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Biomedical subjects

L Rong

Publications and source records attributed to L Rong.

At least 19 recordsLinked to original sources
PubMed2003

Protein crystallization by using porous glass substrate.

The effects of a commercially available porous glass substrate (Corning Porous Glass No.7930) on the heterogeneous nucleation of proteins [hen egg-white lysozyme (HEWL), thaumatin and apoferritin] have been investigated in order to develop an improved method to facilitate the nucleation of protein crystals. It was found that the porous glass substrate could promote the nucleation at lower supersaturations. The induction time for nucleation decreased, and the crystals obtained from porous glass substrates were larger than those from normal glass substrates. Many pores and channels of 10-100 nm in diameter were observed on the porous glass surface by atomic force microscopy (AFM). It is believed that these pores and channels are crucial for facilitating the nucleation process in this work.

Apoferritins↗
PubMed2002

RAGE is a multiligand receptor of the immunoglobulin superfamily: implications for homeostasis and chronic disease.

Receptor for AGE (RAGE) is a member of the immunoglobulin superfamily that engages distinct classes of ligands. The biology of RAGE is driven by the settings in which these ligands accumulate, such as diabetes, inflammation, neurodegenerative disorders and tumors. In this review, we discuss the context of each of these classes of ligands, including advance glycation end-products, amyloid beta peptide and the family of beta sheet fibrils, S100/calgranulins and amphoterin. Implications for the role of these ligands interacting with RAGE in homeostasis and disease will be considered.

Alzheimer Disease↗
PubMed2001

HIV-1 nucleocapsid protein and the secondary structure of the binary complex formed between tRNA(Lys.3) and viral RNA template play different roles during initiation of (-) strand DNA reverse transcription.

In human immunodeficiency virus type 1 (HIV-1), the tRNA(Lys.3) primer and viral RNA template can form a specific complex that is characterized by extensive inter- and intramolecular interactions. Initiation of reverse transcription from this complex has been shown to be distinguished from subsequent elongation by early pausing events, such as at the +1 and +3 nucleotide positions. One major concern regarding the biological relevance of these results is that most kinetic studies of HIV-1 reverse transcription have been performed using tRNA(Lys.3)-viral (v) RNA complexes that were formed by heat annealing. In contrast, tRNA(Lys.3) in viruses is placed onto the primer binding site by nucleocapsid (NC) sequences of the Gag protein. In this study, we have further characterized the initiation features of reverse transcription in the presence of HIV-1 NC protein. In contrast to results obtained with a heat-annealed tRNA(Lys.3).vRNA complex, we found that polymerization reactions catalyzed by HIV-1 reverse transcriptase did not commonly pause at the +1 nucleotide position when a NC-annealed RNA complex was used, and that this was true regardless whether NC was actually still present during reverse transcription. This activity of NC required both zinc finger motifs, as demonstrated by experiments that employed zinc finger-mutated forms of NC protein (H23C NC and ddNC), supporting the involvement of the zinc fingers in the RNA chaperone activity of NC. However, NC was not able to help reverse transcriptase to escape the +3 pausing event. Mutagenesis of a stem structure within the tRNA(Lys.3). vRNA complex led to disappearance of the +3 pausing event as well as to significantly reduced rates of reverse transcription. Thus, this stem structure is essential for optimal reverse transcription, despite its role in promotion of the +3 pausing event.

Amino Acid Motifs↗
PubMed2001

A novel microphysiometer based on MLAPS for drugs screening.

This paper presents a novel microphysiometer for simultaneous measurements of several extracellular ions concentrations in living cells based on MLAPS (multi-light addressable potentiometric sensor). In the microphysiometer, different sensitive membranes are illuminated in parallel with n light sources at different frequencies, the response amplitudes of each frequency component can be measured on-line by parallel processing algorithm. By the experiments, we can analyze the relations of the extracellular environmental H(+), Na(+), K(+), Ca(2+) under the effects of western medicines (dilantin, phenobarbital sodium, penicillin sodium) and Chinese drugs (scutellaria, medlar, hemlock parsley), and estimate the effects of several drugs. As the novel microphysiometer works under regular cell culture conditions, cells can be repeatedly simulated with drugs to complete dose-response curve within a few hours. With the detection of a general parameter (extruded protons and ions), the system can be used to monitor the real-time process of the cells' metabolism, observe the functional responses of different kinds of membrane-bound receptors, evaluate the drugs.

Anticonvulsants↗
PubMed2001

Simple quantification of ultrasonic intensity using aqueous solution of phenolphthalein.

Aqueous phenolphthalein solution under sonication was investigated for use as a chemical dosimeter. The fading time of aqueous phenolphthalein solution under sonication depended on the concentration of phenolphthalein and the pH values of solutions. The fading time was correlated to the ultrasonic intensity in a reaction vessel that is estimated on the basis of decomposition of porphyrin. The relation between the fading time and the ultrasonic intensity for different frequencies is expressed by a single curve. From these results, it is indicated that aqueous solutions of phenolphthalein is useful for simple quantification of ultrasonic intensity for practical use, and one can regard it as one of the ultrasonic intensity indicators.

Phenolphthalein↗
PubMed2001

Hydrophobic amino acids in the human immunodeficiency virus type 1 p2 and nucleocapsid proteins can contribute to the rescue of deleted viral RNA packaging signals.

An RNA fragment of 75 nucleotides, which is located between the primer binding site and the 5' major splice donor site in human immunodeficiency virus type 1, has been shown to participate in specific encapsidation of viral RNA. Compensation studies have identified two second-site mutations, namely, MP2 (a T12I substitution in p2) and MNC (a T24I substitution in the nucleocapsid [NC] protein) that were involved in the rescue of various deletions in the aforementioned RNA region (i.e., BH-D1, BH-D2, and BH-LD3). To study whether the MP2 and MNC point mutations exert their compensatory effects in a cis manner, production of Gag proteins was blocked by insertion of stop codons into LD3, LD3-MP2-MNC, and wild-type BH10 such that the constructs generated, i.e., LD3-DG, LD3-MP2-MNC-DG, and BH-DG, only provided RNA transcripts for packaging. The results of cotransfection experiments showed that the LD3-MP2-MNC-DG viral RNA was packaged as inefficiently as LD3-DG; in contrast, BH-DG was efficiently packaged. Therefore, nucleotide substitutions in MP2 and MNC did not act in a cis manner to correct the packaging deficits in LD3. Next, we deliberately changed the T12 in p2 or the T24 in the NC to each of 19 other amino acids. We found that amino acids with long hydrophobic side chains, i.e., V, L, I, and M, were favored at either position 12 in p2 or at position 24 in NC to compensate for the above-mentioned deletions. Further studies showed that only a few amino acids could not be used at these two sites by the wild-type virus due to decreased RNA levels in the virion or abnormal Gag protein processing. In this case, W, D, and E could not substitute for T12 in p2, and S, D, and N could not substitute for T24 in NC, without affecting viral infectivity. Therefore, the long hydrophobic side chains of V, L, I, and M are necessary for these amino acids to rescue the BH-D1, BH-D2, and BH-LD3 mutated viruses.

Amino Acids↗
PubMed2001

Role of calcium in protein folding and function of Tva, the receptor of subgroup A avian sarcoma and leukosis virus.

Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue cysteine-rich motif called the LDL-A module. In this study, we expressed and purified the wild-type (wt) Tva LDL-A module as well as several mutants and examined their in vitro folding properties. We found that, as for other LDL-A modules, correct folding and structure of the Tva LDL-A module is Ca2+ dependent. When calcium was present during in vitro protein folding, the wt module was eluted as a single peak by reverse-phase high-pressure liquid chromatography. Furthermore, two-dimensional nuclear magnetic resonance (NMR) spectroscopy gave well-dispersed spectra in the presence of calcium. In contrast, the same protein folded in vitro in the absence of calcium was eluted as multiple broad peaks and gave a poorly dispersed NMR spectrum in the presence of calcium. The calcium affinity (Kd) of the Tva LDL-A module, determined by isothermal titration calorimetry, is approximately 40 microM. Characterization of several Tva mutants provided further evidence that calcium is important in protein folding and function of Tva. Mutations of the Ca2+-binding residues (D46A and E47A) completely abrogated the Ca2+-binding ability of Tva, and the proteins were not correctly folded. Interestingly, mutations of two non-calcium-binding residues (W48A and L34A) also exerted adverse effect on Ca2+-dependent folding, albeit to a much less extent. Our results provide new insights regarding the structure and function of Tva in ASLV-A entry.

Alpharetrovirus↗
PubMed2001

Role for human immunodeficiency virus type 1 Tat protein in suppression of viral reverse transcriptase activity during late stages of viral replication.

We have examined the role of the human immunodeficiency virus type 1 (HIV-1) Tat protein in the regulation of reverse transcription. We show that a two-exon but not a one-exon form of Tat markedly suppressed cell-free reverse transcriptase (RT) activity. Conversely, viruses expressing two-exon Tat (pNL43 and pNL101) showed rapid replication kinetics and more efficient endogenous RT activity compared with viruses expressing one-exon Tat (pM1ex). The pM1ex virions, as well as pM1ex-infected cells, also contained higher levels of viral DNA than did either the pNL43 or pNL101 viruses, indicating that reverse transcription might have continued during later stages of viral replication in the absence of the second Tat exon. Moreover, degradation of viral genomic RNA was more apparent in the pM1ex virions. Accordingly, we propose that the two-exon Tat may help augment viral infectivity by suppressing the reverse transcription reaction during late stages of viral synthesis and by preventing the synthesis of potentially deleterious viral DNA products.

Cell Line↗
PubMed2000

The effect of mutations in the HIV-1 nucleocapsid protein on strand transfer in cell-free reverse transcription reactions.

Interactions between the nucleocapsid protein (NC) and reverse transcriptase of HIV-1 have been shown to promote the initiation of reverse transcription. We assayed the effect of NC on later events, using a strand transfer system with donor and acceptor HIV RNA templates and found that the presence of NC resulted in increased synthesis of full-length strand-transferred (FLST) DNA. This effect also occurred with mutated forms of NC that lacked both zinc fingers, or that contained a point mutation (histidine-->cysteine) at amino acid 23. In contrast, NC-derived proteins containing only the proximal or distal zinc fingers, or lacking the N- and C-termini, were all unable to catalyze the synthesis of FLST DNA. Band-shift assays using both the mutated and wild-type forms of these proteins revealed that all the NC proteins promoted strand association between (-) strong-stop DNA [(-)ssDNA] and acceptor RNA. The zinc finger motifs were dispensable for full-length processive reverse transcription, and the N- and C-termini were required; however, all NC domains were dispensable for association of (-)ssDNA and acceptor RNA. This suggests that annealing is a less stringent reaction than DNA polymerization.

Cell-Free System↗
PubMed2000

Deletion mutagenesis downstream of the 5' long terminal repeat of human immunodeficiency virus type 1 is compensated for by point mutations in both the U5 region and gag gene.

We have studied the role of an RNA region at nucleotides (nt) +200 to +233, just downstream of the 5' long terminal repeat, in encapsidation of human immunodeficiency virus type 1 genomic RNA. Three deletion mutations, namely, BH-D0, BH-D1, and BH-D2, were generated to eliminate sequences at positions nt +200 to +219, +200 to +226, and +200 to +233. The result in each case was decreased levels of packaging of viral RNA into the mutated viruses, with the BH-D2 virus being the most severely affected. Consistently, all three deletions resulted in impaired viral infectiousness and the BH-D2 mutation showed the most dramatic impact in this regard. Further analysis revealed additional defects in Gag precursor processing and in the extension efficiency of the tRNA(3)(Lys) primer in reverse transcription reactions performed with these mutated viruses. To shed further light on the function of these deleted sequences in viral replication, the mutated viruses were cultured in MT-2 cells over prolonged periods to enable them to reacquire wild-type replication kinetics. Sequencing of the reverted viruses revealed point mutations in both the noncoding region and the gag gene. In the case of the BH-D0 revertant, two mutations were observed at positions G112A in the U5 region, termed M1, and T24I in the nucleocapsid protein, termed MNC, respectively. Either of these two mutations was able to confer wild-type replication capacity on BH-D0. In the case of BH-D1, each of the M1 mutations, a mutation termed M2, i.e., C227T, just downstream of the primer binding site, a mutation termed MP2 (T12I) in the p2 protein, and the MNC mutation were observed. A combination of either M1 and M2 or MP2 and MNC was able to rescue BH-D1. In the case of the BH-D2 deletion-containing viruses, three point mutations, i.e., M1, MP2, and MNC, were observed and the presence of all three was required to restore viral replication to wild-type levels.

Animals↗
PubMed2000

Roles of Pr55(gag) and NCp7 in tRNA(3)(Lys) genomic placement and the initiation step of reverse transcription in human immunodeficiency virus type 1.

To study in vivo tRNA(3)(Lys) genomic placement and the initiation step of reverse transcription in human immunodeficiency virus type 1, total viral RNA isolated from either wild-type or protease-negative (PR(-)) virus was used as the source of primer tRNA(3)(Lys)/genomic RNA templates in an in vitro reverse transcription assay. At low dCTP concentrations, both the rate and extent of the first nucleotide incorporated into tRNA(3)(Lys), dCTP, were lower with PR(-) than with wild-type total viral RNA. Transient in vitro exposure of either type of primer/template RNA to NCp7 increased PR(-) dCTP incorporation to wild-type levels but did not change the level of wild-type dCTP incorporation. Exposure of either primer/template to Pr55(gag) had no effect on initiation. These results indicate that while Pr55(gag) is sufficient for tRNA(3)(Lys) placement onto the genome, exposure of this complex to mature NCp7 is required for optimum tRNA(3)(Lys) placement and initiation of reverse transcription.

Base Sequence↗
PubMed1999

Substitutions in the receptor-binding domain of the avian sarcoma and leukosis virus envelope uncouple receptor-triggered structural rearrangements in the surface and transmembrane subunits.

The retrovirus avian sarcoma and leukosis virus (ASLV) enters cells via pH-independent membrane fusion. This reaction is catalyzed by the viral glycoprotein Env, composed of a membrane-distal subunit, SU, and a membrane-anchored subunit, TM. Previous mutational analysis of a variable region, central within the SU subunit, indicates that this region constitutes part of the receptor-binding domain for subgroup A envelope (EnvA) and furthermore that basic residues (R210, R213, R223, R224, and K227) within this region are critical determinants of efficient ASLV infection. Substitutions of these basic residues exert effects on both receptor binding and postbinding events in EnvA-mediated entry. In this study, we performed biochemical analysis of the EnvA protein from three of the receptor-binding domain mutants (R213A/K227A, R213A/R223A/R224A, and R213S) to define the role of this domain in early molecular events in the entry pathway. Protease sensitivity assays demonstrated that receptor binding was sufficient to trigger conformational changes in the SU subunit of mutants R213A/K227A and R213S similar to those in the wild-type EnvA, while R213A/R223A/R224A was constitutively sensitive to protease. In contrast, all three receptor-binding domain mutants disrupted receptor-triggered conversion of EnvA to an active, membrane-binding conformation as assessed by liposome flotation assays. Our results demonstrate that mutations in the receptor-binding site can dissociate receptor-triggered conformational changes in the SU subunit from membrane binding. Furthermore, they suggest that communication between the receptor-binding subunit, SU, and the fusogenic subunit, TM, is crucial for efficient activation of the fusogenic state of EnvA. Analysis of these mutants continues earlier observations that binding to the cellular receptor provides the trigger for efficient activation of this pH-independent viral envelope protein.

Alpharetrovirus↗
PubMed1999

Deletion mutagenesis within the dimerization initiation site of human immunodeficiency virus type 1 results in delayed processing of the p2 peptide from precursor proteins.

Previous work has shown that deletions of genomic segments at nucleotide (nt) positions +238 to +253, i.e., construct BH10-LD3, or nt positions +261 to +274, i.e., construct BH10-LD4, within the human immunodeficiency virus type 1 (HIV-1) dimerization initiation site (DIS) destroyed DIS secondary structure and dramatically reduced viral replication capacity. Surprisingly, two point mutations located within the viral peptide 2 (p2) and nucleocapsid (NC) protein termed MP2 and MNC, respectively, were able to compensate for this defect. Since the MP2 mutation involves an amino acid substitution near the cleavage site between p2 and NC, we investigated the effects of the above-mentioned deletions on the processing of Gag proteins. Immunoprecipitation assays performed with monoclonal antibodies against viral capsid (CA) (p24) protein showed that p2 was cleaved from CA with less efficiency in viruses that contained the LD3 and LD4 deletions than in wild-type viruses. The presence of the two compensatory mutations, MP2 and MNC, increased the efficiency of the cleavage of p2 from CA, but neither mutation alone had this effect or was sufficient to compensate for the observed impairment in infectiousness. A virus that contained both of the above-mentioned deletions within the DIS was also impaired in regard to processing and infectiousness, and it could likewise be compensated by the MP2 and MNC point mutations. These results suggest that the DIS region of HIV-1 RNA plays an important role in the processing of Gag proteins.

Animals↗
PubMed1999

Reverse transcriptase inhibitors can selectively block the synthesis of differently sized viral DNA transcripts in cells acutely infected with human immunodeficiency virus type 1.

We have recently reported that the in vitro inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcription by inhibitors of reverse transcriptase (RT) occurred most efficiently when the expected DNA products of RT reactions were long (Quan et al. , Nucleic Acids Res. 26:5692-5698, 1998). Here, we have used a quantitative PCR to analyze HIV-1 reverse transcription within acutely infected cells treated with RT inhibitors. We found that levels of minus-strand strong-stop DNA [(-)ssDNA] formed in acutely infected MT2 cells were only slightly reduced if cells were infected with viruses that had been generated in the presence of either azidothymidine or nevirapine (5 microM) and maintained in the presence of this drug throughout the viral adsorption period and thereafter. Control experiments in which virus inoculation of cells was performed at 4 degrees C, followed directly by cell extraction, showed that less than 1% of total (-)ssDNA within acutely infected cells was attributable to its presence within adsorbed virions. In contrast, synthesis of intermediate-length reverse-transcribed DNA products decreased gradually as viral DNA strand elongation took place in the presence of either of these inhibitors. This establishes that nucleoside and nonnucleoside RT inhibitors can exert similar temporal impacts in regard to inhibition of viral DNA synthesis. Generation of full-length viral DNA, as expected, was almost completely blocked in the presence of these antiviral drugs. These results provide insight into the fact that high concentrations of drugs are often needed to yield inhibitory effects in cell-free RT assays performed with short templates, whereas relatively low drug concentrations are often strongly inhibitory in cellular systems.

Anti-HIV Agents↗
PubMed1999

Mutations within four distinct gag proteins are required to restore replication of human immunodeficiency virus type 1 after deletion mutagenesis within the dimerization initiation site.

Human immunodeficiency virus type 1 (HIV-1) genomic RNA segments at nucleotide (nt) positions +240 to +274 are thought to form a stem-loop secondary structure, termed SL1, that serves as a dimerization initiation site for viral genomic RNA. We have generated two distinct deletion mutations within this region, termed BH10-LD3 and BH10-LD4, involving nt positions +238 to +253 and +261 to +274, respectively, and have shown that each of these resulted in significant diminutions in levels of viral infectiousness. However, long-term culture of each of these viruses in MT-2 cells resulted in a restoration of infectiousness, due to a series of compensatory point mutations within four distinct proteins that are normally cleaved from the Gag precursor. In the case of BH10-LD3, these four mutations were MA1, CA1, MP2, and MNC, and they involved changes of amino acid Val-35 to Ile within the matrix protein (MA), Ile-91 to Thr within the capsid (CA), Thr-12 to Ile within p2, and Thr-24 to Ile within the nucleocapsid (NC). The order in which these mutations were acquired by the mutated BH10-LD3 was MNC > CA1 > MP2 > MA1. The results of site-directed mutagenesis studies confirmed that each of these four substitutions contributed to the increased viability of the mutated BH10-LD3 viruses and that the MNC substitution, which was acquired first, played the most important role in this regard. Three point mutations, MP2, MNC, and MA2, were also shown to be sequentially acquired by viruses that had emerged in culture from the BH10-LD4 deletion. The first two of these were identical to those described above, while the last involved a change of Val-35 to Leu. All three of these substitutions were necessary to restore the infectiousness of mutated BH10-LD4 viruses to wild-type levels, although the MP2 mutation alone, but neither of the other two substitutions, was able to confer some viability on BH10-LD4 viruses. Studies of viral RNA packaging showed that the BH10-LD4 deletion only marginally impaired encapsidation while the BH10-LD3 deletion caused a severe deficit in this regard.

Amino Acid Sequence↗
PubMed1999

[Principle and application of scattering power function method in X-ray fluorescence analysis. III. Continuous scattering radiation].

The study in this paper shows that the relationship between mass attenuation coefficient and continuous scattering radiation at wavelength 0.081 nm is not an inverse proportion supposed as the scattering internal standard method but a power function in a wide range of matix composition. Experimental results are in good agreement with that of theoretical prediction. A new method is recommended for matrix absorption correction using the 1.22 power function of continuous scattering radiation at wavelength 0.081 nm and used for microelement Sr determination in geological samples as an application example. The accuracy of the results by this method is nearly 4 times better than that from the traditional scattering internal method in a wide range of matrix composition.

English Abstract↗
PubMed1998

Characterization of human immunodeficiency virus type-1 (HIV-1) particles that express protease-reverse transcriptase fusion proteins.

We have selectively mutagenized specific residues at the junction between the protease (PR) and reverse transcriptase (RT) genes of human immunodeficiency virus type 1 (HIV-1) to study the effects of PR-RT fusion proteins in the context of a full-length, infectious proviral construct. Mutant viruses derived from COS-7 cells transfected with this construct were analyzed in regard to each of viral replication, maturation, and infectivity. Immunoblot analysis revealed that the mutation prevented cleavage between the PR and RT proteins and that both existed as a PR-RT fusion protein in each of cellular and viral lysates. Interestingly, intracellular PR that existed within the PR-RT fusion protein remained functionally active, whereby HIV-1 precursor proteins were processed efficiently. Furthermore, the RT component of the fusion protein also retained its enzymatic activity as shown in RT assays. Electron microscopy revealed that the mutant viruses containing the PR-RT fusion protein possessed wild-type morphology. These viruses also displayed wild-type sensitivities to inhibitors of each of the HIV-1 PR and RT activities. However, viruses containing the PR-RT fusion protein were 20 times less infectious than wild-type viruses. This defect was further pronounced when mutated Gag-Pol proteins were overexpressed as a consequence of an additional mutation that interfered with frameshifting. Thus, unlike cleavage site mutations at the N terminus of PR, a cleavage site mutation between PR and RT did not affect the enzymatic activities of either PR or RT and viruses containing PR-RT fusion proteins were viable.

Animals↗