Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70.

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.     

Hepititol conformations revisited: C,O versus O,O parallel 1,3-interactions

The molecular and crystal structure of -glycero- -allo-heptitol has been determined. It crystallises in the orthorhombic space-group P2₁2₁2 with a = 8.399(2), b = 23.484(8), c = 4.664(2) Å, = β = γ = 90°, and Z = 4. The heptitol was found to be in the ₃G⁻ form, which has a 1,3-parallel interaction between C-2 and O-5. In solution, as determined by ¹H-n.m.r. spectroscopy, the heptitol and its hepta-acetate also assume conformations which have a C,O parallel interaction. Such an interaction has also been found to occur in several other heptitols and in hexose dialkyl acetals. Because conformers with such an interaction had been disregarded in the past, the conformations of several heptitols had been wrongly assigned.     

Developmental expression of heat shock proteins 60, 70, 90, and A2 in rabbit testis

Currently, no reports exist concerning the expression patterns and developmental changes of heat shock proteins (HSPs) in the reproductive system of the male rabbit. In the present study, the testes of rabbits were collected at post-natal months 1, 2, 3, 4, 5, and 40. HSP60, HSC70, HSP90, and HSPA2 were detected by both Western blot and immunohistochemical methods. The expression levels of HSP60 and HSC70 showed no apparent change during the developmental progress. HSP90 increased at the second month; prior to the third month, HSPA2 was expressed at a low level. Immunohistochemistry localized HSP60 in the cytoplasm of all of the cell types in the testis and in the apical pole of the spermatids. The distribution pattern of HSC70 and HSP90 was similar, both being mainly located in the spermatids of stage VII-VIII and in the cytoplasm of the spermatogonium. HSPA2 staining was mainly observed in the cytoplasm of pachytene spermatocytes and spermatids in testes of 3-, 4-, 5-, and 40-month-old rabbits. These results provide a basic reference point for studying the functions of HSPs in the male rabbit reproductive system and should be beneficial for the future determination of the mechanisms of heat shock on male rabbit fertility.     

The novel organolithium O,C,O-pincer compound

The preparation and characterization of organolithium O,C,O-pincer compound [2,6-(tBuOCH₂)₂C₆H₃]-Li (1) is described. The X-ray diffraction techniques revealed the dimeric structure of 1 consisting of two lithium atoms Li(1) and Li(2) and two monoanionic chelating aryl ligands in the solid state, where each lithium atom is coordinated by two oxygen atoms of two different ligands. The NMR spectroscopy and cryoscopy measurements established monomer-dimer equilibrium of 1 in concentrated solutions of non-coordinated solvents, while the diluted solutions of 1 consist of monomer only.     

Effects of chronic heat stress on the expressions of heat shock proteins 60, 70, 90, A2, and HSC70 in the rabbit testis

Few studies have focused on the expression of heat shock proteins (HSPs) after chronic heat stress. The objective of this study was to investigate the effect of chronic high temperature–humidity index treatment on the expressions of HSP60, HSP70, HSP90, HSPA2 and HSC70, in the Rex rabbit testis and the expressions of these proteins after recovery from the chronic heat shock. Thirty mature male rabbits of the same age were randomly divided into three groups: control, heat stress, and recovery. The western blot results showed that the expressional levels of HSP60, HSP90, and HSC70 increased significantly and HSPA2 was elevated slightly after a 9-week heat treatment. HSP70 was absent in the control testis and had a high level of expression after heat stress. All of these proteins partially reverted back to normal levels after a 9-week recovery. The immunohistochemical results indicated that the expression patterns of HSP60, HSP90, HSPA2, and HSC70 did not change.     

Comparative study of the bimolecular electron transfer of fullerenes (C60/C70) and 9,9-disubstituted fluorenes by laser flash photolysis.

Photoinduced bimolecular electron transfer processes of fullerenes (C60/C70) with fluorene derivatives, namely 9,9-bis(4-amino-3-methylphenyl)fluorene (BAMF), 9,9-bis(4-amino-3-fluorophenyl)fluorene (BAFF) and 9,9-bis(3-amino-4-hydroxyphenyl)fluorene (BAHF) have been comparatively studied in benzonitrile by nanosecond laser photolysis technique in the visible/near-IR regions. By the selective excitation of C60/C70 using 532 nm laser light, it has been proved that electron transfer takes place from the ground state fluorenes to the triplet excited state of C60/C70. The observed rates and efficiencies of the electron transfer processes are found to be significantly associated with the substitution patterns on the 9,9-bisphenylfluorenes, and to be correlative with the free energy changes on the basis of the Rehm-Weller relation.     

Structure of the O-polysaccharide and serological studies of the lipopolysaccharide of Proteus penneri 60 classified into a new Proteus serogroup O70

An alkali-treated lipopolysaccharide of Proteus penneri strain 60 was studied by chemical analyses and 1H, 13C and 31P NMR spectroscopy, and the following structure of the linear pentasaccharide-phosphate repeating unit of the O-polysaccharide was established: 6)-alpha-D-Galp-(1-->3)-alpha-L-FucpNAc-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4NAc-(1-->6)-alpha-D-Glcp-1-P-(O--> Rabbit polyclonal O-antiserum against P. penneri 60 reacted with both core and O-polysaccharide moieties of the homologous LPS. Based on the unique O-polysaccharide structure and serological data, we propose to classify P. penneri 60 into a new, separate Proteus serogroup O70. A weak cross-reactivity of P. penneri 60 O-antiserum with the lipopolysaccharide of Proteus vulgaris O8, O15 and O19 was observed and discussed in view of the chemical structures of the O-polysaccharides.     

Preparation of C60-based active esters and coupling of C60 moiety to amines or alcohols

We report the syntheses of C(60)-based active esters and the coupling of their C(60) moiety to various amines or alcohols. Methano[60]fullerene carboxylic acid was activated by esterification with N-hydroxysuccinimide (NHS) or pentafluorophenol (PFP) and the active esters were isolated. Reactions of the active esters with amines or alcohols proceeded easily to give a variety of compounds having the C(60) moiety.     

Constitutive expression of heat shock proteins Hsp90, Hsc70, Hsp70 and Hsp60 in neural and non-neural tissues of the rat during postnatal development

Heat shock proteins (Hsps) are a group of highly conserved proteins, that are constitutively expressed in most cells under normal physiological conditions. Previous work from our laboratory has shown that neurons in the adult brain exhibit high levels of Hsp90 and Hsc70 mRNA and protein, as well as basal levels of Hsp70 mRNA. We have now investigated the expression of Hsp90, Hsc70, Hsp60 and Hsp70 in neural and non-neural tissues of the rat during postnatal development, a time of extensive cell differentiation. Western blot analysis revealed constitutive expression of these Hsps early in postnatal development. Developmental profiles of these Hsps suggest that they are differentially regulated during postnatal development of the rat. For example, while levels of Hsp90 decrease somewhat in certain developing brain regions, levels of Hsp60 show a developmental increase, and Hsc70 protein is abundant throughout postnatal neural development. Low basal levels of Hsp70 are also observed in the developing and adult brain. A pronounced decrease in Hsp90 and Hsc70 was observed during postnatal development of the kidney while levels of Hsp60 increased. In addition, tissue-specific differences in the relative levels of these Hsps between brain and non-brain regions were found. Immunocytochemical studies demonstrated a neuronal localization of Hsp90, Hsc70 and Hsp60 at all stages of postnatal development examined as well as in the adult, suggesting a role for Hsps in both the developing and fully differentiated neuron. The developmental expression of subunit IV of cytochrome oxidase was similar to that of Hsp60, a protein localized predominantly to mitochondria.     

Expression of Hsp90, Hsp70 and Hsp60 in Trichinella species exposed to oxidative shock

Stress response and phosphorylation of heat shock proteins (HSPs) 60, 70 and 90 were studied in Trichinella nativa, T. nelsoni, T. pseudospiralis and T. spiralis larvae at 30-min intervals following exposure to 20, 100 and 200 mM H2O2. There was a time- and dose-dependent differential survival for the infective stage larvae (L1) of these four Trichinella species. Immunoblotting analysis revealed that constitutive Hsp60 and Hsp70, but not Hsp90, from test Trichinella species are constitutively phosphorylated on serine/threonine residues as they converted to forms with increased sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) mobility by treatment with alkaline phosphatase. After exposure to H2O2, while there was a time-related occurrence of the three HSPs with decreased SDS-PAGE mobility, these HSPs were insensitive to alkaline phosphatase except in the case of exposure to 20 mM H2O2 for Hsp60 from all Trichinella species and Hsp70 from T. spiralis and T. nelsoni. The synthesis of HSPs forms with decreased SDS-PAGE mobility is a susceptibility signal because the lower concentration of peroxide (20 mM) did not cause a decrease on HSPs SDS-PAGE mobility in T. spiralis and T. nelsoni, the two more resistant selected Trichinella species.     

Nanostructured layers from DNA, DNA:AU, DNA:C60 clusters

The idea is requisite to coat DNA, DNA:Au, DNA:C60 clusters from water solution, which can be magnetic and electrical active in biosensor systems and to detect their functional properties by microwave techniques (). Our research has been focused on the application of I-V characteristics and surface microwave resonator methods to recognise and predict these molecular interactions based on primary structure and associated physic-chemical properties. In results we have actually shown that these molecular cluster layers on Si and Al2O3 substrates can conduct, switch electric current and respond on power of microwave (additives Au, C60, determine the conductivity of layers). We also aim to apply these Si and Al2O3 ships for Biochip.     

Interference between Proteins Hap46 and Hop/p60, Which Bind to Different Domains of the Molecular Chaperone hsp70/hsc70

Several structurally divergent proteins associate with molecular chaperones of the 70-kDa heat shock protein (hsp70) family and modulate their activities. We investigated the cofactors Hap46 and Hop/p60 and the effects of their binding to mammalian hsp70 and the cognate form hsc70. Hap46 associates with the amino-terminal ATP binding domain and stimulates ATP binding two- to threefold but inhibits binding of misfolded protein substrate to hsc70 and reactivation of thermally denatured luciferase in an hsc70-dependent refolding system. By contrast, Hop/p60 interacts with a portion of the carboxy-terminal domain of hsp70s, which is distinct from that involved in the binding of misfolded proteins. Thus, Hop/p60 and substrate proteins can form ternary complexes with hsc70. Hop/p60 exerts no effect on ATP and substrate binding but nevertheless interferes with protein refolding. Even though there is no direct interaction between these accessory proteins, Hap46 inhibits the binding of Hop/p60 to hsc70 but Hop/p60 does not inhibit the binding of Hap46 to hsc70. As judged from respective deletions, the amino-terminal portions of Hap46 and Hop/p60 are involved in this interference. These data suggest steric hindrance between Hap46 and Hop/p60 during interaction with distantly located binding sites on hsp70s. Thus, not only do the major domains of hsp70 chaperones communicate with each other, but cofactors interacting with these domains affect each other as well.     

Spore coat genes SP60 and SP70 of Dictyostelium discoideum.

We cloned and sequenced the genes for two of the major proteins found in spore coats of Dictyostelium discoideum. The predicted translation product of each of these genes starts with a hydrophobic signal sequence that is subsequently cleaved. Expression of these spore coat genes is coordinate in prespore cells.     

Differential localization of heat shock proteins 90, 70, 60 and 27 in human decidua and placenta during pregnancy

Little is known about the localization of heat shock proteins (HSPs) in the decidua and placenta during the course of normal pregnancy. In this study, we have examined the localization of the HSPs in decidual and placental tissues obtained from women during the first, second and third trimesters of pregnancy (five in each trimester) by immunohistochemistry using highly specific antisera. HSPs 90, 70, 60 and 27 were detected in decidual stromal cells during each trimester. The intensity of staining did not change during gestation for HSPs 60 and 27, whereas it decreased with advancing gestation for HSPs 90 and 70. HSPs 90 and 60 were localized primarily in the nucleus, whereas HSP 70 was present equally in the nucleus and the cytoplasm; HSP 27 was primarily in the cytoplasm. In the placenta, HSPs 90, 70 and 60 were localized in cytotrophoblast, syncytiotrophoblast, intermediate trophoblast, Hofbauer and endothelial cells. HSPs 90 and 60 were localized primarily in the nucleus, while HSP 70 was in the nucleus and the cytoplasm. In the placenta, HSP 27 was detected only in the intermediate trophoblast and syncytiotrophoblast cells and only in the first two trimesters. These results indicate that there are striking differences in the subcellular localization of HSPs in the decidua and the placenta during normal pregnancy.     

Antiamyloid properties of fullerene C60 derivatives

A comparative estimation of the ability of complexes of fullerene C60 with polyvinylpyrrolidone and fullerene C60 derivatives (the sodium salt of the polycarboxylic derivative of fullerene C60, sodium fullerenolate), has been carried out. The fullerenes destroyed amyloid fibrils of the Abeta(1-42) peptide of the brain and the muscle X-protein. A study of the effect of fullerenes on muscle actin showed that complexes of fullerene C60 with polyvinylpyrrolidone and sodium fullerenolate did not prevent the filament formation of actin, nor did they destroy its filaments in vitro. Conversely, sodium salt of the polycarboxylic derivative of fullerene C60 destroyed actin filaments and prevented their formation. It was concluded that sodium fullerenolate and complexes of fullerene C60 with polyvinylpyrrolidone are the most effective antiamyloid compounds among the fullerenes examined.     

Antioxidant action of sugar-pendant C60 fullerenes

The action of C₆₀ fullerene and its derivatives as a radical-scavenging antioxidant has received much attention, but their reactivity toward free radicals and antioxidant capacity have not been well elucidated yet. In the present study, the reactivity of the two types of water-soluble, sugar-pendant C₆₀ fullerenes, C₆₀-1S and C₆₀-2S, toward peroxyl radical and their effect against human plasma lipid peroxidation were measured. The rate constants for the reaction of C₆₀-1S and C₆₀-2S with peroxyl radicals were obtained from their effect on the bleaching of β-carotene in lipid-SDS micelle system as 4.6 × 10³ and 8.0 × 10³ M^?1 s^?1 at 37 °C, respectively. They inhibited the free radical-induced lipid peroxidation in human plasma in a concentration-dependent manner. These results suggest that the sugar-pendant fullerenes C₆₀-1S and C₆₀-2S act as a radical-scavenging antioxidant with the activity similar to the phenolic antioxidants.     

Interaction of C60 fullerene with lipids

Unsaturated lipids when exposed to air at room temperature undergo a slow autoxidation. When fullerene C₆₀ was dissolved in selected lipids (ethyl oleate, ethyl linoleate, linseed oil and castor oil) the spectrophotometric analysis shows that the oxidation is concentrated to C₆₀ which is converted to an epoxide C₆₀O. Thus, fullerene C₆₀ displays antioxidant activity not only when dissolved in unsaturated lipids but also, more generally, when dissolved in unsaturated solvents subjected to autoxidation like, for example, in cyclohexene. The behaviour of C₆₀ in ethyl oleate has been compared with that of the known antioxidant TMPPD (N,N′,N,N,′-tetramethyl-p-phenylenediamine) in ethyl oleate. The mechanism of the antioxidant action of C₆₀ in lipids has been proposed. The kinetics of C₆₀ oxidation in lipids was determined spectrophotometrically both at room temperature in the dark and under UV irradiation. The oxidized products derived from C₆₀ photo-oxidation in lipids have been identified.     

C60 binds to and deforms nucleotides

Atomistic molecular dynamics simulations are performed for up to 20 ns to monitor the formation and the stability of complexes composed of single- or double-strand DNA molecules and C60 in aqueous solution. Despite the hydrophobic nature of C60, our results show that fullerenes strongly bind to nucleotides. The binding energies are in the range -27 to -42 kcal/mol; by contrast, the binding energy of two fullerenes in aqueous solution is only -7.5 kcal/mol. We observe the displacement of water molecules from the region between the nucleotides and the fullerenes and we attribute the large favorable interaction energies to hydrophobic interactions. The features of the DNA-C60 complexes depend on the nature of the nucleotides: C60 binds to double-strand DNA, either at the hydrophobic ends or at the minor groove of the nucleotide. C60 binds to single-strand DNA and deforms the nucleotides significantly. Unexpectedly, when the double-strand DNA is in the A-form, fullerenes penetrate into the double helix from the end, form stable hybrids, and frustrate the hydrogen bonds between end-group basepairs in the nucleotide. When the DNA molecule is damaged (specifically, a gap was created by removing a piece of the nucleotide from one helix), fullerenes can stably occupy the damaged site. We speculate that this strong association may negatively impact the self-repairing process of the double-strand DNA. Our results clearly indicate that the association between C60 and DNA is stronger and more favorable than that between two C60 molecules in water. Therefore, our simulation results suggest that C60 molecules have potentially negative impact on the structure, stability, and biological functions of DNA molecules.