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Biomedical subjects

Y Jiang

Publications and source records attributed to Y Jiang.

At least 541 records · Page 30Linked to original sources
PubMed1995

BTS1 encodes a geranylgeranyl diphosphate synthase in Saccharomyces cerevisiae.

Protein prenylation utilizes different types of isoprenoids groups, namely farnesyl and geranylgeranyl, to modify proteins. These lipophilic moieties attach to carboxyl-terminal cysteine residues to promote the association of soluble proteins to membranes. Most prenylated proteins are geranylgeranylated. Geranylgeranylation is catalyzed by two different prenyltransferases, the type I and type II geranylgeranyl transferases, both of which utilize geranylgeranyl diphosphate as a lipid donor. In the yeast Saccharomyces cerevisiae, the BET2 gene encodes the beta-subunit of the type II geranylgeranyl transferase. Mutations in this gene cause a defect in the geranylgeranylation of small GTP-binding proteins that mediate vesicular traffic. In an attempt to analyze those genes whose products may interact with Bet2, we isolated a suppressor of the bet2-1 mutant. This suppressor gene, called BTS1, encodes the yeast geranylgeranyl diphosphate synthase. BTS1 is not essential for the vegetative growth of cells; however, disrupting it impedes the geranylgeranylation of many cellular proteins and renders cells cold sensitive for growth. Our findings imply that BTS1 suppresses the bet2-1 mutant by increasing the intracellular pool of geranylgeranyl diphosphate.

Alkyl and Aryl Transferases↗
PubMed1995

Immunoaffinity purification of DNA polymerase delta.

A monoclonal antibody against human DNA polymerase delta (pol delta) was isolated with properties suitable for its utilization for immunoaffinity chromatography. The antibody was immobilized after periodate oxidation and coupled to a hydrazide-activated support. Starting from a partially purified preparation, calf thymus pol delta was purified about 200-fold in a single step. Further purification on ssDNA-cellulose resulted in isolation of a homogeneous preparation. The amount of enzyme isolated, ca. 0.3 mg of pure pol delta from 0.75 kg of calf thymus, is about 15-fold greater than can be achieved by conventional procedures. This procedure provides a significant advance in the isolation of pol delta in allowing its facile isolation from tissues in good yield. The isolated enzyme consisted of two subunits of 125 and 50 kDa. Characterization of the enzyme showed that these two subunits remained associated on glycerol gradient ultracentrifugation even in the presence of 2.8 M urea.

Animals↗
PubMed1995

The morphogenesis of a Chinese strain of HIV-1 forming inclusion bodies in Jurkat-tat III cells.

A rapid/high replicative strain of human immunodeficiency virus type 1 (HIV-1) (BC9101) was isolated directly in the Jurkat-tat III cell line from a Chinese patient with AIDS. The thin-section electron microscopy was performed and revealed high efficiency of replication of BC9101 with some unusual biological properties. Many vacuoles, most of them filled with HIV particles, were found close to the nucleus. Double-cored virions and double budding were frequently observed in the vacuoles and at the vacuolar membrane. Virus particles matured by budding both into intracytoplasmic vacuoles and through the plasma membrane. Inclusion bodies of varying sizes, some consisting of thousands of HIV particles, were found in the cytoplasm. All the illustrated features describing formation of inclusion bodies were compatible with the observation that HIV particles were assembled at and budded from the cytoplasmic vacuole membrane. They were then released from the membrane into the vacuoles, and subsequently, the maturation occurred. Some of the vacuoles accumulated to such a high number of mature virus particles that inclusion bodies were formed. During the disintegration of the cells, the inclusion bodies surrounded by the vacuolar membrane were released from the cells. The nucleotide sequence of the vpu gene of BC9101 was investigated and indicated that the unusual biological properties may due to the lack of a start codon for translation of the vpu protein.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Expression of the catalytic subunit of human DNA polymerase delta in mammalian cells using a vaccinia virus vector system.

The catalytic polypeptide of human DNA polymerase delta was overexpressed in BSC-40 cells (African green monkey kidney cell line) using the vaccinia virus/pTM1 system. The recombinant human DNA polymerase delta was purified to homogeneity in two steps using an immunoaffinity column and a single-stranded DNA-cellulose column. Levels of expression were about 1% of soluble cytosolic protein. The recombinant catalytic subunit was fully active and exhibited enzymatic properties similar to that of the native two-subunit enzyme including the possession of an associated 3' to 5' exonuclease activity. Recombinant pol delta was stimulated by proliferating cell nuclear antigen (PCNA); however, the degree of stimulation was lower than that of the native human enzyme. Analysis of a double mutant of the catalytic subunit, H142R/F144S, showed that it had a greatly reduced sensitivity to PCNA, suggesting that the PCNA binding site of pol delta may be located in this region of the N terminus.

Animals↗
PubMed1995

In vitro effects of HgCl2 on murine lymphocytes. I. Preferable activation of CD4+ T cells in a responder strain.

Mercury-induced autoimmune disorders have been demonstrated in rats and mice injected with HgCl2. We have studied the ability of HgCl2 to activate murine lymphocytes in vitro and found that it induced increased DNA synthesis, which peaked at days 4 to 6. Other metal ions, such as Mg2+ and Zn2+, had no or much less effect. Consistent with the in vivo studies, there were strain differences, and the most significant increase in thymidine uptake was induced in A.SW and BALB/c spleen cells. Both T lymphocytes and adherent cells were required for activation, and anti-CD4 Ab completely abrogated HgCl2-induced proliferation, suggesting the involvement of T helper cells. Cell phenotype analysis demonstrated that HgCl2 induced both proliferation and transformation of CD4+ and CD8+ T cells from BALB/c mice (responder strain). In contrast, only CD8+ T cells from the nonresponder DBA/2 mice were transformed. These findings indicate that helper T cells play a crucial role for the immunologic effects caused by HgCl2 and determine the ability of different mouse strains to respond to HgCl2.

Animals↗
PubMed1995

Nucleotide requirements for activated RNA polymerase II open complex formation in vitro.

The role of nucleotides in activated RNA polymerase II transcription was studied. Permanganate footprinting confirmed that there is a strict nucleotide requirement for forming open promoter complexes that cannot be overcome by the addition of a dinucleotide primer corresponding to the start site sequence. However, higher concentrations of other nucleoside triphosphates can substitute for ATP in catalyzing open complex formation. Opening catalyzed by these nucleotides is inhibited by the ATP analogue adenosine 5'-O-(thio-triphosphate), suggesting that they may function through cross-binding to the ATP site. The KM for ATP for opening and the involvement of other nucleotides in opening differs from the characteristics reported for TFIIH helicase and C-terminal domain kinase activities. This raises the possibility that opening does not involve these activities. The results alleviate very significantly the considerable current uncertainty concerning the role of ATP in the mammalian mRNA transcription initiation pathway.

Enzyme Activation↗
PubMed1995

Comparable sensitivities for detection of HIV-1 reverse transcriptase (RT) and other polymerases by RT assays requiring no radioisotopic materials.

An improved non-radioisotopic (Non-RI) reverse transcriptase (RT) assay with a template-primer-immobilized microtiter plate is described, which has greater sensitivity than the former Non-RI RT assay previously described. Non-RI and commercially available non-radioactive (Non-RA) RT assays were compared for their ability to detect various polymerases. Two RTs from Rous-associated virus 2 (RAV-2) and avian myeloblastosis virus (AMV), one polymerase from Escherichia coli (Pol-I) and one recombinant RT of human immunodeficiency virus type 1 (HIV-1) were assessed. Two HIV-1 samples in a culture supernatant and pelleted virion suspended in Triton X-100 solution were measured. The Non-RI RT assay was one hundred times more sensitive by RAV-2 and Pol-I polymerases, and one thousand times more sensitive by the Non-RA assay than by the AMV RT. The Non-RI RT assay was 10, 16 and 64 times more sensitive than the Non-RA assay for measuring recombinant HIV-1 RT, pelleted virus and virus suspended in culture medium, respectively. To explain the discrepancy, it is shown that free biotin, such as in culture medium, disturbs the assay system of the Non-RA RT assay, but not the Non-RI assay. The present assay can be used to clarify the inhibitory mechanism of an anti-HIV-1 substance.

Animals↗
PubMed1995

Effects of cold storage and water stress on water relations and gas exchange of white spruce (Picea glauca) seedlings.

To determine the effects of lifting time and storage on water-stress resistance of nursery-grown white spruce (Picea glauca (Moench) Voss) seedlings, we compared gas exchange, water relations and mortality of 3-year-old seedlings lifted in October 1991 and stored at -2 degrees C for 3 months with seedlings lifted in January 1992. The seedlings were placed in nutrient solution and subjected to -1.1 or -2.7 MPa water stress induced by polyethylene glycol 3350 for 9 days. Water stress, but not lifting time, had a significant effect on seedling net assimilation, symplastic volume and turgor loss point. In a second experiment, seedlings lifted in October 1991 were stored at -2 degrees C for 7 months and compared with seedlings lifted in May 1992. The seedlings were planted in pots, and their gas exchange and water relation parameters measured in response to gradual water stress. The results suggest that prolonged cold storage retards photosynthetic recovery of seedlings after planting. Higher rates of net assimilation in seedlings lifted in May were not directly related to their water status. Nonstomatal limitations were the primary factor influencing photosynthetic rate. We conclude that the inferior ability of cold-stored seedlings to tolerate water stress was due to poor osmotic adjustment and a lag in recovery of photosynthesis.

Journal Article↗
PubMed1995

Synthesis of alpha-factor analogues containing photoactivatable and labeling groups.

Analogues of alpha-factor, Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing both p-benzoyl phenylalanine (Bpa), a photoactivatable group, and 3-(mono- or di-iodo-4-hydroxyphenyl)propanoic acid (iodinated HPP) or biotin as a tag, were synthesized using solid-phase methodologies on a [phenylacetamido]-methyl (PAM) resin. Bpa was introduced into the peptides using Bpa-hydroxybenzotriazole active ester during peptide chain assembly. Biotinylated alpha-factor analogues were prepared by assembling the desired peptide on the resin, and then reacting a specific amino group either with the symmetrical anhydride of biotin or with biotin using BOP as the activating agent prior to anhydrous hydrogen fluoride cleavage. Iodinated HPP was incorporated by acylating free peptides with Bolton-Hunter reagent (3-[diiodo-4-hydroxyphenyl]propanoic acid hydroxysuccinimide ester) in N,N-dimethylformamide and borate buffer (pH 8.0) solutions. Purification of all peptides to 98% or greater homogeneity was accomplished by high-performance liquid chromatography on a reversed-phase mu-Bondapak C18 column with acetonitrile/water/trifluoroacetic acid as the mobile phase. All products were characterized by amino acid analysis and fast atom bombardment mass spectrometry. Two analogues, alpha-(diiodotyrosine)-His-Bpa-Leu-Gln-Leu-Arg-Pro-Gly-Gln-Pro-Nle-Tyr-O H, and epsilon-(diiodo-HPP)-Lys-His-Bpa-Leu-Gln-Leu-Arg-Pro-Gly-Gln-Pro-Nle-Tyr -OH, were one twentieth to one-fortieth as active as a alpha-factor, and exhibited approximately one order of magnitude lower affinity to the alpha-factor receptor. The results suggest that these two analogues are alpha-factor agonists and that they can be used as probes of the alpha-factor receptor.

Amino Acid Sequence↗
PubMed1995

Image feature analysis and computer-aided diagnosis in mammography: reduction of false-positive clustered microcalcifications using local edge-gradient analysis.

To improve the performance of a computerized scheme for detection of clustered microcalcifications in digitized mammograms, causes of detected false-positive microcalcification signals were analyzed. The false positives were grouped into four categories, namely, microcalcification like noise patterns, artifacts, linear patterns, and others. In an edge-gradient analysis, local edge-gradient values at signal-perimeter pixels of detected microcalcification signals were determined to eliminate false positives that look like subtle microcalcifications or are due to artifacts. In a linear-pattern analysis, the degree of linearity for linear patterns was determined from local gradient values from a set of linear templates oriented in 16 different directions. Threshold values for the edge-gradient analysis and the linear-pattern analysis were determined using a training database of 39 mammograms. It was possible to eliminate 59% and 25%, respectively, of 91 detected false-positive clusters with loss of only 3% of true-positive clusters. The combination of the two methods further improved the scheme in eliminating a total of 73% of the false-positive clusters with loss of 3% of true-positive clusters. Using these thresholds, the two methods were evaluated on another database of 50 mammograms. 62%, 31%, and 80% of the false-positive clusters were eliminated with loss of 3% of true-positive clusters or less, in the edge-gradient analysis, the linear-pattern analysis, and the combination of the two methods, respectively. The edge-gradient analysis and the linear-pattern analysis can reduce the false-positive detection rate, while maintaining a high level of the sensitivity.

Artifacts↗
PubMed1995

MCP-1-stimulated monocytes preferentially utilize beta 2-integrins to migrate on laminin and fibronectin.

Recruitment of monocytes to inflammatory sites involves a series of sequential attachments and detachments to extracellular matrix proteins in response to a chemoattractant gradient. In this study we compared the migration of human peripheral blood monocytes on different extracellular matrix proteins in response to monocyte chemoattractant protein-1 (MCP-1) and N-formylmethionyl-leucyl-phenylalanine. Monocytes migrated more effectively on laminin compared with other extracellular matrix proteins. In contrast, this preference was not observed with neutrophils, suggesting that the monocytes and neutrophils may have differences in their migration on extracellular matrix proteins. To study this further, function-blocking monoclonal antibodies were used to examine mechanistically whether beta 1- and beta 2-integrins were involved in monocyte migration on fibronectin or laminin in response to MCP-1. Monocyte migration on both laminin and fibronectin was blocked 100% (P < 0.05) by intact monoclonal antibody, F(ab') fragments, and F(ab')2 fragments to beta 2-integrins. We also determined that antibodies to beta 2-integrins block monocyte migration that has already been initiated. In contrast, antibody to the beta 1-integrins inhibited monocyte migration by approximately 40% (P < 0.05). Thus monocytes that express both beta 1- and beta 2-integrins require utilization of beta 2-integrins in migration on extracellular matrix proteins. The results also suggest that beta 1-integrins facilitate monocyte migration but that monocyte migration is not absolutely dependent on the interaction of beta 1-integrins with extracellular matrix proteins. In contrast, neutrophil migration is beta 2-integrin dependent and is not facilitated by beta 1-integrins.

CD18 Antigens↗
PubMed1995

Transcription patterns of sequences on human chromosome 21.

The polymerase chain reaction (PCR) was used to screen embryonic, fetal and adult human cDNA libraries for transcription on chromosome 21q22.1-->q22.3. Seventy-three pairs of oligonucleotide primers on chromosome 21, used previously to screen a fetal brain cDNA library, were applied to analyze 41 different cDNA libraries. Only phage eluate (and therefore no DNA isolation) was required for this sensitive screening. Sixty primer pairs were positive with at least one cDNA library, indicating that the majority of primers were derived from transcribed sequences. Even with our most complex human fetal brain cDNA library, we detected only 57% (34/60) of transcribed sequences, illustrating the need to screen multiple human cDNA libraries to determine if transcription occurred. Since only 3/73 clones were present in only one cDNA library, the vast majority of transcribed sequences are present in more than one tissue.

Chromosomes, Human, Pair 21↗