Effects of atomic bomb radiation on the differentiation of B lymphocytes and on the function of concanavalin A-induced suppressor T lymphocytes

The differentiation of peripheral blood B lymphocytes into immunoglobulin-producing cells (Ig-PC) by pokeweed mitogen (PWM) and the function of concanavalin A (Con A)-induced suppressor T lymphocytes were examined to elucidate the late effects of atomic bomb radiation. A total of 140 individuals, 70 with an exposure dose of 100 rad or more and an equal number with an exposure dose of 0 rad matched by sex and age, were selected from the Nagasaki Adult Health Study (AHS) sample. Both the differentiation of peripheral blood B lymphocytes into Ig-PC by PWM and the function of Con A-induced suppressor T lymphocytes tended to be more depressed in the exposed group than in the control group, but a statistically significant difference could not be observed between the two groups. The function of Con A-induced suppressor T lymphocytes tended to decrease with age, but a statistical significance was detected only for percentage suppression against IgM-PC.     

Induction of endoreduplication in Chinese hamsters V79 cells by cytosine arabinoside

Endoreduplication (ER) could be induced very effectively in Chinese hamster V79 cells exposed to cytosine arabinoside (1-β-D-arabinofuranosylcytosine; Ara-C). Cells were cultured for 48 hours in Ara-C containing medium. ER frequency increases rapidly after Ara-C release. About 60% of metaphase cells were endoreduplicated at 8–10 hours after release from Ara-C (5 μg/ml). Induction of ER also depends on Ara-C concentrations.     

Cap structure of U3 small nucleolar RNA in animal and plant cells is different. gamma-Monomethyl phosphate cap structure in plant RNA.

U3 small nucleolar RNA (snoRNA) is an abundant small RNA involved in the processing of pre-ribosomal RNA of eukaryotic cells. U3 snoRNA has been previously characterized from several sources, including human, rat, mouse, frog, fruit fly, dinoflagellates, slime mold, and yeast; in all these organisms, U3 snoRNA contains trimethylguanosine cap structure. In all instances where investigated, the trimethylguanosine-capped snRNAs including U3 snoRNA, are synthesized by RNA polymerase II. However, in higher plants, the U3 snoRNA is synthesized by RNA polymerase III and contains a cap structure different from trimethylguanosine (Kiss, T., and Solymosy, F. (1990) Nucleic Acids Res. 18, 1941-1949; Marshallsay, C., Kiss, T., and Filipowicz, W. (1990) Nucleic Acids Res. 18, 3451-3458; Kiss, T., Marshallsay, C., and Filipowicz, W. (1991) Cell 65, 517-526). In this study, we present evidence that cowpea and, most likely, tomato plant U3 snoRNA contains a methyl-pppA cap structure. These data show that the same U3 snoRNA contains different cap structures in different species and suggest that the kind of cap structure that an uridylic acid-rich small nuclear RNA contains is dependent on the RNA polymerase responsible for its synthesis. In vitro synthesized plant U3 snoRNA, with pppA or pppG as its 5' end, was converted to methyl-pppA/G cap structure in vitro when incubated with extracts prepared from wheat germ or HeLa cells. These data show that the capping machinery is conserved in organisms as evolutionarily distant as plants and mammals. Nucleotides 1-45 of tomato U3 snoRNA, which are capable of forming a stem-loop structure, are sufficient to direct the methyl cap formation in vitro.     

Quantitative identification of the protonation state of histidines in vitro and in vivo

The C[bond]N coupling constants centered at the C(epsilon 1) and C(delta 2) carbons in histidine residues depend on the protonation state and tautomeric form of the imidazole ring, making them excellent indicators of pH or pK(a), and the ratio of the tautomeric states. In this paper, we demonstrate that the intensity ratios for the C(epsilon 1)-H and C(delta 2)-H cross-peaks measured with a constant time HSQC experiment without and with J(C[bond]N) amplitude modulation are determined by the ratios of the protonated and deprotonated forms and tautomeric states. This allows one to investigate the tautomeric state of histidines as well as their pK(a) in situations where changing the pH value by titration is difficult, for example, for in-cell NMR experiments. We apply this technique to the investigation of the bacterial protein NmerA and determine that the intracellular pH in the Escherichia coli cytoplasm is 7.1 +/- 0.1.     

Enhancement of nucleoside phosphorylation activity in an acid phosphatase

Escherichia blattae non-specific acid phosphatase (EB-NSAP) possesses a pyrophosphate-nucleoside phosphotransferase activity, which is C-5'-position selective. Current mutational and structural data were used to generate a mutant EB-NSAP for a potential industrial application as an effective and economical protein catalyst in synthesizing nucleotides from nucleosides. First, Gly74 and Ile153 were replaced by Asp and Thr, respectively, since the corresponding replacements in the homologous enzyme from Morganella morganii reduced the K(m) value for inosine and thus increased the productivity of 5'-IMP. We determined the crystal structure of G74D/I153T, which has a reduced K(m) value for inosine, as expected. The tertiary structure of G74D/I153T was virtually identical to that of the wild-type. In addition, neither of the introduced side chains of Asp74 and Thr153 is directly involved in the interaction with inosine in a hypothetical binding mode of inosine to EB-NSAP, although both residues are situated near a potential inosine-binding site. These findings suggested that a slight structural change caused by an amino acid replacement around the potential inosine-binding site could significantly reduce the K(m) value. Prompted by this hypothesis, we designed several mutations and introduced them to G74D/I153T, to decrease the K(m) value further. This strategy produced a S72F/G74D/I153T mutant with a 5.4-fold lower K(m) value and a 2.7-fold higher V(max) value as compared to the wild-type EB-NSAP.     

Collagen-binding mode of vWF-A3 domain determined by a transferred cross-saturation experiment

The nature of the supramolecular complex between fibrillar collagen and collagen-binding proteins (CBPs) has hindered detailed X-ray and NMR analyses of the ligand-recognition mechanism at atomic resolution because of the lack of appropriate approaches for studying large heterogeneous supramolecular complexes. Recently, we proposed an NMR method, termed transferred cross-saturation (TCS), that enables the rigorous identification of contact residues in a huge protein complex. Here we used TCS to study the supramolecular complex between the A3 domain of von Willebrand factor and fibrillar collagen, which allowed the successful determination of the ligand-binding site of the A3 domain. The binding site of the A3 domain was located at its hydrophobic 'front' surface and was completely different from that of the I domain from the a2 subunit of integrin (alpha2-I domain), which was reported to be the hydrophilic 'top' surface of alpha2-I, although the A3 domain and the alpha2-I domain share a similar fold and possess the identical function of collagen binding.     

Rapid identification of protein-protein interfaces for the construction of a complex model based on multiple unassigned signals by using time-sharing NMR measurements

Protein-protein interactions are necessary for various cellular processes, and therefore, information related to protein-protein interactions and structural information of complexes is invaluable. To identify protein-protein interfaces using NMR, resonance assignments are generally necessary to analyze the data; however, they are time consuming to collect, especially for large proteins. In this paper, we present a rapid, effective, and unbiased approach for the identification of a protein-protein interface without resonance assignments. This approach requires only a single set of 2D titration experiments of a single protein sample, labeled with a unique combination of an (15)N-labeled amino acid and several amino acids (13)C-labeled on specific atoms. To rapidly obtain high resolution data, we applied a new pulse sequence for time-shared NMR measurements that allowed simultaneous detection of a ω(1)-TROSY-type backbone (1)H-(15)N and aromatic (1)H-(13)C shift correlations together with single quantum methyl (1)H-(13)C shift correlations. We developed a structure-based computational approach, that uses our experimental data to search the protein surfaces in an unbiased manner to identify the residues involved in the protein-protein interface. Finally, we demonstrated that the obtained information of the molecular interface could be directly leveraged to support protein-protein docking studies. Such rapid construction of a complex model provides valuable information and enables more efficient biochemical characterization of a protein-protein complex, for instance, as the first step in structure-guided drug development.     

Comparative study of hydrogen peroxide- and 4-hydroxy-2-nonenal-induced cell death in HT22 cells

Several studies have indicated that lipid peroxidation often occurs in response to oxidative stress, and that many aldehydic products including 4-hydroxy-2-nonenal (HNE) are formed when lipid hydroperoxides break down. In order to clarify the mechanism of oxidative stress-induced neuronal death in the nervous system, we investigated H(2)O(2)- and HNE-induced cell death pathways in HT22 cells, a mouse hippocampal cell line, under the same experimental conditions. Treatment with H(2)O(2) and HNE decreased the viability of these cells in a time- and concentration-dependent manner. In the cells treated with H(2)O(2), significant increases in the immunoreactivities of DJ-1 and nuclear factor-kappaB (NF-kappaB) subunits (p65 and p50) were observed in the nuclear fraction. H(2)O(2) also induced an increase in the intracellular concentration of Ca(2+), and cobalt chloride (CoCl(2)), a Ca(2+) channel inhibitor, suppressed the H(2)O(2)-induced cell death. In HNE-treated cells, none of these phenomena were observed; however, HNE adduct proteins were formed after exposure to HNE, but not to H(2)O(2). N-Acetyl-L-cysteine (NAC) suppressed both HNE-induced cell death and HNE-induced expression of HNE adduct proteins, whereas H(2)O(2)-induced cell death was not affected. These findings suggest that the mechanisms of cell death induced by H(2)O(2) different from those induced by HNE in HT22 cells, and that HNE adduct proteins play an important role in HNE-induced cell death. It is also suggested that the pathway for H(2)O(2)-induced cell death in HT22 cells does not involve HNE production.     

Sympathetic Activation Induces Skeletal Fgf23 Expression in a Circadian Rhythm-dependent Manner

The circadian clock network is well known to link food intake and metabolic outputs. Phosphorus is a pivotal nutritional factor involved in energy and skeletal metabolisms and possesses a circadian profile in the circulation; however, the precise mechanisms whereby phosphate metabolism is regulated by the circadian clock network remain largely unknown. Because sympathetic tone, which displays a circadian profile, is activated by food intake, we tested the hypothesis that phosphate metabolism was regulated by the circadian clock network through the modification of food intake-associated sympathetic activation. Skeletal Fgf23 expression showed higher expression during the dark phase (DP) associated with elevated circulating FGF23 levels and enhanced phosphate excretion in the urine. The peaks in skeletal Fgf23 expression and urine epinephrine levels, a marker for sympathetic tone, shifted from DP to the light phase (LP) when mice were fed during LP. Interestingly, β-adrenergic agonist, isoproterenol (ISO), induced skeletal Fgf23 expression when administered at ZT12, but this was not observed in Bmal1-deficient mice. In vitro reporter assays revealed that ISO trans-activated Fgf23 promoter through a cAMP responsive element in osteoblastic UMR-106 cells. The mechanism of circadian regulation of Fgf23 induction by ISO in vivo was partly explained by the suppressive effect of Cryptochrome1 (Cry1) on ISO signaling. These results indicate that the regulation of skeletal Fgf23 expression by sympathetic activity is dependent on the circadian clock system and may shed light on new regulatory networks of FGF23 that could be important for understanding the physiology of phosphate metabolism.