[Dietary nucleotide and its effects on immunity].
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Animals↗
Biomedical subjects
X Zhao
Publications and source records attributed to X Zhao.
[Association and isochromatization of ion-pair of the acid and basic].
In this paper, the method for forming the isochromatic dye ion-pair was described, and the mechanism and the types of the association of the ion-pair studied. The isochromatization of the associated reaction and the solvent effect were discussed by the electron theory with the absorption spectrum and energy diagram. The results of the extraction spectrophotometric method of the isochromatic dye ion-pairs were shown in a table, indicating the high sensitivity of the isochromatic dye ion-pairs. It is proven that the method of the extraction spectrophotometry, the flotation spectrophotometry and the extraction fluorometry was of high and superhigh sensitivity.
English Abstract↗
[Absorption characteristics of the isochromatic dye ion-pair formed by tetrabromofluorescein and buthylrhodamine B].
Absorption properties of tetrabromofluorescein (TBF) and buthylrhodamine B (BRB) and the isochromatic dye ion-pair formed by TBF and BRB were studied. The experiment showed that the proportion of TBF: BRB was 1:2 at pH=7 with Ksp = 2.16 x 10(-18), and the extraction rate was 84.2%. This provides a basis for the application of TBF and BRB to extraction spectrophotometry.
English Abstract↗
Dependence of superoxide anion production on extracellular and intracellular calcium ions and protein kinase C in PMA-stimulated bovine neutrophils.
The involvement of both intracellular and extracellular calcium, as well as the activation of protein kinase C (PKC), in phorbol myristate acetate (PMA)-stimulated respiratory burst in bovine neutrophils has been studied. PMA significantly stimulated the superoxide anion production by these cells. The increased production of superoxide anion was inhibited by BAPTA/AM, an intracellular calcium ([Ca2+]i) chelator, but not affected by EGTA, an extracellular calcium ([Ca2+]0) chelator. PMA also induced PKC activation, and a PKC inhibitor, calphostin C, blocked the stimulatory effect of PMA on superoxide anion production by the neutrophils. Therefore, we conclude that PMA-induced respiratory burst in bovine neutrophils is [Ca2+]i- but not [Ca2+]0-dependent, and also requires PKC activation.
Animals↗
Generation of destabilized green fluorescent protein as a transcription reporter.
The green fluorescent protein (GFP) is a widely used reporter in gene expression and protein localization studies. GFP is a stable protein; this property allows its accumulation and easy detection in cells. However, this stability also limits its application in studies that require rapid reporter turnover. We created a destabilized GFP for use in such studies by fusing amino acids 422-461 of the degradation domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of an enhanced variant of GFP (EGFP). The fusion protein, unlike EGFP, was unstable in the presence of cycloheximide and had a fluorescence half-life of 2 h. Western blot analysis indicated that the fluorescence decay of EGFP-MODC-(422-461) was correlated with degradation of the fusion protein. We mutated key amino acids in the PEST sequence of EGFP-MODC-(422-461) and identified several mutants with variable half-lives. The suitability of destabilized EGFP as a transcription reporter was tested by linking it to NFkappaB binding sequences and monitoring tumor necrosis factor alpha-mediated NFkappaB activation. We obtained time course induction and dose response kinetics similar to secreted alkaline phosphatase obtained in transfected cells. This result did not occur when unmodified EGFP was used as the reporter. Because of its autofluorescence, destabilized EGFP can be used to directly correlate gene induction with biochemical change, such as NFkappaB translocation to the nucleus.
Amino Acid Sequence↗
Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ.
Chaperones of the Hsp70 family bind to unfolded or partially folded polypeptides to facilitate many cellular processes. ATP hydrolysis and substrate binding, the two key molecular activities of this chaperone, are modulated by the cochaperone DnaJ. By using both genetic and biochemical approaches, we provide evidence that DnaJ binds to at least two sites on the Escherichia coli Hsp70 family member DnaK: under the ATPase domain in a cleft between its two subdomains and at or near the pocket of substrate binding. The lower cleft of the ATPase domain is defined as a binding pocket for the J-domain because (i) a DnaK mutation located in this cleft (R167H) is an allele-specific suppressor of the binding defect of the DnaJ mutation, D35N and (ii) alanine substitution of two residues close to R167 in the crystal structure, N170A and T173A, significantly decrease DnaJ binding. A second binding determinant is likely to be in the substrate-binding domain because some DnaK mutations in the vicinity of the substrate-binding pocket are defective in either the affinity (G400D, G539D) or rate (D526N) of both peptide and DnaJ binding to DnaK. Binding of DnaJ may propagate conformational changes to the nearby ATPase catalytic center and substrate-binding sites as well as facilitate communication between these two domains to alter the molecular properties of Hsp70.
Adenosine Triphosphatases↗
Shaping limbs by apoptosis.
Programmed cell death has long been recognized as an important mechanism of normal embryonic development. In the developing limb, massive programmed cell death apparently plays a critical role in controlling the amount of mesodermal tissue, sculpting overall limb shape, and defining the digits. Cell death in the limb bud has been shown to be regulated by a number of factors, including environmental conditions and epithelial-mesenchymal interactions. Removing the ectoderm overlying the interdigital region results in inhibition of cell death and ectopic cartilage formation in the subjacent mesenchyme. Recently, several signaling factors and genes have been implicated in the control of cell death in the limb bud. FGFs may acts as trophic factors that interfere with cell death. There is evidence that BMPs and Msx genes participate in regulating cell death. BMPs may in some cases trigger the cell death cascade, while Msx gene products regulate Bmp expression. A correlation between FGF, BMP and Msx in interdigital cell death is discussed.
Animals↗
Functional analysis of the proximal CCR5 promoter.
Two promoters for the CCR5 gene, termed Pu and Pd, corresponding to the upstream and downstream initiation sites, respectively, have been described. We show here that the proximal promoter, Pd, is used two- to fivefold more frequently than Pu in primary activated T cells and in the transformed T cell line PM1. Because of its importance in CCR5 transcription we characterized the transcriptional activity of this promoter. Pd contains a pair of consensus TATA elements (nt -19 and -31) and several potential regulatory elements and transcription factor-binding sites, including those for STAT, NF-kappaB, AP-1, NF-AT, and CD28RE. Using a transfected reporter vector, we found the promoter to be highly active and cell type specific. By 5' deletion analysis, the minimal CCR5 promoter was localized to a 225-nucleotide region (nt -189 to +36). This region contained the two TATA elements, a CD28RE consensus sequence, an AP-1-binding site, and two STAT-binding sites. The 1.9-kb intron appeared to have a negative influence on reporter gene activity, suggesting the presence of a negative element in this region. In addition, an upstream negative element was detected in the region nt -988 to -588. Mutagenesis of the TATA elements, of the NF-kappaB-, and AP-1-, and STAT-binding sites, and of the CD28RE indicated the importance of each of these in transcription. Finally, the NF-kappaB/Rel family member, p65(RelA), was a potent activator of the CCR5 promoter.
Base Sequence↗
Regional calpain and caspase-3 proteolysis of alpha-spectrin after traumatic brain injury.
Activity of calpains and caspase-3 inferred from proteolysis of the cytoskeletal protein alpha-spectrin into signature spectrin breakdown products (SBDPs) was used to provide the first systematic and simultaneous comparison of changes in activity of these two families of cysteine proteases after traumatic brain injury (TBI) in rats. Distinct regional and temporal patterns of calpain/caspase-3 processing of alpha-spectrin were observed in brain regions ipsilateral to the site of injury after TBI, including large increases of 145 kDa calpain-mediated SBDP in cortex (up to 30-fold), and enduring increases (up to 2 weeks) of 145 kDa SBDP in hippocampus and thalamus. By contrast, 120 kDa caspase-3-mediated SBDP was absent in cortex and showed up to a 2-fold increase in hippocampus and striatum at early (hours) after TBI. Future studies will clarify the pathological significance of large regional differences in activation of calpain and caspase-3 proteases after TBI.
Animals↗
Genotoxicity and chronic health effects of automobile exhaust: a study on the traffic policemen in the city of Lanzhou.
A study on the health and genotoxic effects of occupational exposure to automobile exhaust was carried out among traffic policemen in Lanzhou (China) in 1996. A total of 78 traffic policemen working in the field was the exposed group, and 57 household register policemen working in the office was the control group. The health effects were evaluated by health questionnaires. Significant differences were observed between the exposed and control groups with respect to the morbidity of rhinitis, pharyngitis, trachoma, syndrome of neurasthenia and joint pain, apart from disorders in the digestive system. The percentage of rhinitis and pharyngitis obtained in the two groups was significantly higher among the smokers than among the nonsmokers. The frequencies of micronuclei (MN) and sister-chromatid exchanges (SCEs) were measured in peripheral blood lymphocytes from the two groups of volunteers. Compared with the control group, statistically significant increases of MN and SCE were found for the exposed group. The increase in the induction of MN and SCE among the traffic policemen and household register policemen is enhanced further by smoking.
Adult↗
Giant electrostriction and relaxor ferroelectric behavior in electron-irradiated poly(vinylidene fluoride-trifluoroethylene) copolymer
An exceptionally high electrostrictive response ( approximately 4 percent) was observed in electron-irradiated poly(vinylidene fluoride-trifluoroethylene) [P(VDF-TrFE)] copolymer. The material exhibits typical relaxor ferroelectric behavior, suggesting that the electron irradiation breaks up the coherent polarization domain (all-trans chains) in normal ferroelectric P(VDF-TrFE) copolymer into nanopolar regions (nanometer-size, all-trans chains interrupted by trans and gauche bonds) that transform the material into a relaxor ferroelectric. The expanding and contracting of these polar regions under external fields, coupled with a large difference in the lattice strain between the polar and nonpolar phases, generate an ultrahigh strain response.
Journal Article↗
Temporal relationships between de novo protein synthesis, calpain and caspase 3-like protease activation, and DNA fragmentation during apoptosis in septo-hippocampal cultures.
Caspase 3-like proteases are key executioners in mammalian apoptosis, and the calpain family of cysteine proteases has also been implicated as an effector of the apoptotic cascade. However, the influence of upstream events on calpain/caspase activation and the role of calpain/caspase activation on subsequent downstream events are poorly understood. This investigation examined the temporal profile of apoptosis-related events after staurosporine-induced apoptosis in mixed glial-neuronal septo-hippocampal cell cultures. Following 3 hr exposure to staurosporine (0.5 microM), calpain and caspase 3-like proteases processed alpha-spectrin to their signature proteolytic fragments prior to endonuclease-mediated DNA fragmentation (not evident until 6 hr), indicating that endonuclease activation is downstream from calpain/caspase activation. Cycloheximide, a general protein synthesis inhibitor, completely prevented processing of alpha-spectrin by calpains and caspase 3-like proteases, DNA fragmentation and cell death, indicating that de novo protein synthesis is an upstream event necessary for activation of calpains and caspase 3-like proteases. Calpain inhibitor II and the pan-caspase inhibitor Z-D-DCB each inhibited their respective protease-specific processing of alpha-spectrin and attenuated endonuclease DNA fragmentation and cell death. Thus, activation of calpains and caspase 3-like proteases is an early event in staurosporine-induced apoptosis, and synthesis of, as yet, unknown protein(s) is necessary for their activation.
Animals↗
pH dependency of mu-calpain and m-calpain activity assayed by casein zymography following traumatic brain injury in the rat.
Studies employing casein zymographic assays analyzed the effects of varying pH (from pH 6.8 to pH 8.0) on changes in mu-calpain and m-calpain activity in naive, sham-injured and injured rat cortex 3 h following unilateral cortical impact injury. Mu-calpain activity following cortical impact injury was enhanced between pH values of 7.2 and 7.8, with pH 7.5 being optimal. m-Calpain activity was readily detected only between pH values of 7.2 and 7.4, with pH 7.3 producing the most prominent proteolytic activity. These observations suggest that strict control of pH is an important consideration in assessments of brain pH activation by casein zymography. Moreover, activation of different calpain isoforms, especially after traumatic brain injury, may be differentially influenced by smaller changes in physiological pH than previously recognized.
Animals↗
Solution and crystal structures of a sperm whale myoglobin triple mutant that mimics the sulfide-binding hemoglobin from Lucina pectinata.
The bivalve mollusc Lucina pectinata harbors sulfide-oxidizing chemoautotrophic bacteria and expresses a monomeric hemoglobin I, HbI, with normal O2, but extraordinarily high sulfide affinity. The crystal structure of aquomet Lucina HbI has revealed an active site with three residues not commonly found in vertebrate globins: Phe(B10), Gln(E7), and Phe(E11) (Rizzi, M., Wittenberg, J. B., Coda, A., Fasano, M., Ascenzi, P., and Bolognesi, M. (1994) J. Mol. Biol. 244, 86-89). Engineering these three residues into sperm whale myoglobin results in a triple mutant with approximately 700-fold higher sulfide affinity than for wild-type. The single crystal x-ray structure of the aquomet derivative of the myoglobin triple mutant and the solution 1H NMR active site structures of the cyanomet derivatives of both the myoglobin mutant and Lucina HbI have been determined to examine further the structural origin of their unusually high sulfide affinities. The major differences in the distal pocket is that in the aquomet form the carbonyl of Gln64(E7) serves as a H-bond acceptor, whereas in the cyanomet form the amido group acts as H-bond donor to the bound ligand. Phe68(E11) is rotated approximately 90 degrees about chi2 and located approximately 1-2 A closer to the iron atom in the myoglobin triple mutant relative to its conformation in Lucina HbI. The change in orientation potentially eliminates the stabilizing interaction with sulfide and, together with the decrease in size of the distal pocket, accounts for the 7-fold lower sulfide affinity of the myoglobin mutant compared with that of Lucina HbI.
Amino Acid Sequence↗
Activation of phosphatidylinositol 3-kinase by brain-derived neurotrophic factor gene transfection in septo-hippocampal cultures.
Brain-derived neurotrophic factor (BDNF) has therapeutic potential for treatment of the injured central nervous system. BDNF induces both differentiation and survival of neurons by binding to trkB receptors. This interaction stimulates the intrinsic tyrosine kinase activity of trkB, initiating a signal cascade involving the phosphorylation of intracellular protein on tyrosine, serine, and threonine residues. The purpose of this investigation was to examine the effects of cationic lipid-mediated gene transfection of BDNF on phosphatidylinositol 3 (PI3)-kinase activity in primary septo-hippocampal cell cultures. Thirty-six hours after BDNF gene transfection in the primary CNS cell culture, PI3-kinase activity was significantly increased. The increased PI3-kinase activity was inhibited by wortmannin, a selective and irreversible inhibitor of PI3-kinase. In addition, wortmannin blocked neurofilament increases induced by BDNF gene transfection. This result suggests a possible role of PI3-kinase activation in neuroprotective effects produced by BDNF gene transfection.
Androstadienes↗
Molecular forms of human rhodopsin kinase (GRK1).
The G protein-coupled receptor kinases (GRKs) are critical enzymes in the desensitization of activated G protein-coupled receptors. Six members of the GRK family have been identified to date. Among these enzymes, GRK1 (rhodopsin kinase) is involved in phototransduction and is the most specialized of the family. GRK1 phosphorylates photoactivated rhodopsin, initiating steps in its deactivation. In this study, we found that human retina expressed all GRKs except GRK4. Based on results of molecular cloning and immunolocalization, it appears that both rod and cone photoreceptors express GRK1. This conclusion was supported by the cloning of only GRK1 from cone-dominated chicken retina. Human photoreceptors also transcribe a splice variant of GRK1, which differs in its C-terminal region next to the catalytic domain. This novel variant, GRK1b, is produced by retention of the last intron. mRNA encoding GRK1b is exported to the cytosol; however, the level of the protein is relatively low compared with GRK1 (now called GRK1a), and GRK1b appears to have very low catalytic activity. Thus, these studies suggest that rods and cones, express the same form of GRK1.
Alternative Splicing↗
Null mutation in the rhodopsin kinase gene slows recovery kinetics of rod and cone phototransduction in man.
Rhodopsin kinase (RK), a specialized G-protein-coupled receptor kinase expressed in retina, is involved in quenching of light-induced signal transduction in photoreceptors. The role of RK in recovery after photoactivation has been explored in vitro and in vivo experimentally but has not been specifically defined in humans. We investigated the effects on human vision of a mutation in the RK gene causing Oguchi disease, a recessively inherited retinopathy. In vitro experiments demonstrated that the mutation, a deletion of exon 5, abolishes the enzymatic activity of RK and is likely a null. Both a homozygote and heterozygote with this RK mutation had recovery phase abnormalities of rod-isolated photoresponses by electroretinography (ERG); photoactivation was normal. Kinetics of rod bleaching adaptation by psychophysics were dramatically slowed in the homozygote but normal final thresholds were attained. Light adaptation was normal at low backgrounds but became abnormal at higher backgrounds. A slight slowing of cone deactivation kinetics in the homozygote was detected by ERG. Cone bleaching adaptation and background adaptation were normal. In this human in vivo condition without a functional RK and probable lack of phosphorylation and arrestin binding to activated rhodopsin, reduction of photolyzed chromophore and regeneration processes with 11-cis-retinal probably constitute the sole pathway for recovery of rod sensitivity. The role of RK in rods would thus be to accelerate inactivation of activated rhodopsin molecules that in concert with regeneration leads to the normal rate of recovery of sensitivity. Cones may rely mainly on regeneration for the inactivation of photolyzed visual pigment, but RK also contributes to cone recovery.
Animals↗
Human and rat neutrophils constitutively express neural nitric oxide synthase mRNA.
Freshly isolated rat circulating neutrophils (PMN) constitutively expressed neural nitric oxide synthase (nNOS) mRNA and nNOS protein and exhibited spontaneous basal release of low concentrations of nitrate and nitrite anion (RNI). In contrast, rat peripheral monocytes and macrophages were devoid of nNOS mRNA and protein and did not exhibit basal or spontaneous release of RNI. Constitutive neural NOS mRNA was also found in human PMN. However, nNOS protein was not expressed and spontaneous generation of RNI was absent in the human PMN. Spontaneous release of RNI from rat PMN was inhibited by 7-nitroindazole but not by L-N-iminoethyllysine, which further supported the nNOS origin of the spontaneously produced RNI. Intravenous administration of Escherichia coli endotoxin (0.6 mg/kg) did not acutely affect the content of nNOS mRNA or protein but inhibited nNOS-derived production of RNI in PMN and up-regulated iNOS mRNA and iNOS protein in PMN, macrophages, and monocytes. This communication demonstrates the existence of nNOS mRNA in rat and human PMN and nNOS protein in rat PMN. Moreover, the data also show that the nNOS system in rat PMN is functional and is inhibitable by the nNOS inhibitor 7-nitroindazole. These findings offer an explanation for the spontaneous formation of the PMN-derived relaxing factor resembling nitric oxide (NO). Moreover, since basal production of NO can affect expression of adhesion molecules and cell-cell binding, the nNOS system within the rat may play an important role in PMN function in normal and disease states. Finally and speculatively, if constitutively expressed nNOS mRNA is subject to activation and translation into nNOS protein, nNOS may also play a role in the function of human PMN.
Animals↗