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Biomedical subjects

Y Le

Publications and source records attributed to Y Le.

45 records · Page 3Linked to original sources
PubMed1997

Brief expression of a GFP cre fusion gene in embryonic stem cells allows rapid retrieval of site-specific genomic deletions.

The Cre DNA recombinase of bacteriophage P1 has become a useful tool for precise genomic manipulation in embryonic stem (ES) cells that have been gene modified by homologous recombination. We have re-engineered the cre gene to allow ready identification of living Cre+cells by constructing a functional fusion between Cre and an enhanced green fluorescent protein from Aequorea victoria (GFPS65T). The GFP cre fusion gene product rapidly targeted the nucleus in the absence of any exogenous nuclear localization signal. Moreover, GFPCre catalyzed efficient DNA recombination in both a mouse 3T3 derivative cell line and in murine ES cells. Fluorescence- activated cell sorting (FACS) of transiently GFP cre -transfected ES cells not only allowed rapid and efficient isolation of Cre+cells after DNA transfection but also demonstrated that a burst of Cre expression is sufficient to commit cells to Cre-mediated 'pop-out' of loxP -tagged DNA from the genome. Thus, GFP cre allows rapid identification of living cells in which loxP - flanked DNA sequences are destined to be removed from the genome by Cre-mediated recombination without reliance on recombinational activation or inactivation of a marker gene at the target locus. In addition, the GFP cre fusion gene will prove useful in tracing tissue-specific Cre expression in transgenic animals, thereby facilitating the generation and analysis of conditional gene knockout mice.

Animals↗
PubMed1997

Purification and binding properties of a human ficolin-like protein.

Ficolin was initially identified from porcine uterus as a TGF-beta 1 binding protein and is considered to have an overall structure similar to that of the complement protein C1q and the collectins. Recent studies have shown that human ficolin is synthesized mainly by monocytes in peripheral blood and that it could potentially bind to sugar structures on microorganisms. The aim of the present investigations was to isolate ficolin from human plasma by affinity chromatography on immobilized sugars. A human serum protein was identified in the GlcNAc eluate from GlcNAc-Sepharose which migrated as a polypeptide of approx. 40 kDa on SDS-PAGE under reducing conditions and was, after further purification by FPLC on a mono-Q column, shown to have an identical N-terminal sequence, over the first 14 residues, to P35, a plasma protein having similar sequence and domain organisation to ficolin. This protein, named the ficolin-like protein, was shown to be sensitive to collagenase and similar to P35 in that it was also disulphide-linked into an oligomer of approx. 320 kDa. However, unlike P35, its binding to GlcNAc was independent of Ca2+. Gel-filtration studies showed that this ficolin-like protein also had a molecular weight of approx. 320 kDa under non-dissociating conditions. During the course of this study this ficolin-like protein was found to simply bind to CNBr-activated Sepharose which had been inactivated with Tris, and from which it could be eluted with GlcNAc. This ficolin-like protein was also shown to bind to GlcNAc, but not to mannose and maltose. The functional significance of the unusual binding property of this ficolin-like protein is not clear, but it has facilitated the development of a simple method for its purification.

Acetylglucosamine↗
PubMed1997

Stabilization of yeast artificial chromosome clones in a rad54-3 recombination-deficient host strain.

The cloning and propagation of large fragments of DNA on yeast artificial chromosomes (YACs) has become a routine and valuable technique in genome analysis. Unfortunately, many YAC clones have been found to undergo rearrangements or deletions during the cloning process. The frequency of transformation-associated alterations and mitotic instability can be reduced in a homologous recombination-deficient yeast host strain such as a rad52 mutant. RAD52 is one member of an epistatic group of genes required for the recombinational repair of double-strand breaks in DNA. rad52 mutants grow more slowly and transform less efficiently than RAD + strains and are therefore not ideal hosts for YAC library construction. We have investigated the ability of both null and temperature-sensitive alleles of RAD54 , another member of the RAD52 epistasis group, to prevent rearrangements of human YAC clones containing tandemly repeated DNA sequences. Our results show that the temperature-sensitive rad54-3 allele blocks mitotic recombination between tandemly repeated DYZ3 satellite sequences and significantly stabilizes a human DYZ5 satellite-containing YAC clone. Yeast carrying the rad54-3 mutation can undergo meiosis, have growth and transformation rates comparable with RAD + strains, and therefore represent improved YAC cloning hosts.

Chromosomes, Artificial, Yeast↗
PubMed1996

Biosynthesis of human ficolin, an Escherichia coli-binding protein, by monocytes: comparison with the synthesis of two macrophage-specific proteins, C1q and the mannose receptor.

Ficolin is characterized by the presence of both collagen-like and fibrinogen-like sequences, and potentially has a similar overall structure as the complement protein C1q and the collectins. Previous studies have reported the presence of human ficolin mRNA predominantly in peripheral blood leucocytes. In the present study, the cellular origin of human ficolin was investigated in further detail. Preliminary studies using reverse transcriptase-polymerase chain reaction (RT-PCR) showed that ficolin mRNA was synthesized by U937 cells, a human monocyte cell line. This finding suggested that blood monocytes also normally synthesize human ficolin. Peripheral blood monocytes from adult human donors were harvested at serial time-points (0-20 hr) after adhesion to tissue culture plates, and total RNA was isolated and assayed for ficolin mRNA by RT-PCR. Ficolin mRNA was highly expressed in monocytes throughout the first 20 hr of adhesion. In contrast, C1q and mannose receptor mRNA were not detectable during the first 8 hr of adhesion, but were highly expressed by 20 hr. Cells were harvested at longer time intervals (1, 2, 4, 6 and 8 days) to determine whether ficolin expression was temporally regulated at later stages of monocyte differentiation. Ficolin mRNA levels decreased sharply from day 1 to day 6. In contrast, the levels of both C1q and mannose receptor mRNA showed no changing trend. These results are consistent with the absence of ficolin expression in many macrophage-rich tissues previously reported. The origin of ficolin from monocytes, together with its structural similarity to C1q and the collectins, raises the possibility that ficolin may be another plasma protein capable of binding to surface structures of micro-organisms. Escherichia coli was therefore incubated with human serum, and bound proteins, after elution with sugars, were analysed by Western blotting using an antiserum raised against a synthetic ficolin peptide. The antiserum identified a polypeptide of approximately 42000 MW, which is similar in size to that of ficolin as predicted from its cDNA-derived sequence.

Blotting, Western↗
PubMed1994

Treatment of vitiligo with melanocytic grafting.

This case report documents the treatment of extensive vitiligo with melanocytic grafting using ultra-thin, split-thickness skin grafts. Results, limitations, and disadvantages are discussed.

Adult↗
PubMedDate not supplied

Amphotericin B-associated hypertension.

OBJECTIVE: To report two cases of hypertension related to amphotericin B infusion. CASE SUMMARY: A 63-year-old woman with Candida albicans bacteremia and an 84-year-old man with Aspergillus fumigatus pneumonia developed hypertension within minutes of amphotericin B administration. Both patients' blood pressure returned to baseline soon after the infusion of amphotericin B was stopped. Neither patient was rechallenged. DISCUSSION: A causal relationship may exist between the administration of amphotericin B and these hypertensive episodes. Blood pressure in both patients normalized without treatment on discontinuation of the infusion. The mechanism of amphotericin B-associated hypertension is unclear but could include vasoconstricting properties of the drug or the administration of intravenous NaCl 0.9% prior to amphotericin B infusion. We recommend that intravenous NaCl 0.9% be administered following amphotericin B infusion and that the infusion be stopped if hypertensive episodes arise. CONCLUSIONS: Both acute hypertension and hypotension can occur in patients receiving amphotericin B for systemic fungal infections.

Aged↗
PubMedDate not supplied

A new insight into the role of "old" chemotactic peptide receptors FPR and FPRL1: down-regulation of chemokine receptors CCR5 and CXCR4.

N-formyl peptides, such as fMet-Leu-Phe (fMLP), are some of the first identified and most potent chemoattractants for phagocytic leukocytes. In addition to the bacterial peptide fMLP and the putative endogenously produced formylated peptides, we recently identified a number of other novel peptide agonists that selectively activate the prototype formyl peptide receptor (FPR) and/or its variant FPRL1. These agonists include several synthetic peptide domains derived from the envelope proteins of the human immunodeficiency virus type 1 (HIV-1) and intact human acute phase serum protein serum amyloid A. The activation of FPR and/or FPRL1 in monocytes by these agonists resulted in increased cell migration, calcium mobilisation and the heterologous down-regulation of the expression and function of chemokine receptors, notably CCR5 and CXCR4, two crucial fusion co-receptors for HIV-1. This down-regulation of CCR5 by FPR and FPRL1 agonists was associated with a rapid serine phosphorylation of CCR5. The desensitisation of CCR5 by FPR or FPRL1 agonists, not only inhibited its biological function induced by chemokine ligands, but also interfered with its capacity to act as a fusion co-receptor for monocyte tropic HIV-1. Thus, heterologous desensitisation by FPR and FPRL1 may play an important role in orchestrating the host innate immune responses which generate multiple chemotactic stimulants. Furthermore, the understanding of the structural and biochemical basis of FPR/FPRL1 activation may lead to the development of novel immunoregulatory and anti-HIV agents that emulate the process of heterologous desensitisation.

Chemotaxis, Leukocyte↗