Biomedical subjects

D Sun

Publications and source records attributed to D Sun.

At least 343 records · Page 19Linked to original sources

PubMed1994

Small-for-size liver transplanted into larger recipient: a model of hepatic regeneration.

Orthotopic liver transplantation was performed in 60 recipient rats weighing 200 to 250 gm. Sixty rats of the same strain were used as liver donors, 30 weighing 100 to 140 gm (small for size) and the other 30 weighing 200 to 250 gm (same size). After 1, 2, 3, 4, 7 and 14 days (n = 5 each) DNA synthesis, nuclear thymidine labeling and mitoses were increased in both the small-for-size and same-size groups, but significantly more in the former. These changes were maximal after 48 to 72 hr, similar to but later than the well-known regeneration response after partial hepatectomy, which peaks at 24 hr in rats. Indirect indexes of regeneration of the transplanted livers also were measured: plasma or serum ornithine decarboxylase; insulin and glucagon serum levels; estradiol and testosterone serum levels (and their nuclear and cytosolic receptors); and transforming growth factor-beta, c-Ha-ras and c-jun mRNA expressions. With the small-for-size transplantation, these followed the same delayed pattern as the direct regeneration parameters. The small livers gradually increased in size over the course of 1 to 2 wk and achieved a volume equal to that of the liver originally present in the recipient. In contrast, no significant liver weight gain occurred in the transplanted livers from same-size donors despite the evidence of regeneration by direct indexes, but not by most of the surrogate parameters, including ornithine decarboxylase.

Animals↗

PubMed1993

Inhibition of human immunodeficiency virus type 1 replication by regulated expression of a polymeric Tat activation response RNA decoy as a strategy for gene therapy in AIDS.

We are investigating a strategy for somatic gene therapy to treat human immunodeficiency virus type 1 (HIV-1) infection by intracellular expression of an RNA decoy and a ribozyme. The RNA decoy, consisting of polymeric Tat activation response elements (TARs), is designed to compete for Tat binding in an equilibrium with viral TAR RNA, thereby inhibiting viral replication. The expression of polymeric TAR is regulated by the HIV long terminal repeat (LTR) and transcriptional activation is dependent on the presence of HIV Tat. Our initial studies indicated that plasmids expressing up to 50 tandem copies of TAR RNA (50TAR) inhibited tat-mediated gene expression by > 90% in a transient transfection assay. A HIV LTR-driven 50TAR construct was subcloned into a replication-defective retroviral vector to ensure high-efficiency gene transfer into T lymphocytes. In addition, a gag RNA-specific ribozyme gene was introduced into the 50TAR containing retroviral vector to enhance the inhibitory effect of the construct (designated TAR-Rib). A human T-cell line (Molt3) was infected (transduced) with the TAR-Rib recombinant retrovirus and challenged with either HIV-1 or simian immunodeficiency virus (SIV). HIV-1 replication was inhibited by 99% in the TAR-Rib-transduced T cells and was maintained over a 14-month period, suggesting that this antiviral strategy represses the formation of escape mutants. Interestingly, the TAR-Rib also inhibited SIV replication in transduced T cells, which suggests that polymeric TAR is a general inhibitor of primate lentiviruses; therefore, the macaque model could be used for further in vivo testing of this antiviral gene therapy strategy.

Acquired Immunodeficiency Syndrome↗

PubMed1993

Structure of the altromycin B (N7-guanine)-DNA adduct. A proposed prototypic DNA adduct structure for the pluramycin antitumor antibiotics.

Altromycin B belongs to the pluramycin family of antitumor antibiotics, which also includes kidamycin, hedamycin, pluramycin, neopluramycin, DC92-B, and rubiflavin A. These potent antitumor compounds react with DNA in as yet imprecisely determined ways. In the present investigation, we have used gel electrophoresis methods in combination with nuclear magnetic resonance and mass spectrometry to determine the structure of the altromycin B-DNA adduct. High-resolution gel electrophoresis demonstrated that guanine was the reactive base, and N7 was implicated from experiments in which N7-deazaguanine was used in place of guanine in a strand breakage assay. Experiments using supercoiled DNA demonstrated that altromycin B and related drugs intercalated into DNA, which implicated this as a common mechanism for binding of the pluramycin antibiotics to DNA. The altromycin B-guanine adduct was isolated from calf thymus DNA after thermal depurination of the alkylated DNA. Mass spectrometry confirmed that altromycin alkylated DNA through guanine, and 1H- and 13C-NMR was used to confirm the covalent linkage sites between altromycin B and guanine. On the basis of these results, we propose that altromycin B first intercalates into DNA via a threading mechanism, reminiscent of nogalamycin, to insert the disaccharide into the minor groove and position the epoxide in the major groove in proximity to N7 of guanine. Nucleophilic attack from N7 of guanine leads to an acid-catalyzed opening of the epoxide, resulting in the altromycin B-DNA adduct. On the basis of these results, a general mechanism for the interaction of the pluramycin family of antibiotics with DNA is proposed.

Alkylation↗

PubMed1993

Synthesis and biochemical evaluation of the CBI-PDE-I-dimer, a benzannelated analog of (+)-CC-1065 that also produces delayed toxicity in mice.

A practical synthesis of CBI (2) was developed and applied to the synthesis of benzannelated analogs of CC-1065, including CBI-PDE-I-dimer (13) and CBI-bis-indole [(+)-A'BC]. The CBI-PDE-I-dimer was shown to have similar DNA sequence selectivity and structural effects on DNA as (+)-CC-1065. Of particular importance was the observed duplex winding effect that has been associated with the pyrrolidine ring of the nonalkylated subunits of (+)-CC-1065 and possibly correlated with its delayed toxicity effects. The effect of CBI-PDE-I-dimer was also compared to (+)-CC-1065 in the inhibition of duplex unwinding by helicase II and nick sealing by T4 ligase and found to be quantitatively similar. The in vitro and in vivo potencies of the CBI compounds corresponded very closely to the corresponding CPI derivatives. Finally, CBI-PDE-I-dimer was like (+)-CC-1065 in causing delayed toxicity in mice.

Animals↗

PubMed1993

Major role of antigen-presenting cells in the response of rat encephalitogenic T cells to myelin basic proteins.

The encephalitogenic potential of a segment of myelin basic protein in experimental autoimmune encephalomyelitis is not always mirrored by the ability of the peptide to mediate in vitro activation of encephalitogenic T cells. Recent studies from our laboratory have demonstrated that the responsiveness of Ag-specific T cells in experimental autoimmune encephalomyelitis is determined not exclusively by Ag but also by the nature of the APC. By varying APC during the in vitro selection of T cells, we could generate distinct sets of rat encephalitogenic T cells, as evidenced by the diversity of TCR usage. Here we establish the importance of APC in the activation of rat encephalitogenic T cells by myelin basic protein peptides. Peptides 69-84-Gly and (P80)68-86, which lacked stimulatory activity toward many encephalitogenic T cells in our proliferation assay when standard APC were used, become strongly stimulatory in the presence of less commonly used APC, i.e., an Ia+ T cell clone (LOA) or an Ia-inducible rat glial cell clone (F10). Nonstimulatory APC failed to activate encephalitogenic T cells even when major cytokines were added, suggesting that these cytokines are not among the factors limiting the activating potential of the APC. Thus, whether or not an immunocompetent T cell can be activated by a given Ag in an autoimmune response may be determined by the properties of APC. This finding has implications for current research efforts to identify pathogenic self proteins.

Amino Acid Sequence↗

PubMed1993

A-tract and (+)-CC-1065-induced bending of DNA. Comparison of structural features using non-denaturing gel analysis, hydroxyl-radical footprinting, and high-field NMR.

(+)-CC-1065 is a biologically potent DNA-reactive antitumor antibiotic produced by Streptomyces zelensis. In a previous study we have reported that (+)-CC-1065 produces bending of DNA that has similarities to that intrinsically associated with A-tracts [Lin, C. H., Sun, D., & Hurley, L. H. (1991) Chem. Res. Toxicol, 4, 21-26]. In this article we provide evidence using a combination of non-denaturing gel analysis, hydroxyl-radical footprinting, and high-field NMR for both distinctions between the two types of bends and the importance of junctions in both types of bends. For A-tracts we demonstrate that the locus of bending is at the center of an A-tract and that upon modification of the 3' adenine with (+)-CC-1065 this locus is moved less than 1 base pair to the 3' side, and the bending magnitude is significantly increased. For drug bonding sequences such as 5'-AGTTA* or 5'-GATTA* (where * denotes the drug bonding site), the locus of bending is found to be between the two thymines, and the bending is focused over a 2-base-pair sequence rather than a 5-base-pair sequence, as is the case for the A-tract. An important distinction between an A-tract intrinsic bend and a (+)-CC-1065-induced bend is the effect of temperature. While, as shown previously, the magnitude of A-tract bending increases with decrease in temperature, for drug-induced bending of 5'-AGTTA* the bending magnitude increases with increased temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1993

Long-term treatment of human immunodeficiency virus-infected cells with antisense oligonucleotide phosphorothioates.

The antiviral activity of antisense oligodeoxynucleotide phosphorothioates complementary to the tat gene, the gag mRNA, and the rev mRNA were studied in a long-term infection model. Three antisense oligonucleotides directed to the splice-acceptor site of the tat gene failed to suppress human immunodeficiency virus type 1 replication at 1 microM concentration in long-term culture. In contrast, two oligodeoxynucleotide phosphorothioates (28-mer) complementary to the gag and the rev mRNAs inhibited viral replication for > 80 days, and the antiviral activity was sequence- and length-dependent. In addition, after pretreatment of cells we could reduce the concentration of the antisense oligodeoxynucleotides by > 10-fold and still maintain the inhibition of viral replication. These results suggest that chemotherapy for human immunodeficiency virus type 1 infection with antisense oligodeoxynucleotide phosphorothioates may be achieved by an initial high-dose treatment followed by a lower maintenance dose.

Antiviral Agents↗

PubMed1993

cis-dominant regulation of CD4 and CD8 gene expression in rat/mouse T cell heterohybridomas.

To determine whether expression of CD4 and CD8 molecules on T cells is determined-solely by transacting regulators, we examined heterohybridomas derived from the fusion of a rat CD4+ T cell line and the CD4- CD8- mouse thymoma BW5147. The majority of hybrid offspring expressed rat CD4. However, a fraction of the cell lines obtained expressed not only rat CD4 but also various amounts of mouse CD4 and CD8 molecules from both species. Cloning of two of these heterogeneous lines revealed that expression of all four Ag varied not only between different clones but also within clonal populations. The expression of Ag not present on the parental cells suggested an alteration in the normally stable regulatory mechanisms present in those cells. Moreover, a lack of concordant expression between the rat and mouse loci was observed, indicating that active and silent homologous loci can exist together in single nuclei. Expression of CD4 and CD8 in these cells, therefore, cannot be solely mediated by trans-acting diffusible regulators but must also depend on cis-dominant effects on the loci themselves. The phenotypic heterogeneity of clonal populations was found to result from unpredictable shifts, both positive and negative, in the expression of CD4 and CD8 over time, indicating that the cis-dominant effects were only quasistable. Preliminary examinations of the density of 5-methylcytosine within the CD4 and CD8 loci in various phenotypic populations separated by FACS from within heterogeneous clones revealed a correlation between surface expression of the mouse CD8 protein and a lack of methylation around the mouse CD8 gene. In contrast, the CD4 gene remained extensively methylated regardless of its surface expression.

Animals↗

PubMed1993

Diverse T cell receptor beta chain usage by rat encephalitogenic T cells reactive to residues 68-88 of myelin basic protein.

Encephalitogenic T cells not only cause experimental autoimmune encephalomyelitis (EAE), but they induce resistance against subsequent induction of the disease as well. The T cell receptor (TcR) of encephalitogenic T cells is believed to contribute to their vaccinating activity. Findings in support of this assumption include the apparent restricted use of particular TcR elements, such as V beta 8.2. However, results from other laboratories including ours do not support this idea. We previously showed that rat T cells reactive against the conserved encephalitogenic epitope of myelin basic protein [MBP (87-99)] use the TcR in a heterogeneous fashion (Sun, D. et al., Eur. J. Immunol. 1992. 22: 591). Here we show, in Lewis rats, that the TcR beta chain usage of T cells specific for the dominant MBP (68-88) epitope is not restricted to V beta 8.2. Not only did such cells rely on diverse V beta chains, but some non-V beta 8-bearing cells were highly encephalitogenic. We also show that antigen-presenting cells (APC) play an important role in determining the TcR usage of MBP-specific T cells. Stimulation of MBP (68-88)-specific T cell lines by cloned APC derived from different sources resulted in selection of encephalitogenic T cells bearing different TcR beta chains.

Amino Acid Sequence↗

PubMed1993

Increased expression of chloramphenicol acetyltransferase by carotenoid and retinoid replacement in Drosophila opsin promoter fusion stocks.

Drosophila promoter fusion stocks containing a chloramphenicol acetyltransferase (CAT) reporter gene fused to a 2.8 kb DNA fragment from the Rh1 opsin promoter were carotenoid deprived from egg to adult, and then adults were replaced by feeding carrot juice. CAT activity, determined by radiometric assay, was low in deprived flies; it increased rapidly during the first 3 days of replacement and then declined back to the control level. Retinoic acid increased peak CAT activity as much as carrot juice and more than beta-carotene, all-trans retinol or all-trans retinal. These findings suggest that vitamin A serves not only as rhodopsin's chromophore but also influences Rh1 opsin gene transcription. Three stocks with various deletions in the Rh1 opsin promoter lacked the carrot juice-dependent elevation of CAT activity. All three deletions include the region from -701 to -488, suggesting that this region may contain a vitamin A-responsive DNA sequence.

Animals↗

PubMed1993

Staphylococcal enterotoxin enhances the activation of rat encephalitogenic T cells by myelin basic protein.

It is generally believed that the activation of autoreactive T cells is an essential step in the pathogenesis of autoimmune diseases; however, autologous antigens are often weak immunogens and their detectable levels in vivo are much lower than required for T-cell activation. In experimental autoimmune encephalomyelitis, encephalitogenic T cells specific for myelin basic protein (MBP) react weakly to autologous rat myelin basic protein (RBP) but strongly to guinea pig-derived basic protein, even though both ligands possess the essential epitope (MBP(68-88)). Here we demonstrate that RBP is converted to a strong immunogen in the presence of a small dose of bacterial superantigen, staphylococcal enterotoxin E (SEE). The enhancing effect of SEE on the rat protein was apparent with all encephalitogenic T-cell lines examined in this study, including those not responding to RBP alone. It depended, moreover, on the simultaneous presence of RBP and SEE; delaying the addition of SEE for 8-12 h greatly decreased the potency of RBP. None of a series of major cytokines was able to replace SEE as an enhancer. Our results indicate that two T ligands, one a bacterial superantigen, can interact to enhance the activation of autoreactive T cells. This observation has implications for the involvement of bacterial and viral infections in the pathogenesis of multiple sclerosis.

Amino Acid Sequence↗

PubMed1993

Effects of intraarticular hyaluronan on matrix changes induced in the lateral meniscus by total medial meniscectomy and exercise.

Total medial meniscectomy was performed in 12 adult merino sheep. Immediately after surgery, 8 animals received high-molecular-weight hyaluronan (HA) (1 mL, 10 mg/mL) and 4 were given sterile saline (1 mL) intraarticularly. Injections were given for 5 more weeks. In week 3 an exercise program, consisting of walking 24 km/wk, was initiated. This program was continued until the animals were killed at week 26 postmeniscectomy. At necropsy the lateral menisci were removed and divided into three concentric zones--inner, middle, and outer. Powdered aliquots of tissues from each zone were analyzed for collagen and hexuronate contents using colormetric methods. The glycosaminoglycans (GAGs)--chondroitin-O-sulfate (C-O-S), chondroitin-4-sulfate (C-4-S), chondroitin-6-sulfate (C-6-S), and dermatan sulfate (DS)--were determined using a high-performance liquid chromatography method. The lateral menisci from the joints of animals injected with HA showed higher hexuronate and GAG levels than those of controls. This increase was mainly due to C-6-S, which had highest levels in the inner and middle meniscal zones. In addition, dermatan sulfate levels increased significantly in the middle and outer zones of the lateral menisci compared with the same zones of the meniscus from the saline-treated group. Collagen and C-O-S levels were not statistically different from those of controls. These data suggest that intraarticular administration of high-molecular-weight HA immediately after open total medial meniscectomy may help preserve the proteoglycans in the lateral meniscus remaining in the joint.

Animals↗

PubMed1993

High binding affinity chimeric anti-colorectal carcinoma antibody correlated to enhanced tumor binding and effector function.

The genetically engineered mouse/human chimeric cB72.3m4 and cB72.3m12 antibodies all recognized the tumor-associated TAG72 antigen. The high affinity cB72.3m4 antibody had an approximately 18-fold higher affinity constant for the TAG72 antigen than the low affinity cB72.3m12 antibody. The relationship amongst antibody binding affinity, tumor binding and effector functions was studied by using these two antibodies. The data showed that the high affinity cB72.3m4 antibody was reactive with, on average, 15% more colon adenocarcinoma cells on tissue sections than the low affinity cB72.3m12 antibody, and it did not produce any cross-reactivities with various normal tissues. The high affinity cB72.3m4 antibody was able to mediate more effective ADCC and CDC to the human ovarian cancer cells in vitro than the low affinity cB72.3m12 antibody. This study provides evidence that this high affinity chimeric cB72.3m4 antibody may be useful in both immunodetection and immunotherapy of cancer.

Adenocarcinoma↗

PubMed1993

Cloning, nucleotide sequence, and regulation of the Bacillus subtilis nadB gene and a nifS-like gene, both of which are essential for NAD biosynthesis.

A number of Bacillus subtilis genes involved in NAD biosynthesis have been cloned and sequenced. One of the genes encodes a polypeptide homologous to Escherichia coli L-aspartate oxidase, and its mutation resulted in a nicotinic acid (Nic)-dependent phenotype; this gene was termed nadB. A second open reading frame (orf2) was found downstream of nadB, and an insertional plasmid separating orf2 and nadB also gave a Nic-dependent phenotype. This result suggests that orf2 may also be involved in NAD biosynthesis and that nadB and orf2 are in the same operon. Upstream of nadB was a third gene, transcribed in the opposite direction to that of nadB-orf2. The amino acid sequence derived from the third gene was quite similar to those derived from nifS genes of various nitrogen-fixing bacteria; therefore, the third gene was termed nifS. As with nadB and orf2, mutations in nifS also resulted in a Nic-dependent phenotype. The promoter regions of nadB and nifS overlapped each other and both contained -10 and -35 sequences which resemble those of E sigma A-type promoters. Transcription from both the nifS and nadB promoters, as well as expression of a nadB-lacZ fusion, was repressed by Nic. However, nadB transcription and nadB-lacZ expression were decreased, at most, only slightly by a deletion in nifS. The possible role of the nifS gene product in NAD biosynthesis is discussed.

Amino Acid Oxidoreductases↗

PubMed1993

Cloning and nucleotide sequence of the Bacillus subtilis ansR gene, which encodes a repressor of the ans operon coding for L-asparaginase and L-aspartase.

Previous work has shown that expression of the Bacillus subtilis ans operon which codes for L-asparaginase and L-aspartase, is both increased and made insensitive to repression by NH4+ by the aspH1 mutation. In current work, the gene in which the aspH1 mutation resides has been identified and sequenced; this gene, termed ansR, is immediately upstream of, but transcribed in the opposite direction from, the ans operon. The promoter region of ansR contains -10 and -35 sequences similar to those recognized by RNA polymerase containing the major vegetative-cell sigma factor sigma A, and ansR appears to be monocistronic. The ansR gene codes for a 116-residue protein, but the aspH1 mutant allele has an additional guanine residue at codon 55, resulting in generation of a truncated polypeptide of only 58 residues. Insertional inactivation of ansR resulted in a phenotype identical to that of the aspH1 mutant. The predicted amino acid sequence of the ansR gene product (AnsR) was homologous to that of the repressor of B. subtilis prophage PBSX, and a helix-turn-helix motif, characteristic of many DNA-binding proteins, was present in the AnsR amino-terminal region. These results suggest that ansR codes for a repressor of the ans operon.

Amino Acid Sequence↗

PubMed1993

Inhibition of Rev activity and human immunodeficiency virus type 1 replication by antisense oligodeoxynucleotide phosphorothioate analogs directed against the Rev-responsive element.

The interaction between the Rev protein of human immunodeficiency virus type 1 and its highly structured and conserved RNA target, the Rev-responsive element, is required for virus replication. We demonstrate that antisense oligodeoxynucleotide phosphorothioate analogs directed against the Rev-responsive element effectively inhibit Rev activity, as well as human immunodeficiency virus type 1 replication, and are candidates for antiviral therapy.

Base Sequence↗

PubMed1993

L-arginine analogues blunt prostaglandin-related dilation of arterioles.

The effects of arginine analogues, inhibitors of endothelium-derived nitric oxide synthesis, on dilation of arterioles in response to various vasoactive substances were studied. At 65 mmHg intravascular pressure, isolated arterioles of rat cremaster muscle developed tone spontaneously and achieved control diameters similar to those observed in vivo (84.1 +/- 2.0 microns vs. passive diameter: 161.3 +/- 3.4 microns). Acetylcholine (ACh, 5 x 10(-8) M), sodium nitroprusside (SNP, 5 x 10(-8) M), arachidonic acid (AA, 10(-7) M), prostaglandin E2 (PGE2, 10(-9) M), and adenosine (ADO, 10(-6) M) were added to the Krebs bicarbonate buffer solution, suffusing the vessels. The peak vasodilator effects of all agents were studied before and after the administration of various doses of N omega-nitro-L-arginine (L-NNA; 10(-5), 10(-4), and 10(-3) M), which significantly reduced, in a dose-dependent manner, the basal diameter of arterioles by 3.6, 15.2, and 18.9%, respectively. The lowest concentration of L-NNA significantly inhibited arteriolar dilations to ACh by approximately 26%. Higher concentrations of L-NNA and N omega-monomethyl-L-arginine (L-NMMA; 10(-4) M) caused a further significant reduction in the dilation to ACh (to approximately 47%) and also significantly reduced dilator responses to AA and PGE2. In the presence of the highest concentration of L-NNA (10(-3) M), dilation to SNP and ADO were also significantly reduced. Removal of endothelium abolished dilation to ACh and AA but did not alter that to SNP, PGE2, or ADO.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1993

Endothelial dysfunction augments myogenic arteriolar constriction in hypertension.

To elucidate the underlying reason or reasons for the increased peripheral resistance in hypertension, we investigated the pressure-diameter relation--the myogenic response--of isolated, cannulated arterioles (approximately 50 microns) of cremaster muscle of 12-week-old Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHR), and normal Wistar (NW) rats. All arterioles constricted in response to step increases in perfusion pressure from 20 to 160 mm Hg. This constriction was, however, significantly enhanced from 60 to 160 mm Hg in arterioles of SHR compared with NW or WKY rats. For example, at 80 and 140 mm Hg, respectively, the normalized diameter (expressed as a percentage of the corresponding passive diameter of arterioles of SHR) was 11.8% and 27.6% (P < .05) less compared with those of WKY rats. Endothelium removal eliminated the enhanced pressure-induced tone in SHR. Similarly, indomethacin (10(-5) mol/L, sufficient to block prostaglandin synthesis) or SQ 29,548 (10(-6) mol/L), a thromboxane A2-prostaglandin H2 receptor blocker that inhibited vasoconstriction to the thromboxane agonist U46619, attenuated the enhanced pressure-diameter curve and reversed the blunted dilation to arachidonic acid in SHR. In contrast, the thromboxane A2 synthesis inhibitor CGS 13,080 (5 x 10(-6) mol/L) did not affect the increased pressure-induced tone or the reduced dilation to arachidonic acid in SHR. Thus, the present findings suggest that in early hypertension pressure-induced arteriolar constriction is increased. This seems to be due to an enhanced production of endothelium-derived constrictor factors, primarily prostaglandin H2.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗