector virus particles. Expression targeting can be achieved by limiting the expression of therapeutic genes to the cell type(s) of interest using promoters from genes that are normally active in these cells. This approach to targeting is illustrated using promoters from genes expressed in either the liver, the pancreas or the mammary gland as a means to limit gene expression specifically to the cell types that make up these organs. The successful utilization of new generations of targeted retroviral vectors in the clinic may well pave the way for superior gene delivery systems of the future that seek out their target cell, delivering a therapeutic gene to and expressing it only in such cells.     

Influence of anti-LDL receptor antibody on the transfer of cholesterol from LDL to isolated lymphocytes in normal subjects

Results 2 1/2 years after an enuresis nocturna training are presented, including rate of success, percentage and duration of relapse for 113 children (mean age 11.6 year at the start of the training). The bibliotherapeutic treatment by parents did not require any intervention by a professional. Behaviour of parents in the event of a relapse differed between training conditions. Children in the Arousal condition recovered faster from a relapse, 90% of their parents used the Arousal training again at relapse or did not intervene at all and none of them consulted a professional. Clearly they had confidence in the method of Arousal training: combining the alarm device with reinforcement for correct behaviour at the time the alarm goes off. Parents in control conditions did not use the alarm device as often as the parents in the Arousal condition, but tried other means with less success, including consulting professionals.     

Preparation and stability testing of a hydrogel for topical analgesia

With the commercial availability of a cream (EMLA) containing a eutectic mixture of local anaesthetics, 2.5% (w/w) lidocaine and 2.5% (w/w) prilocaine, effective topical anaesthesia of the intact skin is possible without the need for subcutaneous injections or exposure to high concentrations of local anaesthetics. In our hospital a topical anaesthetic product was designed for the same purpose. The home-made product contains a eutectic mixture of a local anaesthetic (5% w/w) and l-menthol (1% w/w). Prilocaine was used as the local anaesthetic because it is known for its safety and its well investigated analgesic effects. The eutectic mixture of prilocaine and l-menthol was mixed with a carbopol hydrogel (1% w/w). Preliminary testing of this anaesthetic hydrogel in our hospital has yielded satisfactory results. The anaesthetic hydrogel was found to be stable after at least 3 months' storage at ambient temperature.     

In situ blotting: a novel method for direct transfer of native proteins from sectioned tissue to blotting membrane: procedure and some applications

We describe a novel technique for direct transfer of native proteins from unfixed frozen tissues sections to an immobilizing matrix, e.g., nitrocellulose, polyvinyliden difluoride, or positively charged nylon membranes. Proteins are directly blotted onto the membrane, providing optimal accessibility for molecular detection but retaining the anatomic localization at the cellular level. Within 10 min a maximum protein transfer is achieved independent of the protein molecular weight. The total protein bound was 80% of the maximal binding capacity of the blotting membrane and independent of the section thickness. These results indicate that the proteins that bind to the membrane originate from the cut cell monolayer that has direct contact with the blotting membrane. This in situ blotting method provides direct protein mapping from a single cell layer of a tissue section. The procedure includes cryosectioning at 20 microns and collecting sections on a dry blotting membrane at -20 degrees C. For protein transfer the blotted sections are thawed and incubated for 10 min with Tris buffer. After incubation the sections are removed from the membrane by high-pressure spray. The blotted membranes can be subjected to several detection assays. In the present study the presence of several proteins was demonstrated in brain and thymus by immunochemical and enzyme histochemical procedures.     

Isolation and characterization of S-layer proteins from a vent prosthecate bacterium

MWapp 116,000 and 29,000 proteins (p116 and p29), major outer membrane proteins of Hyphomonas jannaschiana reproductive cells, were extracted from cell envelopes by dialysis against EDTA, 2 M urea or distilled water. These proteins were precipitated by divalent cations and resolubilized by EDTA-Na, reflecting alternate monomer, multimer states. From two-dimensional gel electrophoresis it was determined that p116 and p29 had a pl of 4.5. Both were glycoproteins. Results suggest that p116 and p29 are surface layer (S-layer) proteins, with p116 a tetramer of the p29. The S-layer could protect the adherent H. jannaschiana reproductive cell from exoenzyme activity, antibiotics and other bacteriocidal molecules produced in the bacterial films formed on many marine surfaces.     

Analogical transfer from a simulated physical system.

Previous research has consistently found that spontaneous analogical transfer is strongly tied to concrete and contextual similarities between the cases. However, that work has largely failed to acknowledge that the relevant factor in transfer is the similarity between individuals' mental representations of the situations rather than the overt similarities between the cases themselves. Across several studies, we found that participants were able to transfer strategies learned from a perceptually concrete simulation of a physical system to a task with very dissimilar content and appearance. This transfer was reflected in better performance on the transfer task when its underlying dynamics were consistent rather than inconsistent with the preceding training task. Our data indicate that transfer in these tasks relies on the perceptual and spatial nature of the training task but does not depend on direct interaction with the system, with participants performing equally well after simply observing the concrete simulation. We argue that participants generated a spatial, dynamic, and force-based mental model while interacting with the training simulation and tended to spontaneously interpret the transfer task according to this primed model. Unexpectedly, our data consistently show that transfer was independent of reported recognition of the analogy between tasks: Although such recognition was associated with better overall performance, it was not associated with better transfer (in terms of applying an appropriate strategy). Together, these findings suggest that analogical transfer between overtly dissimilar cases may be much more common—and much more relevant to our cognitive processing—than is generally assumed. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Heat transfer from flames in a rotary kiln

Heat flow in the flame zone of a direct-fired rotary kiln has been modeled mathematically. The flame has been assumed to be cylindrical in shape, backmixed radially, and moving axially in plug flow. The length of the flame and the rate of entrainment of secondary air have been characterized by empirical equations reported in the literature. It has been shown that the axial component of radiation can be reasonably neglected since it is relatively small compared to the radial component. The resulting one-dimensional model is capable of predicting the axial temperature profiles of the flame and wall and the axial profiles of heat flux to the solids bed and refractory wall. The model has been employed to study the influence on heat flow to the bed of the following variables: fuel type (fuel oil, natural gas, producer gas), firing rate, temperature of secondary air, pct primary air, and oxygen enrichment. Of the three fuels, combustion of fuel oil gives the longest flame and the greatest heat input to the solids in the flame zone. Raising the secondary-air temperature increases the flame length significantly but has a small effect on the maximum flame temperature and heat flux to the solids. Increasing percent primary air decreases the flame length and increases the peak values of flame temperature and solids heat flux but reduces the quantity of heat received by the solids in the flame zone. Oxygen enrichment results in a shorter flame, higher maximum flame temperature, and increase in the heat transferred to the solids in the flame zone. Formerly Graduate Student, University of British Columbia     

Effect of endogenous proteins on growth and antibody productivity in hybridoma batch cultures

It has been shown that some B-cell hybridomas secrete autocrine factors in vitro which can influence cell metabolic processes. Rather than screen specifically for suspected cytokines, that may or may not affect our cell line, we have examined the lumped effects of intracellular and secreted factors on cell proliferation and monoclonal productivity in hybridoma batch cultures. Firstly, supplements of total soluble intracellular proteins combined with other intracellular metabolites were found to both decrease the specific growth rate and increase the antibody production rate at higher concentrations in batch culture. This is an important consideration in high cell density cultures, such as perfusion systems, where a reduction of growth by the presence of intracellular factors may be compensated by an increase in MAb production. In addition, flow cytometry data revealed that the average cell cycle G1 phase fraction was unaffected by the variation in the maximum specific growth rates during the exponential growth phase, caused by the addition of intracellular factors; this suggests that higher MAb productivity at lower growth rates are not a result of cell arrest in the G1 phase. Secondly, secreted extracellular proteins larger than 10,000 Daltons, which were concentrated from spent culture supernatant, were shown to have no significant effect on growth and specific MAb productivity when supplemented to batch culture at levels twice that encountered late in normal batch culture. This indicates that endogenous secreted cytokines, if at all present, do not play a major autocrine role for this cell line.