Biomedical subjects
Publications and source records attributed to Z Gu.
Six compounds were isolated from the root and stem of Strychnos nitida for the first time. On the basis of chemical properties and spectral data, the compounds were identified as beta-sitosterol, strychnine, brucine, cantieyine, lignoceric acid and palmitic acid.
To further investigate the correlation factors of the prognosis and therapeutic efficiency of sudden deafness, 83 cases (91 ears) from 1993. 1 to 1996. 4 were studied with stepwise regression analysis. The results showed that the prognosis is correlate with the age, the hearing threshold levels of both speech and high frequencies, and the types of the audiometric curve, but not with the illness duration, sex, vertigo, ipsi or bilateral, recurrence, virus infection, cardiac or cerebral vascular diseases, and the medicine or the duration of the treatment. By comparing the results obtained from different statistical analysis, it was suggested that the unification of the criteria of the diagnosis and the therapeutic efficiency as well as the application of the multiple factors regression analysis are essential for the research on sudden deafness.
OBJECTIVE: To evaluate the effect of chemothrapy combined with radiotherapy in advanced nasopharyngeal carcinoma (NPC). METHODS: From Dec. 1989 to Dec. 1990, 76 patients with locally advanced NPC (stage III or IV) were treated with 3 cycles of cisplatin [PDD, 20 mg.(m2)-1.day-1, i.v. on days 1-5] and 5-Fluorouracil (5-Fu, 700 mg.(m2)-1.day-1 by continuous i.v. infusion on days 1-5 followed shortly thereafter by radiotherapy. Eighty-six patients treated with radiotherapy alone in 1989 were taken as control. The methods and time/dose schedule of radiotherapy were similar in the two groups. RESULTS: The response rate to chemotherapy was 89.3% [complete response (CR) 18.4%]. The overall 5-year survival was 48.7% in the combined treatment group and 33.6% in the control group (P > 0.05). The 5-year survival in the combined treatment group was 44.1% and 39.5%, in patients with T2N3 and T2-4N3 and 21.6% and 20.4% in the control group, respectively (P < 0.05). CONCLUSION: This prospective study demonstrates that PDD/5-Fu chemotherapy followed by radiotherapy can improve 5-year survival in patients with T2-4N3 nasopharyngeal carcinoma.
Transmeatal exploratory tympanotomy has been used traditionally for the surgical treatment of conductive deafness. With 8 cases of the middle and inner ear diseases diagnosed and surgical managed successfully with this approach, the extended applications of this procedure were illustrated, which included: 1. conductive deafness with cause undetermined, 2. post traumatic conductive deafness with intact tympanic membrane, 3. suspected primary space occupying lesion of tympanum. 4. perilymphatic fistula, and 5. cerebrospinal fluid otorrhea. The key consideration and advantage of this approach were also discussed in this paper.
The K65R mutation in HIV-1 reverse transcriptase (RT) is associated with viral cross-resistance to 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, and 2',3'-dideoxy-3'-thiacytidine. We have found that in vitro DNA synthesis by K65R RT is significantly more processive than that of wild type (wt) RT. Depending on the template/primer (T/P) used, the total incorporation of nucleotides under single processive cycle conditions was 20-50% higher with K65R RT than with wt RT. With heteropolymeric T/P, the total incorporation of dNMP by K65R and wt RT was similar under continuous DNA synthesis reaction conditions. However, under single processive cycle conditions, the rate of full-length polymerization product synthesis by K65R RT was about 2-fold higher than that by wt RT. We also found a decreased rate of T/P dissociation during K65R RT DNA synthesis, which is consistent with the increased processivity of the enzyme. We postulate that the increased processivity of the K65R RT may be a compensatory response to the decreased affinity of this mutant for certain dNTP substrates, allowing normal viral replication kinetics.
Monotherapy with (-)2',3'-dideoxy-3'-thiacytidine (3TC) leads to the appearance of a drug-resistant variant of human immunodeficiency virus-type 1 (HIV-1) with the methionine-184 --> valine (M184V) substitution in the reverse transcriptase (RT). Despite resulting drug resistance, treatment for more than 48 weeks is associated with a lower plasma viral burden than that at baseline. Studies to investigate this apparent contradiction revealed the following. (i) Titers of HIV-neutralizing antibodies remained stable in 3TC-treated individuals in contrast to rapid declines in those treated with azidothymidine (AZT). (ii) Unlike wild-type HIV, growth of M184V HIV in cell culture in the presence of d4T, AZT, Nevirapine, Delavirdine, or Saquinavir did not select for variants displaying drug resistance. (iii) There was an increase in fidelity of nucleotide insertion by the M184V mutant compared with wild-type enzyme.
We have devised a single-step method that enables purification of HIV-1 recombinant reverse transcriptase directly from bacterial lysates in less than 2 h. Clarified lysates are applied to commercial Q- and S-matrix cartridge columns connected in series. The columns are washed with low-salt buffer to remove unbound protein, then the Q column is removed and reverse transcriptase is eluted from the S column using a salt gradient. The purification has been carried out with both medium-pressure and high-pressure chromatographic systems. Purifications are carried out at room temperature near neutral pH, providing enzyme with high DNA polymerase specific activity. A crucial aspect of the procedure is the use of Tris buffer, a buffer that is normally incompatible in cation-exchange methods. The method is applicable for the purification of the p51/p66 heterodimer and the p5l and p66 homodimer forms of reverse transcriptase. We have used this method to purify wild-type reverse transcriptase and several recombinant proteins containing mutations correlated with dideoxynucleoside drug resistance.
We designed two sets of oligonucleotide primers to amplify the immunoglobulin heavy- and light-chain variable-region genes from genomic DNA by polymerase chain reaction (PCR). The genomic DNA was extracted from hybridoma BDI-1 cells, which secreted a monoclonal antibody (mAb) against human bladder carcinoma. The primers contained special restriction sites that allowed the variable-region genes to be easily cloned for sequencing and expression. The recombinants were sequenced by Sanger's method. It was proved that the full lengths of the VH and VK genes were 366 and 324 bp, respectively. Compared with other published sequences, the VH gene was a member of mouse heavy-chain VH subgroup II and originated from the rearrangement of VH, Dsp2.2 and JH4. The VK gene was VK subgroup IV and from VK and JK4. The VH and VK genes was inserted expression vector pWAI80. By inducement, the ScFv antibodies were expressed and secreted from Escherichia coli. Binding activities against the bladder carcinoma cells were detected. We suggest that ScFv antibody recognized the antigen specifically.
9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA) is an acyclic nucleotide with potent in vitro activity against human immunodeficiency virus type 1 (HIV-1). The present study was undertaken to determine whether HIV-1 resistance to PMEA could be generated by in vitro selection and if so, to determine which mutations in reverse transcriptase (RT) were responsible. HIV-1LAI was serially passaged for 10 months in the presence of increasing concentrations of PMEA up to a maximum of 40 microM. After 40 passages, the 50% inhibitory concentration (IC50) of PMEA had increased almost 7-fold from 4.45 to 30.5 microM. Some cross-resistance to 2',3'-dideoxycytidine (ddC, zalcitabine), 2',3-dideoxyinosine (ddI, didanosine), and 3'-thiacytidine (3TC, lamivudine) was also observed, but no cross-reactive resistance to 3'-azido-3'-thymidine (AZT, zidovudine). Sequencing of the RT encoding region of each of eight pol clones from resistant isolates revealed a Lys-65-->Arg (K65R) substitution. HIV with the K65R mutation inserted by site-directed mutagenesis also had decreased sensitivity to PMEA in H9 cells and a similar cross-resistance profile. Thus, HIV can develop decreased sensitivity to PMEA after long-term in vitro exposure and this change is associated with a K65R substitution. Additional studies will be needed to determine whether a similar mutation in HIV RT develops in patients receiving PMEA or its orally bioavailable prodrug adefovir dipivoxil (bis-POM PMEA).
Rates of rotation for amines in a variety of crystalline environments are reported, and the trends are explained in terms of the strengths of local hydrogen bonding interactions. Proton spin-lattice relaxation times (T1) and deuterium broad-line NMR spectra have been measured for D-, DL- and L- aspartic acid, two polymorphs of glycine, alanine, and leucine in the temperature range from -40 to 110 degrees C. The energy barriers for amine rotation are 27 +/- 2 kJ mol-1 for D- or L-aspartic acid and 22 +/- 2 kJ mol-1 for DL-aspartic acid; these energies are slightly lower than the previously reported value for the L from based on direct proton T1 measurements at 60 MHz. The values for the alpha and gamma forms of glycine were 24 +/- 2 and 30 +/- 2 kJ mol-1 respectively, that for L-alanine was 40 +/- 2 and that for L-leucine was 49 +/- 3 kJ mol-1. These are all in rough agreement with previously reported values (although the differences for the polymorphs of glycine and for L- vs. DL-aspartic acid were not reported). Crystal structures of these amino acids indicate differences in hydrogen bonding environments around the R-NH3+ groups that are probably responsible for the different activation barriers. A molecular mechanics calculation of the rotation energy barriers for L- and DL-aspartic acid based on the crystal structures gave satisfactory agreement with experimental results if a uniform (and arbitrarily chosen) dielectric constant of 2.5 was assumed. Differences between L- and DL-aspartic acids and between two polymorphs of glycine were well represented qualitatively. Including additional neighboring molecules not involved in the hydrogen bonding or including periodic boundary conditions to describe the crystal packing did not significantly affect these results. If vacuum dielectric constants are used, the barriers are uniformly overestimated, and if the experimental macroscopic dielectric constant values are used, the barriers are generally underestimated. Dielectric constants differ substantially from one amino acid to another and significantly affect the estimated barriers; in fact, the bulk dielectric constants appear to be the major difference between the highest and the lowest values. The difficulty of accurately including dielectric relaxation into molecular mechanics calculations resulted in the disagreement between experimental measurements and theoretical calculations.
The bark extracts of Annona squamosa yielded a new bioactive acetogenin, squamotacin (1), and the known compound, molvizarin (2), which is new to this species. Compound 1 is identical to the potent acetogenin, bullatacin (3), except that the adjacent bistetrahydrofuran (THF) rings and their flanking hydroxyls are shifted two carbons toward the gamma-lactone ring. Compound 1 showed cytotoxic selectively for the human prostate tumor cell line (PC-3), with a potency of over 100 million times that of Adriamycin.
The transport of proteins into the nucleus is a receptor-mediated process that is likely to involve between 50-100 gene products, including many that comprise the nuclear pore complex. We have developed an assay in Saccharomyces cerevisiae for the nuclear transport of green fluorescent protein fused to the SV-40 large T antigen nuclear localization signal (NLS-GFP). This assay allows the measurement of relative NLS-GFP nuclear import rates in wild-type and mutant cells under various physiological conditions. Probably the best understood component of the nuclear transport apparatus is Srp1p, the NLS receptor, which binds NLS-cargo in the cytoplasm and accompanies it into the nucleus. When compared to SRP1+ cells, NLS-GFP import rates in temperature-sensitive srp1-31 cells were slower and showed a lower temperature optimum. The in vivo transport defect of the srp1-31 cells was correlated with the purified protein's thermal sensitivity, as assayed by in vitro NLS peptide binding. We show that the kinetics of NLS-directed nuclear transport in wild-type cells is stimulated by the elevated expression of SSA1, which encodes a cytoplasmic heat shock protein 70 (Hsp70). Elevated Hsp70 levels are sufficient to suppress the NLS-GFP import defects in srp1-31 and nup82-3 cells. NUP82 encodes a protein that functions within the nuclear pore complex subsequent to docking. These results provide genetic evidence that Hsp70 acts during both targeting and translocation phases of nuclear transport, possibly as a molecular chaperone to promote the formation and stability of the Srp1p-NLS-cargo complex.
OBJECTIVE: To measure the extent of HIV resistance to (-)-2',3'-dideoxy-3'-thiacytidine (3TC, lamivudine) within the context of monotherapy and to assess the presence of the M184V substitution in the case of 3TC-resistant viruses. Whether the success of 3TC in clinical trials could be due, in part, to an increase in the fidelity of HIV reverse transcriptase conferred by the M184V substitution was also considered. METHODS: Two separate monotherapy studies were evaluated, one involving adults with CD4 counts > or = 300 x 10(6)/l, and the second involving children, some of whom had received antiretroviral treatment previously, while others were drug naive. Peripheral blood and plasma samples were collected regularly, and HIV isolation and determinations of drug median inhibitory concentration values were performed using umbilical cord mononuclear cells as targets. Amplification of the 184 mutation was performed by the polymerase chain reaction, using specific primer pairs. Fidelity determinations using purified, recombinant HIV reverse transcriptase derived from either wild-type virus or viruses that contained the 184V substitution were performed. RESULTS: Phenotypic resistance was detected in almost all subjects at times ranging from 8-20 weeks after initiation of therapy. The 184V substitution was usually detected prior to the occurrence of phenotypic resistance to 3TC. Fidelity determinations revealed that the 184V substitution conferred an approximately 5- to 10-fold increase in HIV reverse transcriptase fidelity. In addition, titres of patient sera tested for their ability to neutralize autologous sequential viral isolates were stabilized in patients receiving 3TC therapy as opposed to other drugs. CONCLUSIONS: Resistance to 3TC developed in virtually all subjects treated with this drug, and was associated with the appearance of an M184V mutation in HIV reverse transcriptase. The clinical benefit of 3TC therapy may be attributable in part to selection of viruses that are less able to replicate and mutate than the wild types.
We have compared the effects of nucleoside analogs in quiescent and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) exposed to human immunodeficiency virus type 1 (HIV-1) with those of their triphosphorylated derivatives in cell-free HIV-1 reverse transcription assays. We observed a substantial decrease in synthesis of early minus-strand proviral DNA products in HIV-1-infected, quiescent PBMC exposed to each of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), and 2',3'-dideoxy-3'-thiacytidine (3TC), in comparison with nontreated, infected controls. In contrast, no such diminution was observed when PHA-stimulated, HIV-1-infected PBMC were treated with the same drugs. This result was attributed to previously reported findings that PHA-stimulated PBMC possessed larger deoxynucleoside triphosphate (dNTP) pools than quiescent cells did. To further investigate this subject, a cell-free HIV-1 reverse transcription reaction involving HIV-1 RNA genomic template, recombinant purified HIV-1 reverse transcriptase, all four dNTPs and either tRNA3Lys or a deoxyoligonucleotide as primer was used to monitor chain termi-nation mediated by 2',3'-dideoxynucleoside triphosphates (ddNTPs) during synthesis of minus-strand strong-stop DNA. Augmented chain termination was observed with decreasing concentrations of both ddNTP and dNTP when the ratio of dNTP to ddNTP was fixed. We also found that both the number and strength of reverse transcription pause sites were increased at low concentrations of dNTPs and when a deoxyoligonucleotide primer was used in place of the cognate primer, tRNA3Lys. Preferential incorporation of ddATP was observed dur-ing reverse transcription opposite a distinct pause site in a short synthetic RNA template. These results con-firm the notion that the antiviral activities of ddNTP are dependent on both cellular dNTP pools and the state of cellular activation. Pausing of HIV-1 reverse transcriptase during reverse transcription, altered by dNTP concentrations, may be a mechanism that controls the position and extent of incorporation of nucleoside analogs.
We have assessed interactions between the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) and a neutralizing monoclonal antibody (1E8) that hinders binding of deoxynucleoside triphosphate (dNTP) substrates. Steady-state reactions with homopolymeric template-primers revealed that 1E8 antagonized inhibition of RT activity mediated by 3'-azido-3'-deoxythymidine triphosphate and 2',3'-dideoxycytidine triphosphate. However, an additive or synergistic inhibition of RT polymerase activity was noted when 1E8 and the nonnucleoside RT inhibitors nevirapine and delavirdine were studied. Chain elongation and dNTP incorporation studies using an HIV-1 genome-derived heteropolymeric template and either oligodeoxynucleotide or tRNA3(Lys) as the primer yielded results consistent with the above observations. 1E8 also increased pausing at certain sites during synthesis of negative-strand, strong-stop DNA, whether or not ddNTP and nonnucleoside RT inhibitors were present.
Kinetic analysis showed that the Ki values and the Ki/Km ratios for mutated, recombinant M184V human immunodeficiency virus type 1 reverse transcriptase (RT) for (-)2'-dideoxy-3'-thiacytidine triphosphate (3TCTP) were 35-fold higher than the equivalent values for wild-type RT but only about twice as high as the equivalent values for each of the triphosphates of ddC (ddCTP) and ddA (ddATP). Fully endogenous RT assays showed that viruses containing the M184V substitution were highly resistant to 3TCTP, with an increase in the 50% inhibitory concentration of 250-fold in comparison with wild-type recombinant virus.
Three groups of extracted human teeth were sectioned longitudinally into experimental and control halves. The enamel surfaces of the three groups of teeth were pretreated with 37% H3PO4 for 1 min, 0.5 M NaClO3 for 2 h, or water rinsing for 10 min. The experimental halves of the three groups received an application of 1% TiF4 for 1 min and were washed with deionized water. The tooth specimens were then immersed in synthetic saliva for 24 h at 37 degrees C before sampling for measurement of fluoride uptake. An acid etching technique was used to determine the uptake of fluoride by measuring both fluoride and calcium concentrations in the solution collected from each etched sample. A significantly higher uptake of fluoride was found in teeth rinsed in water and etched with 37% H3PO4 than in teeth pretreated in 0.5 M NaClO3. It seems that the organic components in enamel play an important role in the fluoride uptake after the topical application of TiF4.
We conducted detailed virological evaluations of 16 HIV-1-infected paediatric patients treated with 3TC (lamivudine) monotherapy. High-level phenotypic resistance against this compound (up to 2,500-fold) was seen in virtually all cases, usually within 8-12 weeks of initiation of therapy. This was concomitant with the appearance of the M184V mutation in viral reverse transcriptase, previously shown to be responsiblefor such resistance. Viral burden fell in virtually all cases after commencement of therapy, and remained below baseline in each instance studied, despite a rebound effect and the appearance of drug resistance. Viral isolates from some patients underwent a switch from a non-syncytium-inducing (NSI) to a syncytium-inducing (SI) phenotype during the course of the study, although no relationship was apparent between dose of drug employed, time to development of drug resistance or time of appearance of SI phenotype.