Biomedical subjects

L J Chen

Publications and source records attributed to L J Chen.

At least 73 records · Page 4Linked to original sources

PubMed1995

Transcription termination at the Escherichia coli thra terminator by spinach chloroplast RNA polymerase in vitro is influenced by downstream DNA sequences.

We have investigated the mechanism of transcription termination in vitro by spinach chloroplast RNA polymerase using templates encoding variants of the transcription-termination structure (attenuator) of the regulatory region of the threonine (thr) operon of Escherichia coli. Fourteen sequence variants located within its d(G+C) stem-loop and d(A+T)-rich regions were studied. We found that the helix integrity in the stem-loop structure is necessary for termination but that its stability is not directly correlated with termination efficiency. The sequence of the G+C stem-loop itself also influences termination. Moreover, the dA template stretch at the 3' end of the terminator plays a major role in termination efficiency, but base pairing between the A and U tract of the transcript does not. From the studies using deletion variants and a series of mutants that alter the sequences immediately downstream from the transcription termination site, we found that termination of transcription by spinach chloroplast RNA polymerase was also modulated by downstream DNA sequences in a sequence-specific manner. The second base immediately following the poly(T) tract is crucial for determining the termination efficiency by chloroplast RNA polymerase, but not of the T7 or E.coli enzymes.

Base Composition↗

PubMed1995

A priori prediction of activity for HIV-1 protease inhibitors employing energy minimization in the active site.

We have observed a high correlation between the intermolecular interaction energy (Einter) calculated for HIV-1 protease inhibitor complexes and the observed in vitro enzyme inhibition. A training set of 33 inhibitors containing modifications in the P1' and P2' positions was used to develop a regression equation which relates Einter and pIC50. This correlation was subsequently employed to successfully predict the activity of proposed HIV-1 protease inhibitors in advance of synthesis in a structure-based design program. This included a precursor, 47, to the current phase II clinical candidate, L-735,524 (51). The development of the correlation, its applications, and its limitations are discussed, and the force field (MM2X) and host molecular mechanics program (OPTIMOL) used in this work are described.

Binding Sites↗

PubMed1995

Genetic regulation of shoot development in Arabidopsis: role of the EMF genes.

To investigate the genetic mechanism regulating Arabidopsis shoot maturation and development, we characterized eight emf mutants that bypassed the vegetative phase of the life cycle. Genetic complementation studies identified two EMF loci; both mapped to chromosome five. Double mutant analysis showed that the early- and late-flowering mutants, co, fb, elf1, elf2, and elf3, could not rescue vegetative development in the emf mutants, confirming the need for both EMF gene activities for rosette development. A series of phenotypes involving successive loss of reproductive organs was also observed in emf single mutants, in emf1-1/emf1-2 transheterozygotes, and in emf1 emf2 double mutants, suggesting that the EMF genes not only specify the rosette (vegetative) but also are involved in inflorescence and flower (reproductive) development. Phenotypic analysis of double mutants between emf and tfl1, lfy, and ag indicated interactions between EMF and genes regulating inflorescence meristem development and floral organ identity. A model depicting the role of the EMF genes in regulating shoot maturation and their interaction with genes that affect phase transitions is presented.

Alleles↗

PubMed1993

[Effect of targeting treatment of mitomycin C immunoconjugate on stomach neoplasm].

An anti-gastric cancer monoclonal antibody MGb2-mitomycin C conjugate via dextran T-70 as intermediate (MGb2-PAD-MMC) was produced, and 28-30 g molecules of MMC were introduced into 1 g molecule of MGb2. Ninety-six hours after ip of 125I-MGb2-PAD-MMC (1.48 MBq/22 micrograms MGb2 per mouse) to nude mice bearing human gastric cancer SGC-7901, the tumor tissue:blood (T/NT) radioactivity ratio was 2.6, very much higher than that of the control 125I-normal IgG-PAD-MMC group (T/NT = 0.20). Single photo computed tomography imaging confirmed the results of biodistribution study. MGb2-PAD-MMC exhibited selective killing action on the SGC-7901 cells in vitro, which was considered to be mediated by monoclonal antibody MGb2. Nude mice inoculated with SGC-7901 xenograft in bilateral subrenal capsule were treated by MMC (ip), human recombinant interferon-alpha (Hu-IFN-alpha im), MGb2-PAD-MMC (ip) and MGb2-PAD-MMC+Hu-IFN-alpha daily for 5 d beginning 4 h after inoculation. The efficacy of the reagents estimated by the reduction of tumor size and calculated by T/C (%), was 28.3%, 16.4%, 47.8%, and 83.1%, respectively. These results demonstrated that the antitumor effect of MGb2-PAD-MMC was superior to free MMC, and that the Hu-IFN-alpha might further enhance the action of MGb2-PAD-MMC.

Adenocarcinoma↗

PubMed1993

[Characterization of a fluorouracil-resistant human gastric carcinoma cell line and its morphological behavior].

A fluorouracil (FU)-resistant human carcinoma cell line (SGC-7901/R) was established in culture by progressively increasing the concentration of FU from 50 ng.ml-1 to 2.5 mg.ml-1. The cell line has been successfully subcultured for more than 150 passages during more than 2 years. Its degree of FU resistance was 139-fold vs that of FU sensitive cell line (SGC-7901/S), and the resistant phenotype was stable when cells were cultured for 23 passages in FU-free medium. The doubling time was 17.3 and 25.6 h for resistant cells and parental cells, respectively. Swiss (nu/nu) nude mice were used for the in vivo experiment, the FU-resistant cell line also exhibited resistance to FU and cross-resistance to mitomycin C. FU inhibited markedly the incorporation of [3H]UR into sensitive cells and only showed a 31.6% inhibition with FU 100 micrograms.ml-1 in resistant cells. For the incorporation of [3H]TdR into DNA, inhibitory rates were seen with different concentrations of FU in resistant cells. By morphologic observation, SGC-7901/R cells showed little secretion but without any tendency to glandular pattern. Their nuclei were allotype with enlarged perinuclear space and a few intranuclear pseudoinclusions. The mitotic phase of cells was found frequently. The phenotype of resistant cell line can be deduced more malignant than that of parental cell line.

Animals↗

PubMed1992

Possible transplacental transmission of human papillomaviruses.

OBJECTIVE: The objective of this study was to examine the possibility of intrauterine human papillomavirus infection of fetuses by transplacental transmission of human papillomavirus before delivery. STUDY DESIGN: Specimens of cervicovaginal cells and peripheral blood mononuclear cells were obtained from 52 consecutive pregnant women in the third trimester of pregnancy. Cord blood specimens were also obtained from the neonates born to these mothers. Presence of human papillomavirus types 16 and 18 deoxyribonucleic acid was analyzed by an in vitro enzymatic deoxyribonucleic acid amplification method. RESULTS: Human papillomavirus type 16 deoxyribonucleic acid was found in 6 (11.5%) cervicovaginal and in 9 (17.3%) peripheral blood mononuclear cell specimens. Seven cord blood specimens from neonates born to mothers who were positive for peripheral blood mononuclear cell human papillomavirus type 16 deoxyribonucleic acid were found to contain human papillomavirus type 16 deoxyribonucleic acid. One cervicovaginal and two peripheral blood mononuclear cell specimens contained human papillomavirus type 18 deoxyribonucleic acid, but none of the cord blood specimens contained human papillomavirus type 18 deoxyribonucleic acid. CONCLUSION: These results seem to suggest possible transplacental transmission of the virus and the potential association of such transmission with the status of human papillomavirus in peripheral blood mononuclear cells.

Base Sequence↗

PubMed1992

Enhanced antitumor activity of daunomycin conjugated with antigastric cancer monoclonal antibody MGb2.

In the present study, an antigastric cancer monoclonal antibody, MGb2, was chosen to prepare an antibody-daunomycin conjugate. Daunomycin was modified by cis-aconitic anhydride, and the derivative was linked to antibody, a carbodiimide reagent being used to produce peptide bonding. Four to five molecules of daunomycin were specifically bound per molecule of antibody, without severely impairing the pharmacological activity of daunomycin and with minimal loss of antibody activity. A tetrazolium dye colorimetric assay indicated that the MGb2-daunomycin conjugate exhibited selective cytotoxicity against human gastric cancer cells SGC-7901 in vitro. The tumor localization in BALB/c nude mice showed that the specific conjugate could recognize the tumor as efficiently as the unconjugated antibody. MGb2-daunomycin conjugate could significantly suppress the growth of human gastric carcinoma GAII inoculated under the renal capsules of BALB/c nude mice. Intraperitoneal injection of MGb2-daunomycin conjugate twice a week for 3 weeks at a dose of 1 mg/kg of drug gave a tumor inhibition rate of 91.58%, far more effective than free daunomycin or an irrelevant conjugate.

Animals↗

PubMed1992

[Feed-back regulation of presynaptic D2 receptors blockaded by l-stepholidine and l-tetrahydropalmatine].

The activity of tyrosine hydroxylase by accumulation of L-dopa content in the striatum under decarboxylase inhibitor (benserazide, 400 mg.kg-1) was measured by HPLC-ECD to ascertain whether l-SPD possesses an agonistic or antagonistic effect on D2 receptor subtype. The accumulated L-dopa content was increased with 1,4-butyrolactone (BL, 750 mg.kg-1) to 1.8 +/- s 0.6 micrograms.g-1 in rat striatum. After the rats were ip l-SPD 5 mg.kg-1 or l-tetrahydropalmatine (l-THP) 10-20 mg.kg-1, the increment of L-dopa level was much more than that in the BL group (P < 0.01) in a dose related manner. On the contrary, apomorphine (ip 5 mg.kg-1) abolished the accumulation of L-dopa induced by BL. Furthermore, l-SPD and l-THP not only reversed the negative feed-back regulation by apomorphine on the accumulated level of L-dopa, but also increased this level over the accumulation by BL. These results showed that l-SPD and l-THP do possess the antagonistic effect on presynaptic DA receptor (D2) without agonistic effect.

Alkaloids↗

PubMed1992

Effects of l-stepholidine on tyrosine hydroxylase activity in rat corpus striatum.

l-Stepholidine (SPD) 2.5 and haloperidol (Hal) 1.0 mg.kg-1 ip increased rat striatal L-3,4-dihydroxyphenylalanine (DOPA) and 3,4-dihydroxyphenylacetic acid (DOPAC) accumulation induced by NSD 1015 (50 mg.kg-1, ip), a decarboxylase inhibitor. SPD (2.5 mg.kg-1, ip) did not alter but apomorphine (2.0 mg.kg-1, ip) decreased the dopamine (DA) content elevated by gamma-butyrolactone (GBL, 750 mg.kg-1, ip) in the rat striatum. Ip injection of either SPD 5.0 or Hal 2.5 mg.kg-1 after NSD 1015 50 mg.kg-1 or NSD 1015 plus GBL 750 mg.kg-1 also augmented tyrosine hydroxylase (TH) activity in the rat striatum. These results suggest that SPD produces an antagonistic effect on presynaptic DA receptors.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1991

Adsorption to starch of a beta-galactosidase fusion protein containing the starch-binding region of Aspergillus glucoamylase.

We have constructed and purified by affinity chromatography three beta-galactosidase (beta Gal) fusion proteins (BSB133, BSBCD8, and BGA134) containing amino acid (aa) sequences from Aspergillus glucoamylase (GA). BSB133, containing the C-terminal 133 aa of GA (aa 484-616), adhered to native starch granules with a much higher affinity (Kad = 18 ml/g starch) than a beta Gal control (Kad = 0.9 ml/g starch). Two other fusion proteins, BSBCD8 and BGA134, similar in size to BSB133, adhered to starch with a relatively low affinity (Kad = 7 ml/g starch, and Kad = 4 ml/g starch, respectively). BSBCD8 differs from BSB133 by a truncation of 8 aa at the C terminus. BGA134 contains 134 aa from an overlapping region of GA (aa 380-513). These results confirm the presence of a strong starch-binding region (SBR) included in the C-terminal 133 aa of GA and indicate that the SBR can confer starch-binding activity on a fusion protein produced in Escherichia coli. In the presence of crude soluble cell extracts, the fusion proteins adsorbed by native starch granules with an affinity similar to that of the purified enzymes. BSB133 that had been adsorbed by starch from crude extracts could be eluted at a high level of purity, similar to that achieved by affinity chromatography. These results suggest that it may be feasible to use native starch as an adsorbent for the recovery and purification of recombinant fusion proteins containing the SBR. Starch has many favorable qualities for this application: it is inexpensive, stable, nontoxic, and easy to recover by centrifugation.

Adsorption↗

PubMed1991

Hepatic bile salt sulfotransferases in the rat: sulfation of 3 beta-hydroxy-5-cholenoate during development.

Bile salt sulfotransferase (BSS) activity for the fetal bile acid, 3 beta-hydroxy-5-cholenoate, was first detected in the fetus at 18-19 days of gestation and was twofold greater than for glycolithocholate. The near-term (20-21 days of gestation) and newborn pup BSS activity was only 5-10% of that in maternal liver. The 3 beta-hydroxy-5-cholenoate sulfotransferase activity rose by the second day of life to levels observed in the mature male, and to activities greater than the mature female by the time of weaning at 3 weeks of age. Sex differences in 3 beta-hydroxy-5-cholenoate sulfotransferase activity developed during adolescence (28-35 days of age), resulting in fivefold greater activity in the mature female compared with the male. Two isoenzyme activities (BSS I and BSS II) were identified in both sexes during development by DEAE-Sephadex A-50 ion-exchange chromatography of liver cytosol. In the fetal and newborn liver, only one isoenzyme activity was distinctly identified for both monohydroxy bile acids, corresponding to BSS I in older rats. After the first week of life, both BSS I and BSS II exhibited activity like glycolithocholate, but only one peak of activity was identified for 3 beta-hydroxy-5-cholenoate, corresponding to BSS I. The 3 beta-hydroxy-5-cholenoate sulfotransferase activity in the mature male was only 20% of the mature female because of a decline in BSS I activity in the male during adolescence. BSS I and II were further purified by taurocholate-Sepharose 4B chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Specific targeting of mitomycin C to tumors by anti-gastric cancer monoclonal antibody.

In the present study, an anti-gastric cancer monoclonal antibody, MGb2, was chosen to prepare antibody-mitomycin C (MMC) conjugate with dextran T-40 as intermediary. Twenty molecules of MMC were introduced into each molecule of antibody while the antigen-binding capacity of the antibody was kept well. The conjugate showed selective cytotoxicity upon human gastric cancer cell line SGC-7901. Radioimmunoimaging and biodistribution studies indicated that after conjugation with MMC via dextran T-40 as intermediary, the tumor localization capacity of the antibody was well retained. When tested in nude mice, inoculated with human gastric carcinoma SGC-7901 in bilateral subrenal capsules, intraperitoneal injection of the conjugate daily for 6 days at a dose of 1 mg/kg gave a tumor inhibitory rate of 68.67%, which was far better than that of free MMC or irrelevant conjugate. No synergetic effect was found in regard to the mixture of MGb2 with MMC.

Animals↗

PubMed1990

Enzymatic sulfation of gastrin in rat gastric mucosa.

An enzyme which catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to gastrin (G17) was identified in rat gastric mucosal cells. The enzyme activity was detected in the 105,000xg supernatant fraction. Formation of gastrin sulfate was shown by using 125I-gastrin and non-radioactive PAPS. The product was sensitive to acid hydrolysis, arylsulfatase treatment and removed by gastrin antibody, but not changed by treatments with chondro-4-sulfatase and chondro-6-sulfatase. The product had a molecular weight of 2050 daltons, close to the molecular weight of G17 sulfate, and, therefore, indicating the sulfated product is not APS derived from the degradation of PAPS. The enzyme activity showed a Km value of 5 microM for PAPS and a pH optimum of 6.0. The activity was not detected in the liver preparation.

Adenine Nucleotides↗

PubMed1990

A primary transcript in spinach chloroplasts that completely lacks a 5' untranslated leader region.

A fifth, previously undetected transcript for the plastid gene encoding the beta subunit of spinach chloroplast ATPase (atpB) has recently been identified in vitro. In this report we show that this transcript is present in vivo and its 5' end is at the translation start codon. When synthesized in vitro using spinach chloroplast RNA polymerase, the 5' end of this novel transcript is located at the beginning of the second codon of the atpB coding sequence. Although this atpB transcript lacks an untranslated leader region, it is an abundant RNA in vivo and is associated with crude polysomal fractions, as are the four larger atpB transcripts. In vitro synthesis of the leaderless transcript does not occur for a plasmid DNA in which the "-35' region of the promoter is deleted.

Adenosine Triphosphatases↗

PubMed1990

Analysis of chloroplast promoters using bidirectional transcription vectors.

Spinach chloroplast RNA polymerase has been shown to efficiently terminate transcription at the threonine attenuator (thra) from Escherichia coli. In this study, efficient transcription termination by the chloroplast RNA polymerase was observed at a second prokaryotic terminator, the histidine attenuator (hisa) from Salmonella typhimurium. Termination occurred regardless of the orientation of either attenuator. In higher-plant chloroplast DNA, the genes for the beta subunit of the ATPase (atpB) and the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL) are adjacent and divergently transcribed. Bidirectional transcription vectors, containing the histidine and threonine terminators, were constructed to analyze the divergently oriented atpB and rbcL promotors. One plasmid construction, pRTT7, contained two tandem copies of the threonine attenuator (pRTT7). Two additional constructs, pRHT1 and pRHT2, each contained oppositely oriented copies of thra and hisa. A DNA fragment containing the rbcL and atpB promoters was inserted between the two terminators present in the pRTT7, pRHT1, and pRHT2 plasmids. Transcription of these recombinant DNAs by spinach chloroplast RNA polymerase resulted in discretely sized rbcL and atpB transcripts. In addition, these bidirectional transcription vectors were used to identify previously uncharacterized chloroplast promoters.

Adenosine Triphosphatases↗

PubMed1990

An in vitro transcription termination system to analyze chloroplast promoters: identification of multiple promoters for the spinach atpB gene.

Promoters for spinach chloroplast genes were cloned 5' to a strong factor-independent transcription terminator from E. coli. These "minigene" constructions were transcribed in vitro by a transcriptionally active extract of spinach chloroplasts. Transcription of supercoiled DNA templates resulted in synthesis of discretely-sized RNAs that were readily quantifiable. The efficiency of transcription was up to 3.5 RNAs per template. The transcription termination system described in this report was used to identify the primary transcripts for the plastid atpB gene. Four in vivo transcripts for the atpB gene have been previously identified with 5' untranslated leaders of approximately 455, 275, 180 and 100 nucleotides, respectively. In this report we show that the "-455", "-275" and "-180" regions function as chloroplast promoters in vitro. In addition, a fourth promoter was found that yields a primary transcript totally lacking an untranslated leader.

Adenosine Triphosphatases↗

PubMed1990

The divergently transcribed rbcL and atpB genes of tobacco plastid DNA are separated by nineteen base pairs.

The in vivo transcripts of the tobacco chloroplast gene for the beta subunit of the ATPase (atpB) were examined. In tobacco, like spinach, the atpB gene encodes multiple transcripts. Six tobacco atpB transcripts are present in vivo, with 5' ends at positions "-90", "-255", "-290", "-490", "-500" and "-610" relative to the translation initiation site. The 5' end of the atpB gene ("-610" position) is 20 base pairs from the 5' end of the rbcL gene, coded for on the complementary strand. The "-255", "-490" and "-610" regions are recognized as promoters in vitro by spinach chloroplast and E. coli RNA polymerases.

Adenosine Triphosphatases↗

PubMed1990

[Therapeutic effects of 9 antitumor drugs on stomach adenocarcinoma (MKN-28) in nude mice].

Therapeutic effects of 9 antitumor drugs were studied on nude mice inoculated with transplanted human stomach tumor (MKN-28). When cisplatin, cyclophosphamide, chlormethine, 5-fluorouracil or mitomycin C was given ip to nude mice bearing MKN-28 at respective dose of 3, 100, 40, 80 and 2 mg/kg once weekly for 3 wk, the tumor inhibition rates of the former 3 drugs were over 80%, while the later 2 were 70-77%. Cytarabine 200 mg/kg twice or thrice weekly for 3 wk gave an inhibition rate of 68-72%. Hydroxycamptothecin 20 mg/kg twice weekly for 3 wk showed a better therapeutic effect than that given at 40 mg/kg once weekly for 3 wk, the tumor inhibition rates were 65 and 32% respectively. No apparent effect of vincristine and methotrexate on MKN-28 was seen. Histopathological observation indicated that in cisplatin group, numerous necrotic areas of tumor cells as well as sparse infiltration of inflammatory cells were found.

Animals↗