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Biomedical subjects

P Lu

Publications and source records attributed to P Lu.

At least 19 recordsLinked to original sources
PubMed1992

Perforin expression in human peripheral blood mononuclear cells. Definition of an IL-2-independent pathway of perforin induction in CD8+ T cells.

Perforin gene expression upon in vitro stimulation was studied at the mRNA level in normal human PBMC and in subpopulations. Freshly isolated PBMC express low levels of perforin mRNA. Increased perforin expression is rapidly induced by the calcium ionophore A23187 and by rIL-2. Phorbolesters (PMA), by comparison, are poor inducers of perforin RNA. Perforin induction by Ca-ionophore, unlike granzyme 2 and IL-2 induction, did not synergize with phorbolesters in PBMC or in purified T cells. Instead, perforin mRNA induction by A23187 in purified T cells requires the presence of adherent cells. Ca-ionophore plus adherent cell-induced perforin occurred in CD8+ T cells and was abolished by depletion of CD8+ T cells but not by depletion of CD4+ T cells. Adherent cells alone did not express perforin under any condition. Perforin mRNA induction by both A23187 and by rIL-2 is independent of de novo protein synthesis. The half-life of perforin mRNA induced by either stimulus is approximately 100 min. Cyclosporin A completely abrogates perforin induction by A23187 but only slightly inhibits the effect of rIL-2 on perforin mRNA expression. These data show that A23187 activates perforin gene expression in CD8+ cells by an IL-2-independent pathway and that the molecular mechanism of perforin expression may be different from the one induced by IL-2. Granzyme 2 (human leukocyte protease-HLP, homologous to murine granzyme B) mRNA expression was studied in comparison to perforin. Granzyme 2 in contrast to perforin responds to the synergistic action of phorbolester and Ca-ionophore in PBMC. In addition, the kinetics of the induction of granzyme and perforin mRNA, by various signals are different. Our data suggest that situations in vivo may exist that allow perforin expression in CD8+ cells in the absence of cytokines by a combination of Ca signals and accessory receptor ligation. The same signals may not be sufficient for granzyme 2 expression in any T cell subpopulation.

Base Sequence↗
PubMed1992

Transcriptional enhancer related DNA sequences: anomalous 1H NMR NOE crosspeaks.

A dynamic heterogeneity which correlates with the function of the operator DNA in the lactose operon of E. coli. was previously observed (1) as a local minimum in the thymine imino proton T1 centered at a GTG/C-CAC sequence. Since this triplet occurs frequently in DNA regulatory regions, it was proposed that these sequences may be part of a structural element for specific protein interaction. We examine here three additional biologically significant 17 base pair duplexes containing GTG/CAC triplets: (1) a sequence from the mouse heavy chain immunoglobulin enhancer, (2) a sequence from the critical core of the Simian Virus 40 (SV40) enhancer, and (3) a sequence from pBR322 plasmid used as control for experiments with the SV40 DNA sequences. The 1H NMR resonance assignment for nearly all the nonexchangeable protons for both eukaryotic enhancer duplexes with the exception of the H5'/H5" protons was accomplished to use for structural analysis of these duplexes. The data presented show several NOE's associated with the GTG/CAC triplets which suggest structural variation from uniform B-DNA. In addition, anomalous broad crosspeaks for the fixed thymine methyl to its own H6 proton in combination with the imino proton kinetics associated with these triplets reinforces the original observation of a sequence dependent dynamic variation.

Animals↗
PubMed1992

Cloning of the cDNA for canine interferon-gamma.

We have taken advantage of conserved regions of cDNA sequences for interferon-gamma (IFN-gamma) from other species to design polymerase chain reaction (PCR) primers capable of amplifying the protein-coding region of the canine mRNA. We report here the cDNA cloning of this region from dog lymphocytes and the cDNA sequence. The predicted amino acid sequence is also reported and compared to the known sequences of these other species. The molecular clone for the canine IFN-gamma will allow direct study of this important cytokine's role in graft rejection in the widely used experimental canine transplant model.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequencing of HLA-DQ gene second exons in Chinese homozygous cells.

Six HLA class I and class II-homozygous Chinese cell lines with unique HLA-Dw types were studied. Since the majority of HLA class II nucleotide sequence polymorphism is localized within the second exons of the genes, we used the polymerase chain reaction (PCR) to amplify these regions in HLA-DQA and DQB genes and subsequently determined the nucleotide sequences. No unique DQA1 or DQB1 alleles were found. However, a new haplotype of DQA1*0601-DQB1*0301-DRB1*1202 was found in two cells; and DQA1*03011 was found in association with DR9 in another two cells. This indicates that new DR-DQ associations may explain the observed new HLA-Dw types. The DQB2 sequences were identical in all six cells and were identical to a sequence previously reported in a DR6 haplotype. The DQA2 sequences from two clones obtained from two cells differed from each other and from previously reported sequences. The results show that the DQA1 and DQB1 alleles in the Chinese individuals studied are as previously reported in Caucasian populations and as such may be typed by restriction fragment-length polymorphism (RFLP) or PCR-sequence-specific oligonucleotide typing (PCR-SSO) or PCR-RFLP using conventional probe or restriction enzyme sets.

Base Sequence↗
PubMed1992

Clinical evaluation of 99mTc-MIBI myocardial tomography for detecting coronary artery disease.

To evaluate the clinical efficacy of 99mTc-MIBI myocardial tomography for detecting coronary artery disease, 34 consecutive patients who underwent both 99mTc-MIBI study and coronary arteriography were included in the present study. Ten without and 24 with significant coronary artery disease were documented by coronary arteriography. The overall sensitivity and specificity of 99mTc-MIBI study for detecting coronary artery disease were 91.8% (22/24) and 80.0% (8/10), respectively. In subset studies, 16 of 17 (94.1%) patients with previous myocardial infarction and 6 of 7 (85.7%) patients with significant CAD but without MI were identified as abnormal. Reversible perfusion abnormalities were shown in 4 of 5 patients undergoing both exercise and rest studies. Rest perfusion abnormalities were shown in 2 patients with unstable angina pectoris who underwent only rest study. In conclusion, 99mTc-MIBI myocardial tomography provides a reliable method for detecting coronary artery disease. Rest study has high sensitivity for identifying patients with MI, and exercise/rest study can identify patients with significant CAD but without MI.

Coronary Disease↗
PubMed1992

Clinical evaluation of 99Tcm-MIBI SPECT in the assessment of coronary artery disease.

To evaluate the clinical efficacy of 99Tcm-MIBI myocardial tomography for detecting coronary artery disease (CAD) 115 consecutive patients who underwent both 99Tcm-MIBI single photon emission computed tomography (SPECT) and coronary arteriography were studied. Thirty-three patients without and 82 with significant coronary artery disease were documented by coronary arteriography. The overall sensitivity and specificity of 99Tcm-MIBI SPECT for detecting coronary artery disease were 96 and 87.9%, respectively. The sensitivity for identifying patients with CAD without myocardial infarction was 88%. The sensitivity of 99Tcm-MIBI SPECT for detecting individual coronary artery lesions was 86% for left anterior descending artery (LAD), 69% for left circumflex artery (Lcx) and 86% for right coronary artery (RCA), lesions respectively. In conclusion, 99Tcm-MIBI SPECT provides a reliable method for detecting CAD.

Adult↗
PubMed1991

In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator.

Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/promoter. In addition to the wild-type lacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3----Tyr) and 61 (Ser61----Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10(2) to 10(4). The expression of these lac repressor fragments in the cell was verified by radioimmunoassays. Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced beta-galactosidase synthesis and methylation protection in vivo. These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain.

Base Sequence↗
PubMed1991

Distribution of taurine-like immunoreactivity in cerebellum of kittens from taurine-supplemented and taurine-deficient mothers.

Using an antibody prepared against taurine conjugated to bovine serum albumin with glutaraldehyde, the distribution of taurine in cerebellum of newborn and 8-week-old kittens from mothers fed 0, 0.02, 0.05, or 1% dietary taurine has been determined. In general, taurine-like immunoreactivity was greater in kittens from mothers fed the greatest amounts of taurine, as was the total cerebellar taurine concentration. The most notable feature in newborn kitten cerebellum was a dense band of staining in the inner molecular layer adjacent to the Purkinje cell layer, which corresponds to the short Purkinje cell dendrites. In cerebellum of 8-week-old kittens, taurine-like immunoreactivity was present in Purkinje cells and their dendrites, most granule cells, and a few interneurons in the molecular layer of the 0.02, 0.05, and 1% groups. The cerebellum of the 0% group was distinctive in that virtually no neurons were reactive, appearing as 'ghosts' against the background, and both white matter and the granule cell layer contained large numbers of reactive astrocytes. The presence of such large numbers of reactive astrocytes and the immunoglobulin within the brain suggests an impairment of the blood-brain barrier in such taurine-deficient kittens.

Animals↗
PubMed1991

Correlation of RFLP typing and MLC reactivity in dogs.

The human recombinant HLA-DRB1 gene probe was used for histocompatibility typing of two families of beagles for the DLA-D equivalent by using restriction fragment length polymorphisms (RFLP). This method was able to determine the segregation of these genes from the parental animals to the individual F1 offspring. Mixed lymphocyte culture (MLC) reactivity as well as serological typing for class I histocompatibility antigens were also performed for comparison. It was found that there was a high correlation between these three methods. We therefore conclude that RFLP typing is an effective procedure for predicting MLC reactivity in dogs and propose that it is a suitable genotyping method for assignment of class II antigen compatibility for donor-recipient pairs in conjunction with organ transplant studies.

Animals↗
PubMed1990

Detection of protein-DNA complex formation by time-resolved fluorescence depolarization of bound ethidium bromide.

We introduce the use of time-resolved fluorescence spectroscopy to probe the interaction between gene regulatory proteins and DNA. Changes in the decay kinetics of fluorescence polarization anisotropy of ethidium bromide bound to DNA segments report changes in hydrodynamic volume and shape which occurs upon complex formation between protein and DNA. We have used the decay of fluorescence polarization anisotropy as a spectroscopic handle on the interaction between several site-specific DNA-binding proteins involved in transcriptional regulation (the cro repressor of coliphage lambda, the lac repressor of Escherichia coli, and the RNA polymerase of coliphage T7) and their target DNA fragments ranging in length from 17 to 36 base pairs. The technique allows one to follow complex formation while varying solution conditions such as temperature, pH, ionic strength, and presence of effector molecules. Macromolecular concentrations ranging from 10(-7) to 10(-4) M can be used, allowing estimates of relative binding affinities. The magnitude of the observed rotational correlation times (phi obs) can be used to infer information about the size and shape of the complexes.

Base Sequence↗
PubMed1990

Lac repressor-operator interaction: DNA length dependence.

The interaction of the E. coli lac operon repressor with its operator DNA has been directly examined as a function of the length of operator-containing DNA. The apparent bimolecular association rate constants were calculated as ka = (kd/KD), where the dissociation equilibrium constant, KD and the dissociation rate constant, kd, were measured by nitrocellulose filter adsorption assays. The values obtained for the overall association rate constants are compared with theoretical association rate curves for specific mechanisms. Association of the repressor with short operator containing DNA fragments (less than 70 base pairs) occurs at rates expected of three-dimensional diffusion. Our data also imply that at longer DNA lengths a combination of three-dimensional diffusion with one-dimensional sliding along with hopping and/or intersegment transfer must be involved to facilitate the repressor operator association.

Base Composition↗
PubMed1990

Fluorescence line-narrowing spectrometry: a versatile tool for the study of chemically initiated carcinogenesis.

An important initiating step in the induction of tumors is believed to be the covalent binding of an active carcinogenic species to a cellular macromolecule, e.g. DNA. Therefore, a spectroscopic technique which allows for positive identification of the intact (macromolecular) DNA adduct and/or isolated damaged nucleosides/nucleotides is highly desirable. It is shown that fluorescence line-narrowing spectroscopy (FLNS) is a rapid, versatile, highly sensitive and selective analytical technique, which can be used directly to characterize DNA adducts and isolated nucleosides. FLNS possesses sufficient resolution to distinguish between the major DNA adducts derived from different enantiomers of benzo[a]pyrene diol-epoxide (BPDE). With the present limit of detection (approximately 1 adducted base per 10(8) normal base pairs for 100 micrograms of DNA), the technique is applicable to in vivo samples. Analysis of liver DNA from fish exposed to benzo[a]pyrene (BP) (100 mg BP/kg fish) showed that a major DNA adduct is derived from syn-BPDE.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1990

lac repressor: crystallization of intact tetramer and its complexes with inducer and operator DNA.

The intact lac repressor tetramer, which regulates expression of the lac operon in Escherichia coli, has been crystallized in the native form, with an inducer, and in a ternary complex with operator DNA and an anti-inducer. The crystals without DNA diffract to better than 3.5 A. They belong to the monoclinic space group C2 and have cell dimensions a = 164.7 A, b = 75.6 A, and c = 161.2 A, with alpha = gamma = 90 degrees and beta = 125.5 degrees. Cocrystals have been obtained with a number of different lac operator-related DNA fragments. The complex with a blunt-ended 16-base-pair strand yielded tetragonal bipyramids that diffract to 6.5 A. These protein-DNA cocrystals crack upon exposure to the gratuitous inducer isopropyl beta-D-thiogalactoside, suggesting a conformational change in the repressor-operator complex.

Base Sequence↗
PubMed1990

Assessment of right ventricular function with nonimaging first pass ventriculography and comparison of results with gamma camera studies.

A new technique has been developed for measuring right ventricular function by nonimaging first pass ventriculography. The right ventricular ejection fraction (RVEF) obtained by non-imaging first pass ventriculography was compared with that obtained by gamma camera first pass and equilibrium ventriculography. The data has demonstrated that the correlation of RVEFs obtained by the nonimaging nuclear cardiac probe and by gamma camera first pass ventriculography in 15 subjects was comparable (r = 0.93). There was also a good correlation between RVEFs obtained by the nonimaging nuclear probe and by equilibrium gated blood pool studies in 33 subjects (r = 0.89). RVEF was significantly reduced in 15 patients with right ventricular and/or inferior myocardial infarction compared to normal subjects (28 +/- 9% v. 45 +/- 9%). The data suggests that nonimaging probes may be used for assessing right ventricular function accurately.

Adult↗
PubMed1990

Preliminary study on the microbiology of Campylobacter pyloridis and gastric histopathology.

Biopsy samples were taken endoscopically from the antral-mucosa of 693 patients with peptic ulcer and chronic gastritis presenting dyspepsia symptoms. Campylobacter pyloridis cultures were positive in 59 of 98 (60.2%) cases and histopathologically the organisms were found in 411 of 693 cases (59.3%). Pathologically, Campylobacter pyloridis was positive in 273 out of 300 patients with chronic superficial gastritis (91.0%), in 102 of 249 patients with chronic atrophic gastritis (40.9%), in 36 out of 144 patients with chronic atrophic gastritis with intestinalization or dysplasia (25.0%). We found that there was a significant association between the presence of Campylobacter pyloridis and chronic superficial gastritis, also the degree of lymphocyte infiltration showed a strong inverse association with the presence of Campylobacter pyloridis, suggesting that a local immune response might exert an important action in the eradication of this organism. These findings support the view that Campylobacter pyloridis, may be etiologically related to chronic gastritis and peptic ulceration, even though its role still remains to be determined.

Campylobacter↗