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J B Lawrence

Publications and source records attributed to J B Lawrence.

At least 55 records · Page 3Linked to original sources
PubMed1994

Acyl transfer reactions associated with cis Golgi apparatus of rat liver.

Isolated Golgi apparatus, highly purified from rat liver, were found to contain an acyl transfer activity capable of restoring the acyl chains of the lysophospholipid products of the action of phospholipase A2 on phosphatidylcholine. The activity was located primarily in cis and medial Golgi apparatus fractions, had a pH optimum of 6.0 to 7.5 and was stimulated by various acyl-CoA derivatives but not by fatty acids plus ATP. The activity, determined from the conversion of [14C]lysophosphatidylcholine to [14C]phosphatidylcholine, was unaffected by EGTA, inhibited by manoalide at high concentrations (0.2 mM), and temperature-dependent. Temperature dependency, however, showed no definite transition temperature over the range 15 to 37 degrees C. The results demonstrated that cis Golgi apparatus membranes have the enzymatic capacity to restore fatty acids lost from phospholipids through the action of phospholipase A. The latter has been previously suggested to occur at the cis Golgi apparatus membranes based on analyses of cell-free transfer of radiolabeled phosphatidylcholine.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1994

Cytogenetic and fluorescence in situ hybridization studies on sporadic and hereditary tumors associated with von Hippel-Lindau syndrome (VHL).

We performed cytogenetic and fluorescence in situ hybridization (FISH) studies on 29 sporadic or familial tumors associated with von Hippel-Lindau [correction of Landau] disease. Four of five renal cell carcinomas with detectable alterations showed clones with chromosome 3 alterations. These changes led to loss of genetic material visible with cytogenetic resolution: either an unbalanced translocation involving 3p or loss of a whole homolog 3, resulting in monosomy of 3p. We have previously mapped the VHL gene to chromosomal region 3p25-p26. We applied FISH using the single copy probes cA233 and cA479, sequences close to the VHL gene, in a search for submicroscopic deletions of 3p. Use of FISH with differentially labeled probes indicated cA479 to be distal to cA233, but both were located within bands 3p25-26. FISH with single copy probes for interphase cytogenetics detected four subclones with deletions in the VHL region in 8/22 tumors, including four tumors which appeared cytogenetically normal. FISH proved to be a powerful tool in tumor genetic studies, especially helpful in detecting tumor subclones in benign and slowly growing tumors.

Adrenal Gland Neoplasms↗
PubMed1994

Dynamic changes in the higher-level chromatin organization of specific sequences revealed by in situ hybridization to nuclear halos.

A novel approach to study the higher level packaging of specific DNA sequences has been developed by coupling high-resolution fluorescence hybridization with biochemical fractionation to remove histones and distend DNA loops to form morphologically reproducible nuclear "halos." Results demonstrate consistent differences in the organization of specific sequences, and further suggest a relationship to functional activity. Pulse-incorporated bromodeoxyuridine representing nascent replicating DNA localized with the base of the chromatin loops in discrete clustered patterns characteristic of intact cells, whereas at increasing chase times, the replicated DNA was consistently found further out on the extended region of the halo. Fluorescence hybridization to unique loci for four transcriptionally inactive sequences produced long strings of signal extending out onto the DNA halo or "loop," whereas four transcriptionally active sequences remained tightly condensed as single spots within the residual nucleus. In contrast, in non-extracted cells, all sequences studied typically remained condensed as single spots of fluorescence signal. Interestingly, two transcriptionally active, tandemly repeated gene clusters exhibited strikingly different packaging by this assay. Analysis of specific genes in single cells during the cell cycle revealed changes in packaging between S-phase and non S-phase cells, and further suggested a dramatic difference in the structural associations in mitotic and interphase chromatin. These results are consistent with and suggestive of a loop domain organization of chromatin packaging involving both stable and transient structural associations, and provide precedent for an approach whereby different biochemical fractionation methods may be used to unravel various aspects of the complex higher-level organization of the genome.

Cell Cycle↗
PubMed1993

Germ line transmission of a yeast artificial chromosome spanning the murine alpha 1(I) collagen locus.

Molecular complementation of mutant phenotypes by transgenic technology is a potentially important tool for gene identification. A technology was developed that allows the transfer of a physically intact yeast artificial chromosome (YAC) into the germ line of the mouse. A purified 150-kilobase YAC encompassing the murine gene Col1a1 was efficiently introduced into embryonic stem (ES) cells via lipofection. Chimeric founder mice were derived from two transfected ES cell clones. These chimeras transmitted the full length transgene through the germ line, generating two transgenic mouse strains. Transgene expression was visualized as nascent transcripts in interphase nuclei and quantitated by ribonuclease protection analysis. Both assays indicated that the transgene was expressed at levels comparable to the endogenous collagen gene.

Animals↗
PubMed1993

Higher level organization of individual gene transcription and RNA splicing.

Visualization of fibronectin and neurotensin messenger RNAs within mammalian interphase nuclei was achieved by fluorescence hybridization with genomic, complementary DNA, and intron-specific probes. Unspliced transcripts accumulated in one or two sites per nucleus. Fibronectin RNA frequently accumulated in elongated tracks that overlapped and extended well beyond the site of transcription. Splicing appears to occur directly within this RNA track, as evidenced by an unambiguous spatial separation of intron-containing and spliced transcripts. Excised introns for neurotensin RNA appear free to diffuse. The transcription and processing site of the fibronectin gene localized to the nuclear interior and was associated with larger transcript domains in over 88 percent of the cells. These results support a view of nuclear function closely integrated with structure.

Animals↗
PubMed1993

A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus.

A quantitative three-dimensional analysis of nuclear components involved in precursor messenger RNA metabolism was performed with a combination of fluorescence hybridization, immunofluorescence, and digital imaging microscopy. Polyadenylate [poly(A)] RNA-rich transcript domains were discrete, internal nuclear regions that formed a ventrally positioned horizontal array in monolayer cells. A dimmer, sometimes strand-like, poly(A) RNA signal was dispersed throughout the nucleoplasm. Spliceosome assembly factor SC-35 localized within the center of individual domains. These data support a nuclear model in which there is a specific topological arrangement of noncontiguous centers involved in precursor messenger RNA metabolism, from which RNA transport toward the nuclear envelope radiates.

Cell Nucleus↗
PubMed1993

Isolation and mapping of human T-cell protein tyrosine phosphatase sequences: localization of genes and pseudogenes discriminated using fluorescence hybridization with genomic versus cDNA probes.

This work reports the isolation, partial characterization, and chromosomal mapping of several human T-cell protein tyrosine phosphatase (PTPase) sequences and provides a direct comparison of the specificity of cDNA versus genomic probes in discriminating the location of genes versus pseudogenes by fluorescence in situ hybridization. In initial attempts to map the T-cell (TC) PTP gene using a 2-kb cDNA, several labeled sites were noted, raising the possibility of multiple related sequences within the genome. To address this, four genomic clones were obtained with homology to the TC PTP cDNA and characterized for their primary structure and their position within the human genome. Based on the presence or absence of an open reading frame and the intron/exon structure, two of these clones were found to be overlapping sequences encoding the true TC PTP gene and two were highly related but distinct processed pseudogenes. The TC PTP gene (gene symbol PTPN2) encoded by clones L17-2 and L5-1 localized to chromosome 18p11.2-p11.3, whereas pseudogenes encoded by clone L17-1, entitled TCPS1 (gene symbol PTPN2P1), and clone L18, entitled TCPS13 (gene symbol PTPN2P2), mapped to chromosomes 1q22-q24 and 13q12-q13, respectively. A direct comparison of the specificity of genomic and cDNA probes demonstrated that under identical conditions the genomic probes (containing both exon and intron sequences) readily identified a single specific site of hybridization, whereas the cDNA identified sites of both the gene and its pseudogenes. While providing mapping and sequencing information on the TC PTPase sequences, this work illustrates a strategy for addressing a recurrent problem in gene mapping studies where highly related sequences exist within the genome.

Amino Acid Sequence↗
PubMed1993

Assignment of the nuclear mitotic apparatus protein NuMA gene to human chromosome 11q13.

A monoclonal antibody that was specific for a nuclear matrix protein was obtained and used to screen a human lambda gt11 expression library. Several partial cDNA clones were isolated and sequenced. The sequence for this protein was shown to be identical to that of NuMA, a 236-kDa nuclear mitotic spindle apparatus protein. NuMA has been recently characterized by two independent studies, and is thought to be part of a family of proteins that is required for the completion of mitosis. In this report, the chromosomal localization and copy number of the NuMA gene are analyzed using cDNA clones. High-resolution in situ hybridization reveals a single pair of signals on sister chromatids of human chromosome 11 at band q13. Stringent Southern analysis of human genomic DNA resulted in simple restriction patterns. These results together indicate that the NuMA gene is present as a single copy on human chromosome 11q13.

Antibodies, Monoclonal↗
PubMed1993

Nuclear RNA tracks: structural basis for transcription and splicing?

Knowledge of how the biochemical machineries governing metabolism and transport of several distinct classes of RNA may be organized and integrated into the structure of the nucleus remains very limited. Recent observations, including advances in the detection of specific nucleotide sequences directly within the nucleus, have heightened the long-standing interest in the structural organization of pre-mRNA transcription and processing.

Journal Article↗
PubMed1992

Pulsed laser and thermal ablation of atherosclerotic plaque: morphometrically defined surface thrombogenicity in studies using an annular perfusion chamber.

Although clinical trials using laser and thermal angioplasty devices have been underway, the effects of pulsed laser and thermal ablation of atherosclerotic plaque on surface thrombogenicity are poorly understood. This study examined the changes in platelet adherence and thrombus formation on freshly harvested atherosclerotic aorta segments from Watanabe-heritable hyperlipidemic rabbits after ablation by two pulsed laser sources (308-nm xenon chloride excimer and 2,940-nm erbium:yttrium-aluminum-garnet [YAG] lasers) and a prototype catalytic hot-tip catheter. Specimens were placed in a modified Baumgartner annular chamber and perfused with citrated whole human blood, followed by quantitative morphometric analysis to determine the percent surface coverage by adherent platelets and thrombi in the treated and contiguous control areas. Pulsed excimer laser ablation of plaque did not change platelet adherence or thrombus formation in the treated versus control zones. However, photothermal plaque ablation with a pulsed erbium:YAG laser resulted in a 67% reduction in platelet adherence, compared with levels in control areas (from 16.7 +/- 2.2% to 5.5 +/- 1.8%; p less than 0.005). Similarly, after plaque ablation using a catalytic thermal angioplasty device, there was a 74% reduction in platelet adherence (from 29.2 +/- 5.1% to 7.7 +/- 1.6%; p less than 0.005) and a virtual absence of platelet thrombi (from 8.6 +/- 2.3% to 0.03 +/- 0.03%; p less than 0.005). This reduced surface thrombogenicity after plaque ablation with either an erbium:YAG laser or a catalytic hot-tip catheter suggests that thermal modifications in the arterial surface ultrastructure or thermal denaturation of surface proteins, or both, may be responsible for reduced platelet adherence. These in vitro findings indicate that controlled thermal plaque ablation by catheter-based techniques may elicit endovascular responses that can reduce early thrombus formation during angioplasty procedures.

Angioplasty, Laser↗
PubMed1991

Molecular cloning, chromosomal mapping, and expression of the cDNA for p107, a retinoblastoma gene product-related protein.

p107 is a cellular protein that forms specific complexes with adenovirus E1A and SV40 large T antigen (T). The genetics of the p107-T/E1A interaction as well as other features of this protein suggests that p107 shares functional properties with the tumor suppressor product, RB. A partial cDNA for human p107 has been cloned. Its sequences map to 20q11.2 and encode a 936 residue protein. Comparison analysis of the p107 protein sequence reveals a major region of RB homology extending over 564 residues. This region in RB is essential to its growth-controlling function. Sequences outside of these two regions are largely unique to each protein. The p107 and RB homology regions can independently bind to T and E1A. Thus, these two proteins display similarities of structure that may, at least in part, explain their known functional similarities and suggest a generic function for p107 in cell cycle regulation.

Adenovirus Early Proteins↗
PubMed1991

DNA and RNA within the nucleus: how much sequence-specific spatial organization?

Spatial organization of various nuclear components is often proposed as a means by which nuclei more efficiently carry out their various tasks. Such functional compartmentalization may involve a sequence-specific packaging and placement of DNA and RNA. Here we review recent insights, allowed primarily by advances in fluorescent in situ hybridization methodology, into the organization of nucleic acids within individual nuclei.

Cell Cycle↗
PubMed1991

Hematologic manifestations of systemic mast cell disease: a prospective study of laboratory and morphologic features and their relation to prognosis.

PURPOSE: Systemic mast cell disease (SMCD) follows an indolent course in most patients, but a significant number of patients die of neoplastic hematologic disorders. Reviews of the literature and retrospective studies in a single institution have defined features that may be associated with a poor prognosis, but prospective studies have been lacking. Therefore, we prospectively analyzed the relationship between clinical, laboratory, and hematopathologic findings and clinical outcome in a series of 46 patients with mast cell disease. This analysis was employed to both define clinically useful prognostic variables and describe the histologic evolution of bone marrow mast cell infiltration and its relationship to hematologic neoplasia. PATIENTS AND METHODS: Forty-six adult patients were referred to the National Institutes of Health (NIH) with clinical and/or pathologic evidence of mast cell proliferation. All patients had bone marrow examinations, and 10 patients underwent serial bone marrow biopsies. The diagnosis of SMCD required pathologic documentation of bone marrow mast cell infiltrates. The patients were followed for up to 13 years at the NIH (up to 30 years after the initial pathologic diagnosis of mast cell disease). Statistical analysis defined the correlation between variables and the presence of diagnostic bone marrow lesions. The Kaplan-Meier method was used to construct survival curves, and the effects of various variables on the survival time were examined. RESULTS: Thirty-two of 46 patients (74%) had a bone marrow biopsy diagnostic for SMCD. The remaining 14 patients were considered to have cutaneous mast cell disease (CMCD). Univariate analysis showed that hepatosplenomegaly, alkaline phosphatase level, absolute lymphocyte count, and age at onset of symptoms were positively correlated with SMCD, whereas hemoglobin level was negatively associated with diagnostic bone marrow lesions. With multivariate analysis, only hemoglobin and absolute lymphocyte count remained as significant independent predictors of bone marrow findings. No CMCD patient died or had significant clinical deterioration in the 1- to 30-year period of follow-up (median = 8.5 years), whereas 10 of 32 SMCD patients (31%) died from 1 to 22 years after diagnosis (median = 2.5 years) (p less than 0.0001). Univariate analysis revealed the following variables as significantly increasing the risk of death in patients with SMCD: later onset of symptoms, absence of cutaneous mastocytosis, thrombocytopenia, elevated lactate dehydrogenase (LDH) level, anemia, bone marrow hypercellularity, qualitative peripheral blood smear abnormalities, elevated alkaline phosphatase level, and hepatosplenomegaly. Multivariate analysis showed that only the age at onset of symptoms and LDH levels were significant independent predictors of survival. Eight of the 10 SMCD patients who died had myeloproliferative or myelodysplastic syndromes or acute nonlymphocytic leukemia. CONCLUSION: Our prospective study has defined a number of important variables in patients with clinical evidence of mast cell proliferation that can predict both the presence of SMCD and the likelihood of fatal disease. Since recent evidence suggests that mast cells derive from a bone marrow hematopoietic progenitor, SMCD may represent a myeloproliferative condition with the propensity to evolve into a neoplastic granulocytic disorder in a significant minority of patients.

Adolescent↗
PubMed1991

Distribution of myosin heavy chain mRNA in embryonic muscle tissue visualized by ultrastructural in situ hybridization.

We have localized myosin heavy chain (MHC) mRNAs in cells of intact embryonic chick muscle using high resolution in situ hybridization. Blocks of muscle were aldehyde-fixed prior to detergent treatment and hybridized with a biotinated cDNA probe, followed by colloidal gold-labeled antibodies, before embedment. Labeling was determined to represent MHC mRNA by extensive quantitative comparisons of electron micrographs from experimental and four different types of control samples. MHC mRNA was localized primarily to peripheral regions of 14-day chick pectoral muscle cells, where the majority of developing myofibrils were found. MHC mRNAs were consistently associated with the nonmyofibrillar cytoskeletal filaments which had diameters ranging from 4 to 10 nm. They were often oriented parallel to the longitudinal axis of the cell. The resolution of the ultrastructural approach allowed us to demonstrate that the mRNA molecules visualized were not directly associated with myofilaments, suggesting that nascent chains read from those messages do not assemble directly into myofilaments simultaneous with translation.

Actin Cytoskeleton↗
PubMed1991

Localizing DNA and RNA within nuclei and chromosomes by fluorescence in situ hybridization.

The enormous potential of in situ hybridization derives from the unique ability of this approach to directly couple cytological and molecular information. In recent years, there has been a surge of success in powerful new applications, resulting from methodologic advances that bring the practical capabilities of this technology closer to its theoretical potential. A major advance has been improvements that enable, with a high degree of reproducibility and efficiency, precise visualization of single sequences within individual metaphase and interphase cells. This has implications for gene mapping, the analysis of nuclear organization, clinical cytogenetics, virology, and studies of gene expression. This article discusses the current state of the art of fluorescence in situ hybridization, with emphasis on applications to human genetics, but including brief discussions on studies of nuclear DNA and RNA organization, and on applications to clinical genetics and virology. Although a review of all of the literature in this field is not possible here, many of the major contributions are summarized along with recent work from our laboratory.

Animals↗