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.     

Phosphatidylinositol transfer proteins: the long and winding road to physiological function

Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring herpesvirus of wild rodents and is genetically related to human herpesvirus 8 and Epstein-Barr virus. The ability of MHV-68 to establish acute and persistent infection within laboratory mice offers a unique opportunity to investigate immunological and virological aspects of gammaherpesvirus pathogenesis.     

Triplet state energy transfer in several proteins

Energy transfer between excited triplet states of aromatic amino acid residues was observed at 1.4 degrees K. The distance necessary for energy transfer between monomeric tyrosine and tryptophan residues was determined to be roughly 63 A. Total phosphorescence decay rate constants for several proteins were determined while emission corresponding to tyrosine and tryptophan residues was monitored. The observed decay rate constants are interpreted in terms of intramolecular interactions of the polypeptide residues.     

Mammalian protein RAP46: an interaction partner and modulator of 70 kDa heat shock proteins

A ubiquitously expressed nuclear receptor-associating protein of approximately 46 kDa (RAP46) was identified recently. Interaction experiments with in vitro-translated proteins and proteins contained in cell extracts revealed that a great variety of cellular regulators associate with RAP46. However, in direct interaction tests by the far-Western technique, only 70 kDa proteins showed up and were identified as members of the 70 kDa heat shock protein (hsp70) family. Interaction is specific since not all members of the hsp70 family bind to RAP46; interaction occurs through their ATP-binding domain. RAP46 forms complexes with hsp70 in mammalian cells and interacts with hsp70 in the yeast two-hybrid system. Consistent with the fact that hsp70 can bind a multitude of proteins, we identified heteromeric complexes of RAP46-hsp70 with some selected proteins, most notably c-Jun. Complex formation is increased significantly by pre-treatment with alkaline phosphatase, thus suggesting modulation of interactions by protein phosphorylation. We observed that RAP46 interferes with efficient refolding of thermally denatured luciferase. Moreover, ATP-dependent binding of misfolded proteins to hsp70 was greatly inhibited by RAP46. These data suggest that RAP46 functions as a regulator of hsp70 in higher eukaryotes.     

Diffusion and transfer of antibody proteins from a sugar-based hydrogel

Diffusion of antibody protein from hydrogel films and hydrogel encapsulated in a microcapillary was studied. Thin hydrogel films were formed by crosslinking 6-acryloyl-B-O-methylgalactoside with N,N'-methylene-bis-acrylamide and the diffusive transport of monoclonal antimouse IgG-FITC into and out of the hydrate films was measured. Diffusion coefficients in 2 and 4% crosslinked hydrogel films were measured. The measured diffusion constants determined for IgG in both the 2 and 4% hydrogel films were comparable to the free diffusion of IgG in bulk water (Dmean approximately 10(-7) cm2/s). In addition, 2% crosslinked hydrogels were prepared in a capillary tube and the transport of antimouse IgG-FITC into and out of the hydrated hydrogel was measured. Kinetic analysis indicated that the protein transport through the capillary hydrogel was faster than would be expected for a simple diffusion process. Finally, by utilizing the diffusion of antibody from the capillary hydrogel, transfer of antibody to a silica surface was demonstrated. A capillary hydrogel loaded with antimouse IgG-FITC was used to transfer the protein to a silica surface forming a 30-micron spot of antibody, which was imaged using fluorescence microscopy. These results may lead to the development of a nonlithographic method of patterning antibodies on surfaces for use in integrated microimmunosensors.     

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.     

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.     

Mammalian heterotrimeric G-protein-like proteins in mycobacteria: implications for cell signalling and survival in eukaryotic host cells

Selectively-infective phage (SIP) is a novel methodology for the in vivo selection of interacting protein-ligand pairs. It consists of two components, (1) a phage particle made non-infective by replacing its N-terminal domains of geneIII protein (gIIIp) with a ligand-binding protein, and (2) an "adapter" molecule in which the ligand is linked to those N-terminal domains of gIIIp which are missing from the phage particle. Infectivity is restored when the displayed protein binds to the ligand and thereby attaches the missing N-terminal domains of gIIIp to the phage particle. Phage propagation is thus strictly dependent on the protein-ligand interaction. We have shown that the insertion of beta-lactamase into different positions of gIIIp, mimicking the insertion of a protein-ligand pair, led to highly infective phage particles. Any phages lacking the first N-terminal domain were not infective at all. In contrast, those lacking only the second N-terminal domain showed low infectivity irrespective of the presence or absence of the F-pilus on the recipient cell, which could be enhanced by addition of calcium. An anti-fluorescein scFv antibody and its antigen fluorescein were examined as a protein-ligand model system for SIP experiments. Adapter molecules, synthesized by chemical coupling of fluorescein to the purified N-terminal domains, were mixed with non-infective anti-fluorescein scFv-displaying phages. Infection events were strictly dependent on fluorescein being coupled to the N-terminal domains and showed a strong dependence on the adapter concentration. Up to 10(6) antigen-specific events could be obtained from 10(10) input phages, compared to only one antigen-independent event. Since no separation of binders and non-binders is necessary, SIP is promising as a rapid procedure to select for high affinity interactions.     

Mammalian sex determination: from gonads to brain

In mammals, sex is determined by the Y chromosome, which encodes a testis-determining factor (TDF). This factor causes the undifferentiated embryonic gonads to develop as testes rather than ovaries. The testes subsequently produce the male sex hormones that are responsible for all male sexual characteristics. In 1990, the sex-determining gene, TDF, was identified and termed SRY in humans (Sry in mice). It encodes a protein containing a high mobility group (HMG) motif, which confers the ability to bind and to bend DNA. Genetic evidence supporting SRY as TDF came from the observation of a male phenotype in XX mice transgenic for a small genomic fragment containing Sry, and from the study of XY sex-reversed individuals who harbor de novo mutations in the SRY coding sequence. Other non-Y-linked genes involved in sex determination were subsequently found by genetic analysis of XY sex-reversed patients not explained by mutations in SRY. These genes are WT1, SF1, DAX1, and SOX9. A regulatory cascade hypothesis for mammalian sex determination, proposing that SRY represses a negative regulator of male development, was recently supported by observation of mice that expressed a DAX1 transgene and developed as XY sex-reversed females. The role of some sex-determining genes, such as DAX1 and SF1, in the development of the entire reproductive axis, a functionally integrated endocrine axis, leads to a new concept. Normal sexual development may result from the functional and developmental integration of a number of different genes that play roles in sex determination, sexual differentiation, and sexual behavior.     

Mammalian mitochondrial ribosomal proteins. N-terminal amino acid sequencing, characterization, and identification of corresponding gene sequences

The integrity of healthy mitochondria is supposed to depend largely on proper mitochondrial protein biosynthesis. Mitochondrial ribosomal proteins (MRPs) are directly involved in this process. To identify mammalian mitochondrial ribosomal proteins and their corresponding genes, we purified mature rat MRPs and determined 12 different N-terminal amino acid sequences. Using this peptide information, data banks were screened for corresponding DNA sequences to identify the genes or to establish consensus cDNAs and to characterize the deduced MRP open reading frames. Eight different groups of corresponding mammalian MRPs constituted from human, mouse, and rat origin were identified. Five of them show significant sequence similarities to bacterial and/or yeast mitochondrial ribosomal proteins. However, MRPs are much less conserved in respect to the amino acid sequence among species than cytoplasmic ribosomal proteins of eukaryotes and bacteria.     

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.     

Acoustic absorption due to proton transfer in solutions of proteins, peptides and amino acids at neutral pH

Measurements of ultrasonic absorption in the frequency range 2 to 60 MHz have been made on solutions of the amino acid histidine, dipeptides glycyl tyrosine and histidyl tyrosine, and proteins lysozyme and bovine serum albumin. Measurements were carried out at 37 degrees C and covered the pH range 6.8 to 7.7. Mechanisms for the observed excess acoustic absorption involving bimolecular and intramolecular proton transfer are considered.     

A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins

BACKGROUND AND PURPOSE: Although the role of gray-scale sonography for neck nodes is well documented, it plays a limited role in the evaluation of nodal response to treatment. This preliminary limited study sought to determine color duplex sonographic changes in successfully treated metastatic nodes from nasopharyngeal carcinoma. METHODS: Fourteen patients with nodal metastases from nasopharyngeal carcinoma were studied. A pretreatment sonogram was obtained for all patients. Patients were divided into two groups of seven: in one group, repeat sonograms were obtained 8 weeks after completion of treatment; in the second group, sonograms were obtained 16 weeks after treatment. The features studied included distribution of intranodal vascularity, resistive and pulsatility indexes, and peak systolic velocity. In 11 patients, follow-up sonograms were obtained 1 year after treatment. RESULTS: The majority (90%) of malignant nodes from nasopharyngeal carcinoma have an increased central and peripheral vascularity, a high resistive index (0.8), and a high pulsatility index (1.8). After radiation therapy to the nodes, a reduction in intranodal vascularity and a statistically significant reduction in the resistive index (0.58 to 0.59) and pulsatility index (0.91 to 0.93) are found. Although a similar reduction in the peak systolic velocity is observed, it is not statistically significant. CONCLUSION: Our preliminary findings suggest that after radiation therapy for malignant nodes in nasopharyngeal carcinoma, a reduction in intranodal vascularity is found, and the resistive and pulsatility indexes may return to benign parameters as early as 8 weeks after completion of treatment.     

A "parallel plate" electrostatic model for bimolecular rate constants applied to electron transfer proteins

A "parallel plate" model describing the electrostatic potential energy of protein-protein interactions is presented that provides an analytical representation of the effect of ionic strength on a biomolecular rate constant. The model takes into account the asymmetric distribution of charge on the surface of the protein and localized charges at the site of electron transfer that are modeled as elements of a parallel plate condenser. Both monopolar and dipolar interactions are included. Examples of simple (monophasic) and complex (biphasic) ionic strength dependencies obtained from experiments with several electron transfer protein systems are presented, all of which can be accommodated by the model. The simple cases do not require the use of both monopolar and dipolar terms (i.e., they can be fit well by either alone). The biphasic dependencies can be fit only by using dipolar and monopolar terms of opposite sign, which is physically unreasonable for the molecules considered. Alternatively, the high ionic strength portion of the complex dependencies can be fit using either the monopolar term alone or the complete equation; this assumes a model in which such behavior is a consequence of electron transfer mechanisms involving changes in orientation or site of reaction as the ionic strength is varied. Based on these analyses, we conclude that the principal applications of the model presented here are to provide information about the structural properties of intermediate electron transfer complexes and to quantify comparisons between related proteins or site-specific mutants. We also conclude that the relative contributions of monopolar and dipolar effects to protein electron transfer kinetics cannot be evaluated from experimental data by present approximations.     

预应力锚索抗滑桩在失稳斜坡治理中的应用

在综合治理潘田南矿山体斜坡的研究方案中，采用了预应力锚索抗滑桩。文中阐述了预应力锚索抗滑桩的特点、设计方法、锚索锚固形式等，并与传统抗滑桩方案进行了比较。其结果表明，预应力锚索抗滑桩是一种抗滑利用率高、加固效果可靠、工程成本低的新型抗滑支挡结构     

Mammalian hibernation: cellular and molecular responses to depressed metabolism and low temperature

Mammalian hibernators undergo a remarkable phenotypic switch that involves profound changes in physiology, morphology, and behavior in response to periods of unfavorable environmental conditions. The ability to hibernate is found throughout the class Mammalia and appears to involve differential expression of genes common to all mammals, rather than the induction of novel gene products unique to the hibernating state. The hibernation season is characterized by extended bouts of torpor, during which minimal body temperature (Tb) can fall as low as -2.9 degrees C and metabolism can be reduced to 1% of euthermic rates. Many global biochemical and physiological processes exploit low temperatures to lower reaction rates but retain the ability to resume full activity upon rewarming. Other critical functions must continue at physiologically relevant levels during torpor and be precisely regulated even at Tb values near 0 degrees C. Research using new tools of molecular and cellular biology is beginning to reveal how hibernators survive repeated cycles of torpor and arousal during the hibernation season. Comprehensive approaches that exploit advances in genomic and proteomic technologies are needed to further define the differentially expressed genes that distinguish the summer euthermic from winter hibernating states. Detailed understanding of hibernation from the molecular to organismal levels should enable the translation of this information to the development of a variety of hypothermic and hypometabolic strategies to improve outcomes for human and animal health.     

Repair of soft tissue to bone using a biodegradable suture anchor

A decrease in blood androgen levels is well documented in women who experience natural or surgical menopause. This change may be associated with various negative effects on bone metabolism in addition to psychosocial and sexuality aspects of life. A review of published information on androgen replacement therapy shows that major benefits may be achieved; unfortunately, only minimal quality information is available to help clinicians make decisions about this type of therapy. In this review, we point out the potential benefits and risks to bone, lipid and carbohydrate metabolism, and sexuality; in addition, we discuss potential risks of neoplasms and virilizing somatic changes. Long-term, physiologic, and well-designed androgen replacement studies should be performed to obtain the knowledge needed to guide therapy in this important area.