Recombinant homo- and hetero-oligomers of an ultrastable chaperonin from the archaeon Pyrodictium occultum show chaperone activity in vitro

The archaeon Pyrodictium occultum is one of the most thermophilic organisms presently known. Previous experiments provided support for the significant contribution of a high-molecular-mass protein complex to the extreme thermotolerance of P. occultum. This protein complex, the 'thermosome', is composed of two subunits, alpha and beta, which form a hexadecameric double ring complex. In order to obtain the thermosome in amounts sufficient for structural and functional investigations, we produced the two subunits jointly and separately in Escherichia coli BL21(DE3). In all three cases, we isolated soluble, high-molecular-mass double-ring complexes from E. coli BL21(DE3). On electron micrographs, the recombinant complexes were indistinguishable from each other and from the natural thermosome. To characterize the quaternary structure of the recombinant particles, we used native gel electrophoresis, analytical gel filtration, and analytical ultracentrifugation. Spectral analysis, using absorption, fluorescence emission and far-UV circular dichroism spectroscopy were applied to compare the three recombinant protein complexes with the natural thermosome from P. occultum. All three recombinant complex species exhibit ATPase activity. Furthermore, we could demonstrate that the recombinant complexes slow down the aggregation of citrate synthase, alcohol dehydrogenase, and insulin. Thus, we conclude that the recombinant protein complexes exhibit a chaperone-like activity, interacting with non-native proteins; they do so at temperatures far below the lower physiological limit of growth.     

Chemical structure and thermal properties of initiator tRNA from Euphausia sperba in comparison with those of other eucaryotic initiator tRNAs

The nucleotide sequence of initiator tRNA (tRNAiMet) from Euphausia sperba, which was harvested in the Antarctic Sea, was determined to be pA-G-C-A-G-A-G-U-m1G-m2G-C-G-C-A-G-U-G-G-A-A-G-C-G-U-m2G-C-U-G-G-G-C-C-C-A-U-t6 A-A-C-C-C-A-G-A-G-m7G-U-C-G-G-U-A-G-A-psi-C-G-m1A-A-A-C-U-A-C-U-C-U-C-U-G-C-U-A -C-C-AOH by using post-labeling methods recently developed. The nucleotide sequence was very similar to that of mammalian tRNAiMet except for changes in six bases and three modifications: C16, U55, D47 and m5C48 are replaced by U16, psi 55 and unmodified U47 and C48, respectively. A50-U64 and G52-C62 base pairs of mammalian tRNAiMet are reversed in Euphausia tRNAiMet. In addition, the G49-C65 pair of the former is replaced by a less stable G49-U65 pair in Euphausia tRNAiMet. The sequence homology was compared between Euphausia tRNAiMet and over ten different species of eucaryotic tRNAiMet so far sequenced. The melting temperature of Euphausia tRNAiMet was 72.5 degrees C, which is 4.2 degrees C and 8.3 degrees C lower than those of rat liver and yeast tRNAiMet's, respectively. The origin of the thermal instability of Euphausia tRNAiMet is discussed in comparison of its secondary structure compared with those of other eucaryotic tRNAiMet's.     

The potato mitochondrial initiator methionine tRNA gene and its flanking regions: an illustration of the diversity of mitochondrial genome rearrangements among plant species

The initiator methionine transfer RNA (tRNA(fMet)) gene was identified on a 347 bp Eco RI-Hind III DNA fragment of the potato mitochondrial (mt) genome. The sequence of this gene shows 1 to 7 nucleotide differences with the other plant mt tRNAs(fMet) or tRNA(fMet) genes studied so far. Whereas the tRNA(fMet) gene is present as a single copy in the potato mt genome, a tRNA 'pseudogene' corresponding to 60% of a complete tRNA (from the 5' end to the variable region) and located at 105 nucleotides upstream of the tRNA(fMet) gene on the opposite strand was shown to be repeated at least three times. Furthermore, the physical environment of the tRNA(fMet) gene in the mt genome is very different among plants, which suggests that the tRNA(fMet) gene region has often been implicated in recombination events of plant mt genomes leading to important rearrangements in gene order.     

Stimulation of corticotrophin-releasing hormone release by the obese (ob) gene product, leptin, from hypothalamic explants

Recent data have suggested that adipocytes synthesize and secrete a 16 kDa peptide which acts centrally to regulate weight gain by suppressing appetite and activating the sympathetic nervous system. To exert such effects, it may function as an endogenous ligand in the CNS, since specific receptors (OB-R) have been recently reported to be widely distributed in the brain. We have speculated that this peptide, now known as leptin, may act centrally by stimulating the release of corticotrophin-releasing hormone (CRH), a recognized potent inhibitory modulator of appetite. We tested in vitro the effect of murine leptin on CRH secretion in the dose range of 0.1 pM-100 nM. The static rat hypothalamic incubation system used involved fresh hypothalamic explants maintained in EBSS with consecutive 20 min incubations, and estimation of CRH concentrations in the medium by a specific and sensitive radioimmunoassay. The effect of heat-denatured leptin at a dose of 1 nM and 10 nM, was also investigated. Any possible modulation of leptin effects by adrenergic pathways was then explored by coincubating hypothalami with leptin 10 nM and equimolar concentrations of the alpha 1-adrenergic antagonist prazosin or the beta-adrenergic antagonist propranolol. The active leptin, but not the heat-inactivated peptide, caused a dose-dependent stimulation of CRH release in vitro (p < 0.05- < 0.0001 vs control), with a plateau effect at a dose of 10 nM. The addition of either prazosin or propranolol was without effect on leptin-dependent CRH stimulation. These findings are consistent with the reported presence of leptin receptors in the rat brain, and suggest that leptin may act to regulate appetite at least in part by directly modulating the secretion of CRH from the hypothalamus. It would also appear that such effect occurs via a non-adrenergic mechanism.     

A comparative study of the thermal stability of phenylalanyl tRNA synthetase from Escherichia coli MRE-600 and Thermus thermophilus HB 8

The thermal stability of phenylalanyl-tRNA-synthetase (PTS) from E. coli and T.thermophilus HB 8 was studied in solution at various conditions by scanning microcalorimetry. It has been shown that the value of heating rate, concentration of the enzyme and Mg2+ ions in the solution affects the parameters of thermal denaturation of both enzymes. The higher thermal stability of PTS from T. thermophilus was observed as well as the independence of its properties upon broad variations of experimental conditions. The role of thermostability of the enzymes are discussed with respect to the biological properties of E. coli and T.thermophilus.     

Modified nucleosides in the first positions of the anticodons of tRNA(Leu)4 and tRNA(Leu)5 from Escherichia coli

Minor leucine tRNA species, tRNA(Leu)4 and tRNA(Leu)5, from Escherichia coli B have been reported to recognize leucine codons UUA and UUG [Goldman, E., Holmes, W. M., and Hatfield, G. W. (1979) J. Mol. Biol. 129, 567-585]. In the present study, these two tRNA(Leu) species were purified from E. coli A19, and the nucleotide sequences were determined by a post-labeling method. tRNA(Leu)5 was found to correspond to the tRNA gene reported as su degrees6 tRNA [Yoshimura, M., Inokuchi, H., and Ozeki, H. (1984) J. Mol. Biol. 177, 627-644]. The first letter of the anticodon was identified to be 2'-O-methylcytidine (Cm). tRNA(Leu)4 was identified as the minor leucine tRNA that has been sequenced previously (tRNA(Leu)UUR) [Yamaizumi, Z., Kuchino, Y., Harada, F., Nishimura, S., and McCloskey, J. A. (1980) J. Biol. Chem. 255, 2220-2225]. There was an unidentified modified nucleoside (N*) in the first position of the anticodon of tRNA(Leu)4. Nucleoside N* was isolated to homogeneity (1 A260 unit). By 1H NMR spectroscopy, nucleoside N was found to be a 2'-O-methyluridine derivative with a substituent having a -CH2NH2+CH2COO- moiety in position 5 of the uracil ring. On the basis of these NMR analyses together with mass spectrometry, the chemical structure of nucleoside N* was determined as 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um). Nucleoside N* was thus found to be a novel type of naturally occurring modified uridine. Because of the conformational rigidity of Cm and cmnm5Um in the first position of the anticodon, these tRNA(Leu) species recognize the leucine codons UUA++ and UUG correctly, but never recognize the phenylalanine codons UUU and UUC.     

Structural characteristics of the pedicle and its role in screw stability

STUDY DESIGN: Cross-sectional regional bone mineral density of the pedicle was measured by peripheral quantitative computed tomography. Biomechanical tests were performed to clarify the role of the pedicle in screw stability. OBJECTIVES: To identify the structural characteristics of the pedicle that supports pedicle screw stability and the differences in these characteristics between normal and osteoporotic vertebrae. SUMMARY OF BACKGROUND DATA: The pedicle screw is an essential component of many systems used to align the spine. The contribution of the pedicle to screw stability, however, has not been fully investigated. METHODS: Trabecular, subcortical, and cortical bone mineral density and the area of the pedicle were measured by peripheral quantitative computed tomography. Bone mineral density also was recalculated in four circumferential layers. These parameters were compared between normal and osteoporotic individuals. The relative contribution of the pedicle to screw stability was evaluated by caudocephalad and pull-out loading in a vertebra with or without its body. RESULTS: Inner trabecular, middle subcortical, and outer cortical bone mineral density and cortical bone area in the pedicle were significantly lower in osteoporotic vertebrae than those in normal vertebrae. In the pedicle, bone mineral density increased close to the outer layer. Bone mineral density not as thick even in the outer layer in osteoporotic subjects. Approximately 80% of the caudocephalad stiffness and 60% of the pullout strength of the pedicle screw depended on the pedicle rather than on the vertebral body. CONCLUSION: Screw stability depends on the structural characteristics of the pedicle. The pedicle was denser in the subcortical bone, in which the threads of the screw engage, than in trabecular bone. In osteoporosis, bone mineral density was not as dense even in the outer layer, and the cortex was thinner than normal. A larger screw would not enhance screw stability and may break the thin cortex in osteoporotic vertebrae.     

Overexpression, purification, DNA binding, and dimerization of the Escherichia coli uvrD gene product (helicase II)

We have subcloned the Escherichia coli uvrD gene under control of the inducible phage lambda PL promoter and report a procedure for the large-scale purification of helicase II protein. Yields of approximately 60 mg of > 99% pure helicase II protein, free of detectable nuclease activity, are obtained starting from 250 g of induced E. coli cells containing the overexpression plasmid. Overproduction of helicase II protein at these levels is lethal in E. coli. The extinction coefficient of helicase II protein was determined to be epsilon 280 = 1.06 (+/- 0.05) x 10(5) M-1 (monomer) cm-1 [20 mM Tris-HCl (pH 8.3 at 25 degrees C), 0.2 M NaCl, and 20% (v/v) glycerol, 25 degrees C]. We also present a preliminary characterization of the dimerization and DNA binding properties of helicase II and a systematic examination of its solubility properties. The apparent site size of a helicase II monomer on ss-DNA is 10 +/- 2 nucleotides as determined by quenching of the intrinsic tryptophan fluorescence of the protein upon binding poly(dT). In the absence of DNA, helicase II protein can self-assemble to form at least a dimeric species at concentrations > 0.25 microM (monomer) and exists in a monomer-dimer equilibrium under a variety of solution conditions. However, upon binding short oligodeoxynucleotides, the dimeric form of helicase II is stabilized, and dimerization stimulates the ss-DNA-dependent ATPase activity, suggesting that the dimer is functionally important. On the basis of these observations and similarities between helicase II and the E. coli Rep helicase, which appears to function as a dimer [Chao, K., & Lohman, T. (1991) J. Mol. Biol. 221, 1165-1181], we suggest that the active form of helicase II may also be a dimer or larger oligomer.     

A mitochondrial tRNA(Val) gene mutation (G1642A) in a patient with mitochondrial myopathy, lactic acidosis, and stroke-like episodes

We studied a patient with the diagnosis of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) for mitochondrial DNA mutations in muscle. Established MELAS mutations were excluded. Mitochondrial DNA was further analyzed for mutations in the 22 tRNA genes by single-strand conformation polymorphism (SSCP) analysis; a tRNA(Val) mutation (G1642A) was found. The structure of the altered tRNA, the heteroplasmy, and the absence of the mutation in the mother and in 100 control subjects suggests that the tRNA(Val) mutation is associated with the MELAS syndrome.     

Molecular cloning and nucleotide sequence of the superoxide dismutase gene and characterization of its product from Bacillus subtilis

Bacillus subtilis was found to possess one detectable superoxide dismutase (Sod) in both vegetative cells and spores. The Sod activity in vegetative cells was maximal at stationary phase. Manganese was necessary to sustain Sod activity at stationary phase, but paraquat, a superoxide generator, did not induce the expression of Sod. The specific activity of purified Sod was approximately 2, 600 U/mg of protein, and the enzyme was a homodimer protein with a molecular mass of approximately 25,000 per monomer. The gene encoding Sod, designated sodA, was cloned by the combination of several PCR methods and the Southern hybridization method. DNA sequence analysis revealed the presence of one open reading frame consisting of 606 bp. Several putative promoter sites were located in the upstream region of sodA. The deduced amino acid sequence showed high homology with other bacterial manganese Sods. Conserved regions in bacterial manganese Sod could also be seen. The phenotype of double mutant Escherichia coli sodA sodB, which could not grow in minimal medium without supplemental amino acids, was complemented by the expression of B. subtilis sodA.     

蓬莱大柳行金矿区水沟——时金河断裂构造特征及其找矿意义

以构造地质及构造地球化学等研究为依据，重新厘定了水沟－时金河断裂的规模，弄清了它的引张－压扭－张扭的构造性质，探讨了断裂形成时代、演化历史及其对岩浆作用和成矿作用的控制规律。认为该断裂具有导容矿双重性。导矿作用发生在断裂的开放构造环境及相关部位，并在其两侧形成石英脉金矿床。容矿作用发生在断裂的封闭构造环境及相关部位，在断裂内形成蚀变岩型金矿床。实现了找矿勘探方向由派生断裂向主干断裂的战略转移，为扩     

Heme binding by a bacterial repressor protein, the gene product of the ferric uptake regulation (fur) gene of Escherichia coli

The fur gene product, Fur, of Escherichia coli is a repressor when it binds Fe(II). Since heme and iron metabolism are closely linked and Fur is rich in histidine, a ligand for heme, the binding of heme to Fur was investigated. The oxidized Fur-heme complex is stable and low spin with a Soret maximum at 404 nm and no 620-nm band. CO coordinates with the reduced heme-Fur complex, causing a shift from 412 nm to 410 nm, and stabilizes it, increasing the half-life from 5 to 15 min. Circular dichroism (CD) spectra in the Soret region show heme bound in an asymmetric environment in Fur, both in the oxidized and reduced-CO forms. Quenching of tyrosine fluorescence by heme revealed rapid, tight binding (Kd < 1 microM) with an unusual stoichiometry of 1 heme:1 Fur dimer. Fur binds Mn(II), a model ligand for the endogenous Fe(II), much more weakly (Kd > 80 microM). Far-ultraviolet CD spectroscopy showed that the alpha-helix content of apo-Fur decreases slightly with heme binding, but increases with Mn(II) binding. Competition experiments indicated that heme interacts with Fur dimers at the same site as Mn(II) and can displace the metal. In contrast to Mn(II), Zn(II) did not quench the tyrosine fluoroescence of Fur, affected the CD spectrum less than Mn(II), but did bind in a manner which prevented heme from binding. In sum, Fur not only binds heme and Zn(II) with sufficient affinity to be biologically relevant, but the interactions that occur between these ligands and their effects on Mn(II) binding need to be taken into account when addressing the biological function of Fur.