The kidney as a novel target tissue for protein adduct formation associated with metabolism of halothane and the candidate chlorofluorocarbon replacement 2,2-dichloro-1,1,1-trifluoroethane.

Hydrochlorofluorocarbons (HCFCs) have been identified as chemical replacements of the widely used chlorofluorocarbons (CFCs) that are implicated in stratospheric ozone depletion. Many HCFCs are structural analogues of the anesthetic agent halothane and may follow a common pathway of biotransformation and formation of adducts to protein-centered and other cellular nucleophiles. Exposure of rats to a single dose of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) or of the candidate CFC substitute HCFC 123 (2,2-dichloro-1,1,1-trifluoroethane) led to the formation of trifluoroacetylated protein adducts (CF3CO-proteins) not only in the liver, but also in the kidney as a novel target tissue for protein trifluoroacetylation. CF3CO-proteins in the kidney amounted to about 5% of those formed in the liver of the same animal. The amount of CF3CO-proteins formed within the kidney was roughly reflected by the capacity of metabolism of halothane or HCFC 123 by rat kidney microsomes in vitro which amounted to about 10% of that observed with liver microsomes. By immunohistochemistry, CF3CO-proteins in the kidney were mainly localized in the tubular segments of the cortex. In the liver, the density of CF3CO-proteins decreased from the central vein towards the portal triad. In vitro incubation of rat liver microsomes with halothane or HCFC 123 resulted in extensive formation of CF3CO-proteins and reproduced faithfully the pattern of liver CF3CO-proteins obtained in vivo. CF3CO-proteins generated in vitro were immunochemically not discernible from those generated in vivo. Glutathione (5 mM) and cysteine (5 mM) virtually abolished CF3CO-protein formation; the release of Br- from halothane and Cl- from HCFC 123 was reduced to much lesser a degree. S-Methyl-glutathione, N-acetyl-cysteine, methionine, and N-acetyl-methionine only slightly affected the formation of CF3CO-proteins or metabolism of either substrate. The data suggest that metabolism and concomitant CF3CO-protein formation of halothane or of candidate CFC replacements like HCFC 123 is not restricted to the liver but also takes place in the kidney. Furthermore, an in vitro system for CF3CO-protein formation has been developed and used to show that protein-centered and glutathione-centered nucleophilic sites compete for intermediates of metabolism of halothane or of HCFC 123.     

Exposure to perflubron is associated with decreased Syk phosphorylation in human neutrophils.

Liquid ventilation with perflubron is associated with reduced neutrophil recruitment into the lung during acute injury. Perflubron also reduces chemotactic responses, the respiratory burst, and cytokine production in neutrophils and in alveolar macrophages in vitro. In the current studies, the effect of perflubron on neutrophil chemotaxis to formyl-Met-Leu-Phe (fMLP) and phagocytosis of opsonized sheep erythrocytes (EA) correlated with decreased phosphorylation of Syk, an important intracellular second messenger in pathways regulating neutrophil functional responses. Brief (5 min) exposure of neutrophils to perflubron resulted in a dose-dependent reduction in chemotaxis to fMLP and reduced phagocytosis of EA but no apparent morphological changes as seen by electron microscopy. Concurrently, there was a reduction in both total cytosolic tyrosine phosphorylation and Syk phosphorylation. Binding studies indicated that this effect was neither a result of impaired ligand-receptor affinity nor a change in the number of fMLP receptors available on the neutrophil surface. These results suggest that perflubron nonspecifically affects cellular activation as measured by tyrosine phosphorylation perhaps by interfering with transmembrane signal transduction.     

The heterotrimeric protein Go is required for the formation of heart epithelium in Drosophila.

The gene encoding the alpha subunit of the Drosophila Go protein is expressed early in embryogenesis in the precursor cells of the heart tube, of the visceral muscles, and of the nervous system. This early expression coincides with the onset of the mesenchymal-epithelial transition to which are subjected the cardial cells and the precursor cells of the visceral musculature. This gene constitutes an appropriate marker to follow this transition. In addition, a detailed analysis of its expression suggests that the cardioblasts originate from two subpopulations of cells in each parasegment of the dorsal mesoderm that might depend on the wingless and hedgehog signaling pathways for both their determination and specification. In the nervous system, the expression of Goalpha shortly precedes the beginning of axonogenesis. Mutants produced in the Goalpha gene harbor abnormalities in the three tissues in which the gene is expressed. In particular, the heart does not form properly and interruptions in the heart epithelium are repeatedly observed, henceforth the brokenheart (bkh) name. Furthermore, in the bkh mutant embryos, the epithelial polarity of cardial cells was not acquired (or maintained) in various places of the cardiac tube. We predict that bkh might be involved in vesicular traffic of membrane proteins that is responsible for the acquisition of polarity.     

The mechanism of adduct formation between NAD+ and pyruvate bound to pig heart lactate dehydrogenase.

1. The rate of adduct formation between NAD+ and enol-pyruvate at the active site of lactate dehydrogenase is determined by the rate of enolization of pyruvate in solution. 2. The proportion of enol-pyruvate solutions is less than 0.01%. 3. The overall dissociation constant of adduct formation is less than 5 X 10(-8) M for pig heart lactate dehydrogenase at pH 7.0. 4. The unusual kinetics for adduct formation previously observed in the case of rabbit muscle lactate dehydrogenase [Griffin & Criddle (1970) Biochemistry 9, 1195--1205] may be attributed to the concentration of enol-pyruvate in solution being considerably less than the concentration of enzyme.     

Design and biophysical characterization of a monomeric four-alpha-helix bundle protein Aα4 with affinity for the volatile anesthetic halothane

A monomeric four-α-helix bundle protein Aα₄ was designed as a step towards investigating the interaction of volatile general anesthetics with their putative membrane protein targets. The alpha helices, connected by glycine loops, have the sequence A, B, B′, A′. The DNA sequence was designed to make the helices with the same amino acid sequences (helix A and A′, B and B′, respectively) as different as possible, while using codons which are favorable for expression in E. coli. The protein was bacterially expressed and purified to homogeneity using reversed-phase HPLC. Protein identity was verified using MALDI–TOF mass spectrometry. Far-UV circular dichroism spectroscopy confirmed the predominantly alpha-helical nature of the protein Aα₄. Guanidinium chloride induced denaturation showed that the monomeric four-α-helix bundle protein Aα₄ is considerably more stable compared to the dimeric di-α-helical protein (Aα₂-L38M)₂. The sigmoidal character of the unfolding reaction is conserved while the sharpness of the transition is increased 1.8-fold. The monomeric four-α-helix bundle protein Aα₄ bound halothane with a dissociation constant (K_d) of 0.93 ± 0.02 mM, as shown by both tryptophan fluorescence quenching and isothermal titration calorimetry. This monomeric four-α-helix bundle protein can now be used as a scaffold to incorporate natural central nervous system membrane protein sequences in order to examine general anesthetic interactions with putative targets in detail.     

Caveolin-1 expression is associated with plaque formation in hypercholesterolemic rabbits.

Caveolin-1, the major structural protein of caveolae, is present in several cell types known to play a role in the development of atherosclerosis. In this study, the distribution and expression of caveolin-1 in the arterial walls were studied in hypercholesterolemic rabbits. Immunohistochemical results indicated that the staining intensity of caveolin-1 reached a high level in the arterial intima at 5 weeks after high-cholesterol-diet treatment and decreased to a very low level at 8 weeks when atheromatous plaques appeared. Western blot analysis showed that in rabbits fed a high-cholesterol diet for 5 weeks, the expression of caveolin-1 reached its highest level and then decreased from 8 to 12 weeks. The proliferative activity of smooth muscle cells (SMCs) decreased to the lowest level at 5 weeks and then increased at 8 and 12 weeks. Nitric oxide synthase activity gradually decreased in animals fed a high-cholesterol diet throughout the experiment. These studies demonstrate that the change in abundance of caveolin-1 is associated with SMC proliferation in the formation of atheromatous plaque after hypercholesterolemia insult.     

Exposure to environmental radionuclides is associated with altered metabolic and immunity pathways in a wild rodent

Wildlife inhabiting environments contaminated by radionuclides face putative detrimental effects of exposure to ionizing radiation, with biomarkers such as an increase in DNA damage and/or oxidative stress commonly associated with radiation exposure. To examine the effects of exposure to radiation on gene expression in wildlife, we conducted a de novo RNA sequencing study of liver and spleen tissues from a rodent, the bank vole Myodes glareolus. Bank voles were collected from the Chernobyl Exclusion Zone (CEZ), where animals were exposed to elevated levels of radionuclides, and from uncontaminated areas near Kyiv, Ukraine. Counter to expectations, we did not observe a strong DNA damage response in animals exposed to radionuclides, although some signs of oxidative stress were identified. Rather, exposure to environmental radionuclides was associated with upregulation of genes involved in lipid metabolism and fatty acid oxidation in the livers – an apparent shift in energy metabolism. Moreover, using stable isotope analysis, we identified that fur from bank voles inhabiting the CEZ had enriched isotope values of nitrogen: such an increase is consistent with increased fatty acid metabolism, but also could arise from a difference in diet or habitat between the CEZ and elsewhere. In livers and spleens, voles inhabiting the CEZ were characterized by immunosuppression, such as impaired antigen processing, and activation of leucocytes involved in inflammatory responses. In conclusion, exposure to low dose environmental radiation impacts pathways associated with immunity and lipid metabolism, potentially as a stress‐induced coping mechanism.     

Pressure-induced formation of inactive triple-shelled rotavirus particles is associated with changes in the spike protein Vp4

Rotaviruses are non-enveloped, triple-shelled particles that cause enteritis in animals and humans. The interactions among the different viral proteins located in the three concentric layers make the rotavirus particle an excellent model for physico-chemical and biological studies of viral assemblage. SA11-4S rotaviruses subjected to high pressure were inactivated by more than five log units. After pressure treatment, the particles were recovered with slight structural changes when compared to the control. Electron microscopy suggested subtle changes in the viral outer layer in some pressurised particles. Fluorescence spectroscopy showed that much more dramatic changes were produced by urea denaturation than by pressure. Based on the fluorescence spectrum, the genome resistance to ribonuclease, and the absence of changes in hydrodynamic properties, there was little or no disruption of the capsid under pressure. On the other hand, hemagglutination assays indicated that the main component affected by pressure was the spike protein VP4, thus accounting for changes in interaction with host cells and greatly reduced infectivity. The changes leading to inactivation did not cause removal of VP4 from the outer capsid, as verified by size-exclusion chromatography. Antibodies raised against pressurised material were as effective as antibodies raised against the intact virus, based on their neutralisation titre in plaque reduction assays, enzyme-linked immunosorbent assays and direct interaction with the particle, as measured by gel-filtration chromatography. Therefore, the new conformation of the pressurised particle did not result in loss of immunogenicity. We propose that pressure alters the receptor-binding protein VP4 by triggering changes similar to those produced when the virus interacts with target cells. As the changes in VP4 conformation caused by pressure occur prior to virus exposure to target cells, it leads to non-infectious particles and may lead to the exposure of previously occult epitopes, important for vaccine development.     

A development-specific protein in Myxococcus xanthus is associated with the extracellular fibrils.

We have been using monoclonal antibodies (MAbs) as probes to study developmentally relevant cell surface antigens (CSA) that may be required for cellular interactions in Myxococcus xanthus. Three independently isolated MAbs, G69, G357, and G645, isolated by Gill and Dworkin recognize a CSA detectable only on developing cells (J. S. Gill and M. Dworkin, J. Bacteriol. 168:505-511, 1986). The CSA is made within the first 30 min of submerged development and increases until myxosporulation. The CSA is also produced at low levels after 24 h in shaken-starved cultures and during glycerol sporulation. No antigen can be detected in lysed, vegetative cells, and expression of the antigen is blocked in the presence of rifampin or chloramphenicol. The antigen is expressed in submerged, developmental cultures of asg, bsg, csg, dsg, and mgl mutants and is not expressed in a dsp mutant. All of the three MAbs immunoprecipitate the same protein of approximately 97,000 Da from lysed developmental cells. Competitive immunoprecipitations suggest that they recognize at least two different epitopes on the CSA. The epitopes recognized by MAbs G69, G357, and G645 are sensitive to protease digestion, whereas the epitopes recognized by MAbs G357 and G645 are resistant to periodate oxidation. The epitope recognized by MAb G69 is sensitive to periodate oxidation. Fractionation of lysed developing cells shows that most of the antigen is localized in the pellet after centrifugation at 100,000 x g. To determine whether the antigen is expressed on the cell surface, we labeled developing whole cells with either MAb G69, G357, or G645 and gold-labeled anti-mouse immunoglobulin G. Low-voltage scanning electron microscopy of labeled cells shows that the antigen is associated with the fibrillar matrix that surrounds the cells and that the antigen is retained on isolated, developmental fibrils from M. xanthus. The CSA has been designated dFA-1, for developmental fibrillar antigen 1.     

Phragmoplastin, a dynamin-like protein associated with cell plate formation in plants.

Cytokinesis in a plant cell is accomplished by the formation of a cell plate in the center of the phragmoplast. Little is known of the molecular events associated with this process. In this study, we report the identification of a dynamin-like protein from soybean and demonstrate that this protein is associated with the formation of the cell plate. Plant dynamin-like (PDL) protein contains 610 amino acids showing high homology with other members of the dynamin protein family. Western blot experiments demonstrated that it is associated with the non-ionic detergent-resistant fraction of membranes. Indirect immunofluorescence microscopy localized PDL to the cell plate in dividing soybean root tip cells. Double labeling experiments demonstrated that, unlike phragmoplast microtubules which are concentrated on the periphery of the forming plate, PDL is located across the whole width of the newly formed cell plate. Based on the temporal and spatial organization of PDL in the phragmoplast, we termed this protein 'phragmoplastin'. The data suggest that phragmoplastin may be associated with exocytic vesicles that are depositing cell plate material during cytokinesis in the plant cell.     

RFP is a DNA binding protein associated with the nuclear matrix.

We reported that the RFP gene encodes a protein with putative zinc finger domains and was involved in the activation of the ret proto-oncogene. To further characterize the RFP protein, we developed a polyclonal antibody against the product synthesized from a fragment of the RFP cDNA expressed in Escherichia coli. Western blot analysis showed that RFP was identified as a 58 kDa protein in cell lysates from four human and rodent cell lines and from mouse testis. In addition, a unique 68 kDa protein was detected in the testis. Using AH7974 (rat ascites hepatoma) and Raji (human Burkitt lymphoma) cells, we demonstrated strong association of RFP with the nuclear matrix. Furthermore, RFP solubilized from the nuclear matrix had DNA-binding activity although it appears to bind more preferentially to double-stranded DNA than to single-stranded DNA. These results thus suggest that RFP may play a role in molecular processes which occur in the nuclear matrix.     

Angiogenesis is associated with collagenous protein synthesis and degradation in the chick chorioallantoic membrane.

Extracts of the chick embryo chorioallantoic membrane (CAM) obtained from 7-20 day old embryos, contained enzyme activity that could degrade type IV collagen. Peak enzyme activity was observed on days 8-10 of embryogenesis, which coincides with the stage of maximum angiogenesis. This activity decreased to lowest values at days 13-15 and increased thereafter up to day 20. Maximum rate of collagen biosynthesis in CAM was observed between days 7 and 10, with a drastic decrease at day 12, when vascular density has reached a plateau. The type IV collagen-degrading activity of CAM was of the metalloprotease type, since it was inhibited by 1,10-phenanthroline and EDTA but was also partially inhibited by serine and thiol protease inhibitors.     

RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus.

The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.     

Pore formation associated with the tail-tip protein pb2 of bacteriophage T5.

Upon binding of bacteriophage T5 tails to purified FhuA receptor protein the tail-tip protein pb2 became extremely sensitive to trypsin and other proteases. However, when T5 tails were bound to FhuA integrated into liposomes, pb2 was found to retain some resistance to trypsin. Electron microscopic examination of tail-liposome complexes supported the idea that trypsin resistance of pb2 in such complexes was caused by insertion of the tail-tip into the liposomes. pb2 was isolated from tails by treatment with sodium dodecyl sulfate and was further purified by gel filtration using a fast protein liquid chromatography system. pb2 obtained with this procedure was most likely monomeric. It was extremely sensitive to trypsin. When reconstituted into black lipid bilayer membranes, it formed pores with an average single-channel conductance of 4.6 nanosiemens in 1 M KCl. Zero-current potential measurements showed only a very slight preference, if any, for cations over anions. The data are compatible with pb2 forming a large water-filled transmembrane channel. The functioning during infection of pb2 in cytoplasmic membrane depolarization and phage DNA uptake into the cell is discussed.     

Hydrolysis of GTP associated with the formation of tubulin oligomers is involved in microtubule nucleation.

Hydrolysis of GTP is known to accompany microtubule assembly. Here we show that hydrolysis of GTP is also associated with the formation of linear oligomers of tubulin, which are precursors (prenuclei) in microtubule assembly. The hydrolysis of GTP on these linear oligomers inhibits the lateral association of GTP-tubulin that leads to the formation of a bidimensional lattice. Therefore GTP hydrolysis interferes with the nucleation of microtubules. Linear oligomers are also formed in mixtures of GTP-tubulin and GDP-tubulin. The hydrolysis of GTP associated with heterologous interactions between GTP-tubulin and GDP-tubulin in the cooligomer takes place at a threefold faster rate than upon homologous interactions between GTP-tubulins. The implication of these results in a model of vectorial GTP hydrolysis in microtubule assembly is discussed.     

Effects of the volatile anesthetic halothane on fertilization and early development in the sea urchin Lytechinus variegatus: evidence that abnormal development is due to polyspermy

The volatile anesthetic halothane, when present at fertilization, dose-dependently increases the incidence of abnormally developing sea urchin embryos at the first cell division. Microscopic examinations of eggs stained with aceto-orcein or the DNA fluorochrome bisbenzimide and direct observations on isolated sperm aster complexes show that halothane induces polyspermy (multiple sperm entry) when present at fertilization. Experimental evidence suggests that anesthetic-induced polyspermy involves impairment of both the fast (electrically mediated) and slow (morphological) blocks to multiple sperm entry. These observations clearly show that relatively brief exposures to halothane at fertilization cause polyspermy and that this effect is almost certainly responsible for the ensuing abnormal development observed at the first cell division.     

A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability.

In order to study the structural and functional organization of the eukaryotic nucleolus, we have started to isolate and characterize nucleolar components of the yeast Saccharomyces cerevisiae. We have identified a major 38 kd nucleolar protein (NOP1), which is located within nucleolar structures resembling the dense fibrillar region of mammalian nucleoli. This 38 kd protein is conserved in evolution since affinity-purified antibodies against the yeast protein stain the nucleolus of mammalian cells in indirect immunofluorescence microscopy and the yeast protein is decorated by antibodies directed against human fibrillarin. Affinity-purified antibodies against the yeast NOP1 efficiently precipitate at least seven small nuclear RNAs involved in rRNA maturation. We have cloned the gene encoding the yeast NOP1 protein. Haploid cells carrying a disrupted copy of the gene are not viable, showing that NOP1 is essential for cell growth. The gene codes for a 34.5 kd protein which contains glycine/arginine rich sequence repeats at the amino terminus similar to those found in other nucleolar proteins. This suggests that NOP1 is in association with small nucleolar RNAs, required for rRNA processing and likely to be the homologue of the mammalian fibrillarin.     

Wiskott-Aldrich syndrome protein is associated with the adapter protein Grb2 and the epidermal growth factor receptor in living cells.

Src homology domains [i.e., Src homology domain 2 (SH2) and Src homology domain 3 (SH3)] play a critical role in linking receptor tyrosine kinases to downstream signaling networks. A well-defined function of the SH3-SH2-SH3 adapter Grb2 is to link receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), to the p21ras-signaling pathway. Grb2 has also been implicated to play a role in growth factor-regulated actin assembly and receptor endocytosis, although the underlying mechanisms remain unclear. In this study, we show that Grb2 interacts through its SH3 domains with the human Wiskott-Aldrich syndrome protein (WASp), which plays a role in regulation of the actin cytoskeleton. We find that WASp is expressed in a variety of cell types and is exclusively cytoplasmic. Although the N-terminal SH3 domain of Grb2 binds significantly stronger than the C-terminal SH3 domain to WASp, full-length Grb2 shows the strongest binding. Both phosphorylation of WASp and its interaction with Grb2, as well as with another adapter protein Nck, remain constitutive in serum-starved or epidermal growth factor-stimulated cells. WASp coimmunoprecipitates with the activated EGFR after epidermal growth factor stimulation. Purified glutathione S-transferase-full-length-Grb2 fusion protein, but not the individual domains of Grb2, enhances the association of WASp with the EGFR, suggesting that Grb2 mediates the association of WASp with EGFR. This study suggests that Grb2 translocates WASp from the cytoplasm to the plasma membrane and the Grb2-WASp complex may play a role in linking receptor tyrosine kinases to the actin cytoskeleton.     

MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube.

The Mesp1 gene encodes the basic HLH protein MesP1 which is expressed in the mesodermal cell lineage during early gastrulation. Disruption of the Mesp1 gene leads to aberrant heart morphogenesis, resulting in cardia bifida. In order to study the defects in Mesp1-expressing cells during gastrulation and in the specification of mesodermal cell lineages, we introduced a (beta)-galactosidase gene (lacZ) under the control of the Mesp1 promoter by homologous recombination. The early expression pattern revealed by (beta)-gal staining in heterozygous embryos was almost identical to that observed by whole mount in situ hybridization. However, the (beta)-gal activity was retained longer than the mRNA signal, which enabled us to follow cell migration during gastrulation. In heterozygous embryos, the Mesp1-expressing cells migrated out from the primitive streak and were incorporated into the head mesenchyme and heart field. In contrast, Mesp1-expressing cells in the homozygous deficient embryos stayed in the primitive streak for a longer period of time before departure. The expression of FLK-1, an early marker of endothelial cell precursors including heart precursors, also accumulated abnormally in the posterior region in Mesp1-deficient embryos. In addition, using the Cre-loxP site-specific recombination system, we could determine the lineage of the Mesp1-expressing cells. The first mesodermal cells that ingressed through the primitive streak were incorporated as the mesodermal component of the amnion, and the next mesodermal population mainly contributed to the myocardium of the heart tube but not to the endocardium. These results strongly suggest that MesP1 is expressed in the heart tube precursor cells and is required for mesodermal cells to depart from the primitive streak and to generate a single heart tube.