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Biomedical subjects

L Huang

Publications and source records attributed to L Huang.

At least 559 records · Page 31Linked to original sources
PubMed1993

Activity of calf thymus DNA helicase E on cis-diamminedichloroplatinum (II)-damaged DNA.

Calf DNA helicase E (hel E) is a moderately processive, 3' to 5' helicase, active on nicked DNA, that we have proposed to have a role in DNA repair (Turchi, J. J., Murante, R. S., and Bambara, R. A. (1992) Nucleic Acids Res. 20, 6075-6080). Here we have examined its activity on a series of cis-diamminedichloroplatinum (II) (cis-DDP)-modified DNA substrates. Hel E was capable of efficiently displacing a primer strand containing, in an internal position, a cis-DDP-modified dGG. In a two-primer model system, calf DNA polymerase epsilon could successfully extend an upstream primer through a cis-DDP-modified down-stream primer, to the end of the complementary template strand, in a reaction dependent on hel E. However, the translocation of hel E was blocked by cis-DDP modification of the template strand. Primer displacement was completely prevented if the modified site was located just upstream of the primer. The DNA-dependent ATPase activity of helicase E was also reduced by cis-DDP modification of the template DNA. Substrate competition experiments indicated that cis-DDP-modified DNA templates did not sequester hel E. Substrate titration experiments suggested that there is a short delay without ATP hydrolysis before dissociation of helicase E from cis-DDP-modified template sites. Interestingly, hel E could displace a primer if the cis-DDP modification was on the template within the annealed region. Possible explanations for this are discussed. Taken together, these results are consistent with the proposal that hel E participates in DNA repair by displacing segments of damaged DNA.

Adenosine Triphosphatases↗
PubMed1993

Direct gene transfer with DNA-liposome complexes in melanoma: expression, biologic activity, and lack of toxicity in humans.

Direct gene transfer offers the potential to introduce DNA encoding therapeutic proteins to treat human disease. Previously, gene transfer in humans has been achieved by a cell-mediated ex vivo approach in which cells from the blood or tissue of patients are genetically modified in the laboratory and subsequently returned to the patient. To determine the feasibility and safety of directly transferring genes into humans, a clinical study was performed. The gene encoding a foreign major histocompatibility complex protein, HLA-B7, was introduced into HLA-B7-negative patients with advanced melanoma by injection of DNA-liposome complexes in an effort to demonstrate gene transfer, document recombinant gene expression, and determine the safety and potential toxicity of this therapy. Six courses of treatment were completed without complications in five HLA-B7-negative patients with stage IV melanoma. Plasmid DNA was detected within biopsies of treated tumor nodules 3-7 days after injection but was not found in the serum at any time by using the polymerase chain reaction. Recombinant HLA-B7 protein was demonstrated in tumor biopsy tissue in all five patients by immunochemistry, and immune responses to HLA-B7 and autologous tumors could be detected. No antibodies to DNA were detected in any patient. One patient demonstrated regression of injected nodules on two independent treatments, which was accompanied by regression at distant sites. These studies demonstrate the feasibility, safety, and therapeutic potential of direct gene transfer in humans.

Aged↗
PubMed1993

Characterization of a gene encoding a Ca(2+)-ATPase-like protein in the plastid envelope.

By screening an Arabidopsis expression library with an antiserum against chloroplast envelope proteins, we have isolated a partial cDNA with an open reading frame that encodes a polypeptide similar to P-type cation-transporting ATPases. The corresponding genomic clone was isolated and the complete coding sequence was deduced after identification and mapping of introns. The gene has been designated PEA1 (plastid envelope ATPase) and the predicted polypeptide PEA1p. PEA1p has 946 amino acids and a molecular mass of 104 kDa. This protein is 40-44% identical to various mammalian plasma membrane Ca(2+)-ATPases but lacks the C-terminal calmodulin binding domain present in the mammalian polypeptides. When aligned with mammalian plasma membrane Ca(2+)-ATPases, PEA1p has a 70- to 80-amino acid N-terminal region that extends beyond the N terminus of these enzymes. This extension has some similarity to the transit peptide of the plastid envelope phosphate translocator and may function to target the protein to the plastid. Antibodies raised against a portion of PEA1p recognize a single 90- to 95-kDa polypeptide in chloroplast inner envelope preparations. Transcript abundance as determined by RNase protection was found to be 7- to 9-fold higher in roots than in leaves. Possible roles for a plastid envelope calcium pump are suggested.

Amino Acid Sequence↗
PubMed1993

Superantigen-driven peripheral deletion of T cells. Apoptosis occurs in cells that have lost the alpha/beta T cell receptor.

Injection of lymphoid cells expressing minor lymphocyte-stimulating antigen-1 (Mls-1a) induces tolerance to the superantigen, and partial deletion of Mls-1a-reactive T cells. We have identified a transient population of T cells that have lost the alpha/beta T cell receptor at the time when Mls-1a-reactive T cells start to disappear during the process of tolerance induction. Apoptosis was directly demonstrated in this TCR-alpha/beta negative T-cell population. This indicates a peripheral T-cell deletion pathway, characterized by TCR down-regulation, apoptosis, and clonal deletion. The consequence of Mls-1a-induced TCR down-regulation appears to be different in CD4+ cells and in CD8+ cells. Although most of the CD4+ cells that have lost TCR expressing alpha- and beta-chains appear to be undergoing apoptosis, many of their CD8+ counterparts may be able to re-express the Ag receptor. This argues for the involvement of coreceptors in the induction of apoptosis during peripheral deletion.

Animals↗
PubMed1993

Anti-HIV-1 activity and cellular pharmacology of various analogs of gossypol.

We previously reported that the racemic mixture and both enantiomers of gossypol inhibit the replication of human immunodeficiency virus-type 1 (HIV-1) (Lin et al., Antimicrob Agents Chemother 33: 2149-2151, 1989). The present study evaluates the activities of a variety of analogs of gossypol as well as a few non-gossypol analogs. Compounds 2, 3, 10, and 13 were slightly more inhibitory than (-)-gossypol to the replication of HIV-1 in cell culture. Compounds 4 and 8 were cytotoxic to human peripheral blood monocyte (PBM) cells, and compounds 2 and 3 were cytotoxic to Vero cells but not PBM cells. The effects of the two enantiomers of gossypol on the cell volume and migration of H9 cells through the cell cycle were evaluated during 72 hr of incubation. The (-)-enantiomer of gossypol was more toxic to H9 cells than the (+)-enantiomer of gossypol as evidenced by cell destruction. Prior to cell destruction, there appeared to be no significant effect on cell cycle distribution with either enantiomer.

Animals↗
PubMed1993

Amperometric monitoring of chemical secretions from individual pancreatic beta-cells.

The goal of the work was to develop and test an amperometric method for measuring insulin secretion from individual pancreatic beta-cells. The electrode used was a carbon fiber microelectrode modified with a polynuclear ruthenium oxide/cyanoruthenate film. The chemically modified electrode allowed anodic detection of insulin in physiological buffers with a detection limit of 0.5 microM. To measure secretion, an electrode was positioned 1 micron away from a beta-cell that had been stimulated with K+ or glucose. Recordings made from the cells consisted of a series of current spikes averaging 38 ms full width at half-height. The spikes decreased in height and increased in width as the electrode was pulled away from the cell. Spikes were only observed if a modified electrode was used and its potential was sufficient to oxidize insulin. The area under the spikes correspond to approximately 600 zmol of insulin, which is within the expected range for vesicular insulin content. Spike area was independent of stimulation method. The results support the hypothesis that the electrode was anodically detecting a substance secreted from the cells by exocytosis. The results support, but do not prove, that insulin was the primary substance detected.

Cells, Cultured↗
PubMed1993

Cytoplasmic expression of a reporter gene by co-delivery of T7 RNA polymerase and T7 promoter sequence with cationic liposomes.

Expression of bacteriophage T7 RNA polymerase in mammalian cells can efficiently drive the transcription of a foreign gene controlled by the T7 promoter (Elroy-Stein et al., Proc. Natl. Acad. Sci. USA. 86, 6126-6130, 1989). We have tested the hypothesis that purified T7 RNA polymerase can be co-delivered into mammalian cells together with a reporter gene (chloramphenicol acetyltransferase, CAT) controlled by the T7 promoter (pT7-EMC-CAT) using DC-chol cationic liposomes. Indeed, significant level of CAT activity was observed in human lung adenocarcinoma (A549-1) cells which had been incubated with a complex of T7 RNA polymerase, pT7-EMC-CAT DNA and DC-chol cationic liposomes. The expression was specific in that T3 RNA polymerase could not replace the T7 RNA polymerase, and that co-delivered T7 RNA polymerase did not enhance the expression of a CAT gene controlled by the SV40 early promoter. The system was optimized in terms of enzyme, DNA and liposome concentrations. Time course experiment indicated that the expression of the T7 system was about 8-10 hours sooner than the SV40 system, consistent with the notion that T7 RNA polymerase does not enter into the nucleus and the transcription takes place in the cytoplasm of the transfected cells. The expression of the T7 system was transient; it declined after 30 hours post transfection, probably due to turnover of the phage enzyme in the mammalian cells. The expression system described here should be useful for gene transfer experiments which require a fast but transient expression of a foreign gene. We have also compared our delivery system with a commercial reagent, Lipofectin, which has been used to deliver T3 or T7 RNA polymerase with a reporter plasmid encoding the T3 or T7 promoter.

Bacteriophage T7↗
PubMed1993

Immunotherapy of malignancy by in vivo gene transfer into tumors.

The immune system confers protection against a variety of pathogens and contributes to the surveillance and destruction of neoplastic cells. Several cell types participate in the recognition and lysis of tumors, and appropriate immune stimulation provides therapeutic effects in malignancy. Foreign major histocompatibility complex (MHC) proteins also serve as a potent stimulus to the immune system. In this report, a foreign MHC gene was introduced directly into malignant tumors in vivo in an effort to stimulate tumor rejection. In contrast to previous attempts to induce tumor immunity by cell-mediated gene transfer, the recombinant gene was introduced directly into tumors in vivo. Expression of the murine class I H-2Ks gene within the CT26 mouse colon adenocarcinoma (H-2Kd) or the MCA 106 fibrosarcoma (H-2Kb) induced a cytotoxic T-cell response to H-2Ks and, more importantly, to other antigens present on unmodified tumor cells. This immune response attenuated tumor growth and caused complete tumor regression in many cases. Direct gene transfer in vivo can therefore induce cell-mediated immunity against specific gene products, which provides an immunotherapeutic effect for malignancy, and potentially can be applied to the treatment of cancer and infectious diseases in man.

Animals↗
PubMed1993

Primary structure and characterization of an Arabidopsis thaliana calnexin-like protein.

A cDNA clone (pTE-83) encoding a protein (CNX1p) related to the microsomal Ca(2+)-binding protein, calnexin, was isolated from an Arabidopsis thaliana expression library. Southern and Northern hybridization indicated that CNX1 is a single-copy gene encoding a message of 1900 nucleotides. The open reading frame encodes a polypeptide with 530 amino acids, a molecular mass of 60.5 kDa, and overall 48% identity to dog calnexin. Both animal calnexin and CNX1p contain a large luminal domain followed by a single potential membrane-spanning domain near the C terminus and a small C-terminal domain exposed to the cytoplasm. The in vitro translation product from the cloned cDNA yielded a polypeptide of 67 kDa that was co-translationally imported into dog microsomes and processed to a 64-kDa product. Antibodies generated against the C-terminal half of the protein cross-react with an identically sized protein present in the microsomal fraction from Arabidopsis. Both the imported and native proteins are cleaved by trypsin to a 59-kDa product indicating that the gene product was indeed correctly processed and translocated into dog microsomes and that the membrane topology of CNX1p resembles that of dog calnexin. The presence of a calnexin-like protein within the plant kingdom indicates that this protein is widespread and involved in processes fundamental to all eukaryotes.

Amino Acid Sequence↗
PubMed1993

Effect of chemically modified GM1 and neoglycolipid analogs of GM1 on liposome circulation time: evidence supporting the dysopsonin hypothesis.

The sugar group of the ganglioside GM1 has been modified by periodate oxidation, reduction or reductive amination. The negative charge of sialic acid of GM1 has also been removed by methylation or reductive hydrolysis. A series of neoglycolipid analogs of GM1 were synthesized by coupling the GM1 oligosaccharide (GM1OS) to dioleoylphosphatidylethanolamine (DOPE) via different spacer arms. The individual GM1 derivatives were incorporated into egg phosphatidylcholine/cholesterol liposomes and tested in mice in order to see whether they were effective in prolonging liposome circulation. The oxidized GM1 did not show any ability to prolong circulation. However, the lost activity after oxidation was completely recovered by the subsequent reduction step. A series of aminated GM1 derivatives were prepared via oxidation followed by reductive amination with various substituted amines. beta-Alanyl GM1 showed a comparable activity to the native GM1, while other aminated GM1S showed reduced activity in terms of prolonging circulation of liposomes. Blocking the negative charge of sialic acid by methylation did not greatly lessen the activity, and removing the carboxyl group of sialic acid by reductive hydrolysis reduced the activity by only approx. 20%. Among the neoglycolipid analogs of GM1 only GM1OS directly conjugated to DOPE was effective in prolonging the circulation, whereas conjugates with a spacer of various length were not effective. These results emphasize the importance of the molecular structure of GM1 for its functional ability to prolong the liposome circulation. Furthermore, modifications which abolish the cholera toxin-binding activity of GM1 also decrease the ability to prolong the circulation time of the liposomes, and vice versa. Such strong correlation further supports the idea that the specific recognition of GM1 oligosaccharide by putative dysopsonin(s) is responsible for the ability to prolong the liposomes circulation time.

Amination↗
PubMed1993

Effects of the anticancer drug cis-diamminedichloroplatinum(II) on the activities of calf thymus DNA polymerase epsilon.

DNA polymerase (pol) epsilon is essential for DNA replication and is thought to be a component of DNA repair systems in eukaryotic cells. The activities of pol epsilon have been examined using a series of synthetic oligonucleotides designed with cis-diamminedichloroplatinum(II) (cis-DDP)-modified specific guanine residues. Pol epsilon was incapable of synthesis over cis-DDP-modified single guanine or adjacent guanine residues present in the template strand. Both single and double guanines modified by cis-DDP present at the 3'-OH end of a primer strand completely inhibited the synthetic activity of pol epsilon and, in addition, sequestered pol epsilon at the platinated 3'-OH termini. The sequestering of pol epsilon on cis-DDP modified DNA may interfere with the function of this enzyme in DNA repair in vivo. The intrinsic 3' to 5' proofreading exonuclease activity of pol epsilon was also examined. Pol epsilon was capable of degrading a single-strand template with internal cis-DDP-modified guanines up to, but not through, the platinated nucleotides. A single platinated guanosine was sufficient to block the 3' to 5' exonuclease activity of pol epsilon. These results suggest that cis-DDP-DNA adducts inhibit DNA synthesis mediated by DNA polymerase epsilon and that platinated sites can arrest the nuclease of pol epsilon, a function exhibited during DNA repair.

Animals↗
PubMed1993

Immunocytochemical and immunogold localization of two prolactin isoforms in the same pituitary cells and in the same granules in the tilapia (Oreochromis mossambicus).

The tilapia pituitary secretes two forms of prolactin (tPRL) and a single growth hormone (tGH). The tPRLs share only 69% sequence identity and are designated tPRL177 and tPRL188 to indicate the number of amino acid residues in each isoform. Our aim was to develop specific antisera for detection of these three related polypeptides. Ten peptides corresponding to unique epitopes on the tPRLs and two peptides of tGH were synthesized using solid-phase methods, conjugated to carrier proteins, and used as immunogens for antibody production in rabbits. Select antisera for the tPRLs were highly specific, exhibiting only 1% cross-reactivity to the alternate tPRL under noncompetitive ELISA conditions at dilutions used in immunocytochemical analysis. The anti-tGH specifically bound to cells in the proximal pars distalis. Production of both tPRLs by a single cell type was indicated by the binding of both anti-tPRL177 and anti-tPRL188 to the same cells in the rostral pars distalis. Ultrastructural analysis of PRL-producing cells stained sequentially using the two different anti-tPRL antibodies labeled with immunogold of two size classes indicated that both tPRLs appear in the same granules. These findings suggest that the biological significance of two forms of PRL in the adult tilapia is not a function of differential regulation of two different classes of PRL cells or differential release of unique secretory granules.

Amino Acid Sequence↗
PubMed1993

Monophosphoryl lipid A enhances specific CTL induction by a soluble protein antigen entrapped in liposomes.

Exogenous soluble antigen loaded in membranous vehicles including splenocytes and liposomes can induce a CD8+ cytotoxic T-lymphocyte (CTL) response in mice. Plain liposomes of simple composition, however, are not as effective as cellular vehicles such as splenocytes. In the present study it is shown that incorporation of monophosphoryl lipid A (MPL), a semisynthetic bacterial adjuvant, into liposomes enhanced the ability of liposomal ovalbumin (OVA) to prime for a specific CTL response. With the MPL formulation, the minimal antigen dose required for a detectable CTL induction was reduced about fivefold, and this approximated the required minimal dose of OVA loaded in the splenocytes. Moreover, liposomes containing MPL could induce a considerable level of CTL activity by either an intravenous, intramuscular or subcutaneous immunization protocol, whereas liposomes without MPL could only elicit such a response by an intravenous injection route. Subcutaneous injection of a mixture of liposomes containing MPL and liposomes containing antigen also elicited specific CTL activity. However, simultaneous subcutaneous administration of liposomal MPL and liposomal OVA at two distant sites did not prime the mice for a CTL response. These results indicate that MPL, although not necessarily incorporated in the same liposomes, must be in close proximity to the antigen to exert its adjuvant activity. Based on the results of this model antigen study, it is suggested that an optimal CTL inductive vaccine should include immunomodulatory adjuvant in addition to a class I pathway delivery vehicle such as liposomes.

Adjuvants, Immunologic↗
PubMed1993

Growth of lactobacilli, Staphylococcus aureus and Escherichia coli in normal and mastitic milk and whey.

The growth of three lactobacilli (Lactobacillus acidophilus, L. bulgaricus and L. casei), Staphylococcus aureus and Escherichia coli was followed in normal and mastitic milk and whey using the standard plate count method. L. acidophilus, L. bulgaricus 6032 and L. casei 6028 grew well in normal milk, but had decreased growth in mastitic milk if not pre-adapted in mastitic whey. S. aureus 26003 and E. coli 44102 showed enhanced growth in mastitic milk as compared with their growth in normal milk. These mastitis pathogens grew faster than the lactobacilli in both the normal milk and the mastitic milk. Among the lactobacilli, L. acidophilus and L. bulgaricus grew faster than L. casei in both types of milk samples. All the bacteria tested grew well in the normal and mastitic whey samples. However, they seemed to have enhanced growth in mastitic whey with the exception of L. casei. Pre-adaptation of the bacteria in mastitic whey in subculture markedly improved the growth of both pathogenic and non-pathogenic bacteria in mastitic milk. All the bacteria showed decreased replication in mastitic milk as compared with mastitic whey.

Animals↗
PubMed1993

Zaragozic acids D and D2: potent inhibitors of squalene synthase and of Ras farnesyl-protein transferase.

Two new zaragozic acids, D and D2, have been isolated from the keratinophilic fungus Amauroascus niger. Zaragozic acids D [4] and D2 [5] are related to the previously described zaragozic acids A [1], B [2], and C [3] and are potent inhibitors of squalene synthase. Furthermore, all the zaragozic acids (A, B, C, D, and D2) are also active against farnesyl transferase. Zaragozic acids D and D2 inhibit farnesyl transferase with IC50 values of 100 nM, while zaragozic acids A and B are less potent.

Alkyl and Aryl Transferases↗
PubMed1993

Immunotargeting of liposomes containing lipophilic antitumor prodrugs.

Potential therapeutic applications of recently developed liposomes with a reduced affinity to the reticuloendothelial systems and a prolonged circulation time as targeting systems for lipophilic prodrugs were examined. In these studies, liposomes composed of phosphatidylcholine and cholesterol, additionally containing monosialoganglioside (GM1) or polyethylene glycol conjugated to phosphatidylethanolamine (PEG-PE), were used. Three antitumor lipophilic prodrugs, N-trifluoroacetyl-adriamycin-14-valerate (AD32), araC-diphosphate-diglyceride (araCdPdG), and 3',5'-o-dipalmitoyl-5-fluoro-2'-deoxyuridine (dpFUdR), were used to examine the effect of lipophilic prodrug incorporation into long-circulating liposomes and immunoliposomes on their biodistribution in mouse. Biodistribution studies with antibody-free liposomes containing lipophilic prodrugs showed that the activities of GM1 or PEG2000-PE in prolonging the circulation time of liposomes appeared to be preserved in the presence of each of the three lipophilic prodrugs at a drug/lipid molar ratio of 3:97. The effect of lipophilic prodrug incorporation on target binding of immunoliposomes was then examined using a mouse model. Incorporation of AD232 or dpFUdR into immunoliposomes, directed to the normal endothelium, did not affect the targetability of immunoliposomes, suggesting a potential effectiveness of these lipophilic prodrug-containing immunoliposomes in therapy for lung tumors. On the contrary, incorporation of araCdPdG resulted in significantly reduced target binding of immunoliposomes by yet unknown mechanism(s).

Animals↗
PubMed1993

An Arabidopsis mutant with a reduced level of cab140 RNA is a result of cosuppression.

We analyzed a mutant of Arabidopsis with a severely reduced level of cab140 RNA. This mutant, named lct for low level of cab140 transcript, was obtained during a selection for phytochrome signal transduction mutants. The selection was based on reduced expression of the tumor morphology shoots gene (tms2), an introduced counter-selectable marker under the control of the cab140 promoter. Expression of the introduced cab140::tms2 gene was also greatly reduced in lct, but surprisingly, expression of other phytochrome-regulated genes was not comparably affected. Furthermore, the lct phenotype could not be separated genetically from the T-DNA insert; thus, we suggest that this phenotype was caused by cosuppression of the introduced construct and the endogenous cab140 gene, and that the mutation causing the cosuppression was located on the T-DNA insert. In vitro nuclear transcription experiments demonstrated that the suppression was occurring at the level of transcription. We also found that the suppressed cab140 genes were not significantly more methylated than the nonsuppressed cab140 genes.

Amidohydrolases↗