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Biomedical subjects

X Ji

Publications and source records attributed to X Ji.

At least 127 records · Page 7Linked to original sources
PubMed1996

Positive and negative cooperativities at subsequent steps of oxygenation regulate the allosteric behavior of multistate sebacylhemoglobin.

Cross-linked human hemoglobin (HbA) is obtained by reaction with bis(3,5-dibromosalicyl) sebacate. Peptide maps and crystallographic analyses confirm the presence of the 10 carbon atom long sebacyl residue cross-linking the two beta82 lysines of the beta-cleft (DecHb). The Adair's constants, obtained from the oxygen binding isotherms, show that at the first step of oxygenation normal hemoglobin and DecHb have a very similar oxygen affinity. In DecHb negative binding cooperativity is present at the second step of oxygenation, which has an affinity 27 times lower than at the first step. Positive cooperativity is present at the third binding step, whose affinity is 380 times that of the second step. The fourth binding step shows a weak negative cooperativity with an affinity one-half that of the third step. Crystals of deoxy-DecHb diffracted to 1.9 angstroms resolution. The resulting atomic coordinates are very similar to those of Fermi et al. [(1984) J. Mol.Biol. 175, 159-174] and Fronticelli et al. [(1994) J. Biol Chem. 269, 23965-23969] for deoxy-HbA. The electron density map of deoxy-DecHb indicates the presence of the 10 carbon bridge between the beta82 lysines. Molecular modeling confirms that insertion of the linker into the T structure requires only slight displacement of the two beta82 lysines. Instead, insertion of the linker into the R and R2 structures [Shaanan (1983) J. Mol. Biol. 171, 31-59; Silva et al. (1992) J. Biol. Chem. 267, 17248-17256] is hindered by serious sterical restrictions. The linker primarily affects the partially and fully liganded states of hemoglobin. The data suggest in DecHb concerted conformational changes at each step of oxygenation.

Allosteric Regulation↗
PubMed1996

Crystallographic, molecular modeling, and biophysical characterization of the valine beta 67 (E11)-->threonine variant of hemoglobin.

The crystal structure of the mutant deoxyhemoglobin in which the beta-globin Val67(E11) has been replaced with threonine [Fronticelli et al. (1993) Biochemistry 32, 1235-1242] has been determined at 2.2 A resolution. Prior to the crystal structure determination, molecular modeling indicated that the Thr67(E11) side chain hydroxyl group in the distal beta-heme pocket forms a hydrogen bond with the backbone carbonyl of His63(E7) and is within hydrogen-bonding distance of the N delta of His63(E7). The mutant crystal structure indicates only small changes in conformation in the vicinity of the E11 mutation confirming the molecular modeling predictions. Comparison of the structures of the mutant beta-subunits and recombinant porcine myoglobin with the identical mutation [Cameron et al. (1993) Biochemistry 32, 13061-13070] indicates similar conformations of residues in the distal heme pocket, but there is no water molecule associated with either of the threonines of the beta-subunits. The introduction of threonine into the distal heme pocket, despite having only small perturbations in the local structure, has a marked affect on the interaction with ligands. In the oxy derivative there is a 2-fold decrease in O2 affinity [Fronticelli et al. (1993) Biochemistry 32, 1235-1242], and the rate of autoxidation is increased by 2 orders of magnitude. In the CO derivative the IR spectrum shows modifications with respect to that of normal human hemoglobin, suggesting the presence of multiple CO conformers. In the nitrosyl derivative an interaction with the O gamma atom of Thr67(E11) is probably responsible for the 10-fold increase in the rate of NO release from the beta-subunits. In the aquomet derivative there is a 6-fold decrease in the rate of hemin dissociation suggesting an interaction of the Fe-coordinated water with the O gamma of Thr67(E11).

Biophysical Phenomena↗
PubMed1996

Characterization and sequence analysis of the human homeobox-containing gene GBX2.

Polymerase chain reaction (PCR) was used to amplify portion of homeobox genes present in a human 11-week fetal brain cDNA library. One of these PCR products was determined by sequencing to be the Gastrulation and brain specific-2 gene (GBX2). Screening this human fetal brain cDNA library with probes specific for GBX2 led to the identification of a 2151-bp cDNA clone. The nucleotide sequence of the cDNA clone encodes for a protein of 347 amino acid residues. The amino acid sequence of the GBX2 homeodomain is identical (100%) to the that of homologous gene, Gbx2, expressed in the developing mouse embryo and virtually identical (97%) to a gene expressed in the developing chicken embryo, CHox7. The 5' end of the GBX2 gene contains a CpG island in the untranslated region and a trinucleotide (CCG)8 repeat in the coding region. The amino-terminal end of the GBX2 protein is proline-rich, with 30 proline residues in one stretch of 120 amino acids. A single 2.2-kb transcript was detected by Northern analysis in the developing human CNS as well as in other tissues. The human genomic clone for GBX2 was also isolated, characterized, and mapped to 2q36(d)-q37 by somatic cell hybrid analysis and fluorescence in situ hybridization. These studies provide a framework for designing future experiments that are needed to determine the functional significance of this gene in CNS development.

Amino Acid Sequence↗
PubMed1996

Effect of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein on HIV-1 reverse transcriptase activity in vitro.

Conversion of human immunodeficiency virus type 1 (HIV-1) genomic RNA to viral DNA is a requisite step in the virus life cycle. This conversion is catalyzed by reverse transcriptase (RT) associated with a large nucleoprotein complex composed of several viral proteins including nucleocapsid (NC). To better characterize the biochemical mechanisms of viral DNA synthesis, we overexpressed and purified recombinant HIV-1 NC and studied its effect on the activity and processivity of HIV-1 RT during polymerization of HIV-1 template sequences in vitro. The effect of NC on steady-state RT activity was dependent on the order of addition of reaction components. Addition of NC prior to formation of RT-primer.template-dNTP ternary complexes inhibited primer extension and reduced total product yields by slowing steady-state RT turnover. In contrast, addition of NC to preformed ternary complexes resulted in efficient primer extension and increased RT processivity at specific DNA template sites. NC stimulated polymerization (2-4 times) through eight of 13 sites examined in the cRRE region of HIV-1 env and increased the rate of polymerization through the D3/CTS region of HIV-1 pol 10 times. The data suggest that NC affects RT processivity by facilitating polymerization through regions of template secondary structure. Thus, NC functions as a single-strand binding (SSB)-like accessory replication factor for RT in vitro and may be part of a multicomponent retroviral replication complex.

Base Sequence↗
PubMed1996

The basic motif-leucine zipper transcription factor Nrl can positively regulate rhodopsin gene expression.

The retinal protein Nrl belongs to a distinct subfamily of basic motif-leucine zipper DNA-binding proteins and has been shown to bind extended AP-1-like sequence elements as a homo- or heterodimer. Here, we demonstrate that Nrl can positively regulate the expression of the photoreceptor cell-specific gene rhodopsin. Electrophoretic mobility-shift analysis reveals that a protein(s) in nuclear extracts from bovine retina and the Y79 human retinoblastoma cell line binds to a conserved Nrl response element (NRE) in the upstream promoter region of the rhodopsin gene. Nrl or an antigenically similar protein is shown to be part of the bound protein complex by supershift experiments using Nrl-specific antiserum. Cotransfection studies using an Nrl-expression plasmid and a luciferase reporter gene demonstrate that interaction of the Nrl protein with the -61 to -84 region of the rhodopsin promoter (which includes the NRE) stimulates expression of the reporter gene in CV-1 monkey kidney cells. This Nrl-mediated transactivation is specifically inhibited by coexpression of a naturally occurring truncated form of Nrl (dominant negative effect). Involvement of Nrl in photoreceptor gene regulation and its continued high levels of expression in the adult retina suggest that Nrl plays a significant role in controlling retinal function.

Animals↗
PubMed1996

Expression of the bZIP transcription factor gene Nrl in the developing nervous system.

Proteins of the Maf/Nrl subfamily of bZIP transcription factors are involved in the regulation of tissue-specific gene expression. The Nrl gene, initially identified from a subtracted retinal library, is expressed in all cell layers of the adult retina, including photoreceptors. The Nrl protein has high sequence homology with Maf proteins, binds to an AP-1 like sequence element, and in photoreceptors appears to be involved in regulating the expression of rhodopsin. In the present study, we investigated the expression of Nrl in the developing and adult mouse using in situ hybridization and RT-PCR. We demonstrate that beginning at embryonic day 12.5 Nrl is expressed throughout the developing central and peripheral nervous system, with the exception of the nasal epithelium. The spatial pattern of hybridization suggests that Nrl is transcribed in post-mitotic, differentiating neurons, the developing cephalic mesenchyme and lens. Nrl expression is downregulated postnatally in the brain, and becomes restricted to neocortex and brainstem in the adult. High levels of Nrl transcripts, however, persist in the mature photoreceptors and other retinal neurons. Our studies suggest a role for the Nrl protein in neuronal differentiation and in mature neurons of the adult retina.

Animals↗
PubMed1996

Characterization of the calcium overload in cultured neonatal rat cardiomyocytes under metabolic inhibition.

We studied the calcium compartmentation of the Ca overload induced by 50 min of metabolic inhibition (MI) in cultured neonatal rat cardiomyocytes. MI was achieved by application of 1 mM iodoacetic acid (IAA) and 10 mM 2-deoxyglucose (2-DOG) with omission of glucose in the perfusate. MI per se abolished spontaneous beating, but 10 mM caffeine pulses at 15, 30 and 45 min were able to induce contractile responses. The contractile responses were abolished in the presence of Thg. After 20 min of MI, cells began to accumulate Ca. After 50 min of MI, the Ca exchanged via Na/Ca exchange (Na/Ca exchange-dependent Ca content) was increased by 3.21 +/- 0.15 mmol/kg dry weight (n = 5). The net increase remaining exchangeable via Na/Ca exchange represents 80% of the global net 45Ca uptake after 50 min in MI which was 3.98 mmol/kg dry weight, obtained in the "on-line" experiments. The release of this component of Ca is completed within 150s via Na/Ca exchange. There also was a 50% increase in the Ca content of the so-called "slow" compartment, 0.44 +/- 0.08 mmol/kg dry weight (n = 10) after 50 min under MI. The t1/2 of this compartment was 24.8 +/- 3.8 min typical of a mitochondrial origin in these cells. Thus 92% of the net Ca increase after 50 min MI remains exchangeable-80% of this is completed within 150s via Na/Ca exchange and 20% more slowly, t1/2 about 25 min. Cell ATP content, 27.35 +/- 2.07 nmoles/mg protein, was decreased after 10 min of MI to 2.25 +/- 0.29 nmoles/mg protein (n = 10) and remained close to this level after 20 min of MI (2.11 +/- 0.17 nmoles/mg protein; n = 6). It is to be noted that our results indicate no compromise of the Na/Ca exchange system after 50 min of MI although the global ATP is depleted in these cells.

Animals↗
PubMed1996

Comparison of 5-hydroxy-2, 3-dihydrophthalazine-1, 4-dione and luminol as co-substrates for detection of horseradish peroxidase in enhanced chemiluminescent reactions.

The utility of 5-hydroxy-2, 3-dihydrophthalazine-1, 4-dione (HDP) as a co-substrate for the chemiluminescent detection of horseradish peroxidase was assessed. Several substituted aryl boronic acid derivatives (4-phenyl, 4-iodo) acted as potent enhancers of the peroxidase catalyzed reaction. Addition of chelating agents (EDTA) and surfactants (Tween-20 and [poly (vinylbenzyl)tributylphosphonium chloride-poly (vinylbenzyl) trioctylphosphonium chloride copolymer]) modulated background light emission and the intensity and duration of the signal from both HDP and luminol. However, HDP was found to be inferior to luminol in the peroxidase assay. Comparative studies revealed that at 500 amol of peroxidase the S/B was ten-fold higher using a commercial luminol-based signal reagent as compared with an HDP-EDTA-Tween-20 reagent (S/B t = 0 min 21.8 vs 1.7, S/B t = 10 min 17.8 vs 2.0).

Boronic Acids↗
PubMed1996

[Alkaloids in the flowers of Sophora viciifolia Hance].

Six alkaloids have been isolated from the flowers of Sophora viciifolia for the first time. Based on of physico-chemical methods and spectroscopic analysis, their structures have been identified as oxymatrine, oxysophocarpine, sophocarpine, matrine, sophoramine and sophoridine.

Alkaloids↗
PubMed1996

[The value of the clinical test of glucocorticoid receptors of peripheral blood leukocytes in patients with chronic pulmonary heart disease].

In order to inquire into the functional state of adrenal cortex in patients with pulmonary heart disease, the number of glucocorticoid receptors(GCR) of peripheral blood leukocytes in patients with chronic pulmonary heart disease was determined with radioligand-binding assay and the corresponding plasma cortisol levels were assessed with radioimmune assays. The results showed that the number of GCR in the patients was significantly reduced (P < 0.01) and it was increased when their health state was improved. However, it was still lower than that in healthy subjects (P < 0.01). The number of GCR in the patients was greatly increased when these patients were treated with oxygen (P < 0.01). No difference in plasma cortisol was found between the patients and the healthy subjects (P > 0.05). These results indicate that the function of adrenal cortex may be improved by the compensation mechanism of the patients, but the lower GCR number was the result of lacking of oxygen in the patients. The number of GCR may be improved by inhalation of oxygen. Therefore oxygen therapy is helpful in raising the activity of glucocorticoid receptors and controlling the development of the disease.

Adult↗
PubMed1995

Three-dimensional structure, catalytic properties, and evolution of a sigma class glutathione transferase from squid, a progenitor of the lens S-crystallins of cephalopods.

The glutathione transferase from squid digestive gland is unique in its very high catalytic activity toward 1-chloro-2,4-dinitrobenzene and in its ancestral relationship to the genes encoding the S-crystallins of the lens of cephalopod eye. The three-dimensional structure of this glutathione transferase in complex with the product 1-(S-glutathionyl)-2,4-dinitrobenzene (GSDNB) has been solved by multiple isomorphous replacement techniques at a resolution of 2.4 A. Like the cytosolic enzymes from vertebrates, the squid protein is a dimer. The structure is similar in overall topology to the vertebrate enzymes but has a dimer interface that is unique when compared to all of the vertebrate and invertebrate structures thus far reported. The active site of the enzyme is very open, a fact that appears to correlate with the high turnover number (800 s-1 at pH 6.5) toward 1-chloro-2,4-dinitrobenzene. Both kcat and kcat/KmCDNB exhibit pH dependencies consistent with a pKa for the thiol of enzyme-bound GSH of 6.3. The enzyme is not very efficient at catalyzing the addition of GSH to enones and epoxides. This particular characteristic appears to be due to the lack of an electrophilic residue at position 106, which is often found in other GSH transferases. The F106Y mutant enzyme is much improved in catalyzing these reactions. Comparisons of the primary structure, gene structure, and three-dimensional structure with class alpha, mu, and pi enzymes support placing the squid protein in a separate enzyme class, sigma. The unique dimer interface suggests that the class sigma enzyme diverged from the ancestral precursor prior to the divergence of the precursor gene for the alpha, mu, and pi classes.

Amino Acid Sequence↗