Biomedical subjects

L B Nabors

Publications and source records attributed to L B Nabors.

9 recordsLinked to original sources

PubMed2001

HuR, a RNA stability factor, is expressed in malignant brain tumors and binds to adenine- and uridine-rich elements within the 3' untranslated regions of cytokine and angiogenic factor mRNAs.

Tumors of the central nervous system (CNS) often have sustained expression of labile genes, including angiogenic growth factors and immunosuppressive cytokines, which promote tumor progression. Stabilization of the RNA transcripts for these genes, such as vascular endothelial growth factor (VEGF), is an important molecular pathway for this up-regulation. HuR, a member of the Elav family of RNA-binding proteins, has been implicated in this pathway through its binding to adenine and uridine (AU)-rich stability elements (ARE) located in the 3' untranslated regions (3'-UTRs) of the mRNA. Whereas three of the Elav family members (Hel-N1, HuC, and HuD) are restricted to young and mature neurons, HuR is more broadly expressed, including proliferating cells of the developing CNS. Because RNA stabilization of labile genes may promote tumor growth, we analyzed and compared the expression pattern of HuR in 35 freshly resected and cultured CNS tumors to determine whether there was any correlation with tumor grade or histological type. We found that HuR mRNA was consistently expressed in all of the tumors, regardless of cell origin or degree of malignancy. Using a novel HuR-specific polyclonal antibody, we found that strong HuR protein expression was limited to high-grade malignancies (glioblastoma multiforme and medulloblastoma). Within the glioblastoma multiforme, prominent HuR expression was also detected in perinecrotic areas in which angiogenic growth factors are up-regulated. To further define its role as a potential RNA stabilizer, we analyzed whether HuR could bind to the stability motifs within the 3'-UTRs of cytokines and growth factors linked to brain tumor progression. We used a novel ELISA-based RNA binding assay and focused on the 3'-UTRs of angiogenic factors VEGF, COX-2, and (interleukin) IL-8 as well as the immunomodulating factors IL-6, transforming growth factor (TGF)-beta and tumor necrosis factor (TNF)-alpha as potential RNA ligands. Our results indicated overall a very high binding affinity to these RNA targets. A comparison of these ligands revealed a hierarchy of binding affinities with the angiogenic factors, and TGF-beta showing the highest (Kd of 1.8-3.4 nM), and TNF-alpha the lowest (Kd of 18.3 nM). The expression pattern of HuR, coupled with the RNA binding data, strongly suggests a role for this protein in the posttranscriptional regulation of these genes in CNS tumors.

3' Untranslated Regions↗

PubMed2001

Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells.

Cyclooxygenase-2 (COX-2) expression is normally tightly regulated. However, constitutive overexpression plays a key role in colon carcinogenesis. To understand the molecular nature of enhanced COX-2 expression detected in colon cancer, we examined the ability of the AU-rich element-containing (ARE-containing) 3' untranslated region (3'UTR) of COX-2 mRNA to regulate rapid mRNA decay in human colon cancer cells. In tumor cells displaying enhanced growth and tumorigenicity that is correlated with elevated COX-2, vascular endothelial growth factor (VEGF), and IL-8 protein levels, the corresponding mRNAs were transcribed constitutively and turned over slowly. The observed mRNA stabilization is owing to defective recognition of class II-type AREs present within the COX-2, VEGF, and IL-8 3'UTRs; c-myc mRNA, containing a class I ARE decayed rapidly in the same cells. Correlating with cellular defects in mRNA stability, the RNA-binding of trans-acting cellular factors was altered. In particular, we found that the RNA-stability factor HuR binds to the COX-2 ARE, and overexpression of HuR, as detected in tumors, results in elevated expression of COX-2, VEGF, and IL-8. These findings demonstrate the functional significance rapid mRNA decay plays in controlling gene expression and show that dysregulation of these trans-acting factors can lead to overexpression of COX-2 and other angiogenic proteins, as detected in neoplasia.

3' Untranslated Regions↗

PubMed2000

Analysis of the 5' end of the mouse Elavl1 (mHuA) gene reveals a transcriptional regulatory element and evidence for conserved genomic organization.

mHuA (Elavl1) belongs to a highly conserved family of genes encoding RNA-binding proteins and has been linked to cell growth and proliferation through its regulation of mRNA stability. Here, we use an RNase protection assay to demonstrate that the mHuA transcript is relatively abundant in a range of mouse tissues, with the highest levels being found in lung and embryonic stem cells. We then cloned and mapped an 18 kb DNA fragment which encompasses the 5' end of the mHuA gene. The genomic organization in this region is similar to the neural-restricted family members, Hel-N1 (ELAVL2) and mHuD (Elavl4). The first exon is lengthy and untranslated, and the second exon, which includes the methionine start site, ends between the ribonucleoprotein motifs of the first RNA binding domain. Mapping of the mHuA transcript by primer extension demonstrated three potential transcription-initiation sites which were detected consistently among different tissues and cell lines. Analysis of the sequence flanking these sites revealed the presence of transcriptional elements including TATA, CREB, c-ets, and AP1 sites. Transfection analysis of this promoter region using a luciferase-reporter-gene assay indicated strong transcriptional activity both in HeLa and in mouse macrophage (RAW) cells which is consistent with the ubiquitous expression pattern of mHuA. Thus, while the genomic organization of mHuA is similar to the neural-restricted members of the Elav family, the promoter element differs substantially both by sequence analysis and transcriptional activity in non-neural cell types.

Amino Acid Sequence↗

PubMed1999

Hu antigen specificities of ANNA-I autoantibodies in paraneoplastic neurological disease.

Despite a broad clinical spectrum, paraneoplastic enecephalomyelitis/sensory neuronopathy (PEM/SSN) is characterized by the presence of a common autoantibody, referred to as anti-Hu or type I anti-neuronal nuclear antibody (ANNA-1). The target of these antibodies is a family of four Hu antigens: three (Hel-N1, HuC, HuD) are neural-specific, while the fourth (HuR) is ubiquitous. Here, we have analysed by enzyme-linked immunosorbent assay (ELISA) the immunoreactivity of all four Hu antigens in serum from 75 patients with ANNA-1 autoantibodies and looked for clinical correlations. IgG in all the patients' sera bound to each of the four antigens, and the titers correlated with those of the ANNA-I immunofluorescence assay. Median titers for the neural-specific antigens (range: 56, 892-90,051) were significantly higher than for HuR (36,799). Patients with gastrointestinal dysmotility or subacute sensory neuronopathy had the highest median titers to all four antigens, while patients with sensorineural deafness had the lowest titers. The results indicate a heterogeneous immune response to individual Hu antigens in patients with PEM/SSN, and that the titers to these antigens as a group, rather than individually, correlate with clinical profile. Furthermore, these results suggest that ELISA analysis of a single neural-specific Hu antigen is sufficient for serological screening in PEM/SSN.

Antigens, Surface↗

PubMed1998

HuR, a novel target of anti-Hu antibodies, is expressed in non-neural tissues.

Paraneoplastic encephalomyelitis (PEM) is characterized by a diverse set of clinical signs that are limited to the nervous system. The serologic hallmark of PEM is the presence of circulating autoantibodies, collectively referred to as 'anti-Hu,' which immunoreact specifically with members of the Elav protein family. Until recently, the ELAV antigens were only detected in neurons, thus strongly supporting a role for anti-Hu antibodies in the selective neural tissue injury in PEM. The identification of HuR, however, a new member with a broad, non-neural pattern of RNA expression, raises several fundamental questions regarding PEM. First, why are non-neural tissues spared in PEM? Second, why is PEM predominantly associated with neuroendocrine tumors? To begin addressing these questions, we sought to determine whether the antibody response to HuR differs from the neural-specific counterparts in patients with PEM, and to characterize the protein expression pattern of this novel antigen in peripheral tissues and tumors. Using sera from 11 patients with Hu-positive PEM, we found that the majority of samples (73%) were weakly or non-reactive for recombinant HuR on Western blot, in contrast to consistently strong immunoreactivity with the neural-specific members HuD and Hel-N1. We also demonstrate that HuR is expressed at the protein level in both non-neural tissues and non-neuroendocrine tumors. These findings suggest that immunoreactive differences among Elav family members may contribute to the neural-restrictive pattern of tissue injury in patients with PEM.

Aged↗

PubMed1997

Magnetic resonance cisternography in the diagnosis of delayed iatrogenic cerebrospinal fistula: a case report.

A 52-year-old woman presented with a clinical picture consistent with bacterial meningitis 3 years after functional endoscopic sinus surgery. Diagnosis of a cerebrospinal fluid (CSF) fistula was made clinically, and the site of the fistula was confirmed using magnetic resonance cisternography. The utilization of this technique in the diagnosis of CSF disorders is gaining popularity. Its usefulness in the context of other imaging modalities is discussed.

Cerebrospinal Fluid Rhinorrhea↗

PubMed1991

A unique neuronal organization in the cat pretectum revealed by antibodies to the calcium-binding protein calbindin-D 28K.

The pretectum is an important center for visual reflexes. However, the location, boundaries, and connections of individual nuclei of the pretectum are incompletely understood. In cat, the traditionally defined nuclear boundaries have been placed in doubt by recent evidence showing that the retinal input to the pretectum forms four continuous projection zones that do not match the cytoarchitectural boundaries of individual pretectal nuclei defined by previous studies. We now show that antibodies to the calcium-binding protein calbindin-D 28K (CaBP) label clusters of neurons within the pretectum that match the zones of retinal termination. Four obvious cell clusters within the pretectum were labeled by CaBP antisera. Computer three-dimensional reconstruction of these cell clusters revealed that they form four distinct but continuous zones that run the rostrocaudal length of the pretectum in a medial-to-lateral direction. By combining anterograde HRP labeling of retinal terminals with CaBP immunocytochemistry, these CaBP-labeled cell clusters were found virtually to overlap the retinal projection zones. The CaBP-labeled neurons included both multipolar and fusiform morphologies, and most were medium- to large-sized cells. HRP retrograde transport studies showed that many CaBP-labeled neurons in the clusters projected to the LGN, while none projected to the inferior olive (IO). GABA-immunoreactive neurons were also found within the CaBP cell clusters, but these neurons were smaller than most CaBP-labeled neurons, and none were retrogradely labeled following HRP injections into the LGN or IO. Two-color antibody double-labeling experiments did not reveal any GABA neurons within the clusters that colocalized CaBP. In summary, calbindin is a precise marker of neuron clusters that overlap the retinal projection zones in the cat pretectum. Many of these CaBP neurons project to the LGN, and none contain GABA.

Animals↗

PubMed1988

Quantitative immunocytochemistry using an image analyzer. I. Hardware evaluation, image processing, and data analysis.

In this review we describe how video-based image analysis systems are used to measure immunocytochemically labeled tissue. The general principles underlying hardware and software procedures are emphasized. First, the characteristics of image analyzers are described, including the densitometric measure, spatial resolution, gray scale resolution, dynamic range, and acquisition and processing speed. The errors produced by these instruments are described and methods for correcting or reducing the errors are discussed. Methods for evaluating image analyzers are also presented, including spatial resolution, photometric transfer function, short- and long-term temporal variability, and measurement error. The procedures used to measure immunocytochemically labeled cells and fibers are then described. Immunoreactive profiles are imaged and enhanced using an edge sharpening operator and then extracted using segmentation, a procedure which captures all labeled profiles above a threshold gray level. Binary operators, including erosion and dilation, are applied to separate objects and to remove artifacts. The software then automatically measures the geometry and optical density of the extracted profiles. The procedures are rapid and efficient methods for measuring simultaneously the position, geometry, and labeling intensity of immunocytochemically labeled tissue, including cells, fibers, and whole fields. A companion paper describes non-biological standards we have developed to estimate antigen concentration from the optical density produced by antibody labeling (Nabors et al., 1988).

Animals↗

PubMed1988

Quantitative immunocytochemistry using an image analyzer. II. Concentration standards for transmitter immunocytochemistry.

Image analyzers can measure both the optical density and geometry of immunocytochemically labeled cells and fibers, as reviewed in a companion paper (Mize et al., 1988). In this paper, we report a procedure which allows us to estimate the concentration of a neurotransmitter based upon the optical density of antibody labeling produced by immunocytochemistry. To accomplish this, we developed a standard which binds conjugated neurotransmitters. Several artificial media for the standard were compared, including agar, gelatin, and agar-gelatin. A 3% agar matrix was found to be most suitable because it cut well and was nearly transparent. The agar sections were activated with cyanogen bromide/acetonitrile to promote coupling to the antigen. To test the standard, we used gamma-aminobutyric acid (GABA) conjugated to bovine serum albumin (BSA) as the antigen. The antibody was directed against this conjugate. Activated agar sections were incubated in serial dilutions of the tritium-labeled GABA/BSA conjugate. The radioactivity of some of these sections was measured to estimate the amount of coupled antigen. The remaining sections were incubated in the GABA antibody and processed for immunocytochemistry. The optical density of these sections was measured with an image analyzer. A linear relationship was found between GABA concentration and optical density over a range of at least 0.01 to 1 nmol/mg of agar. These results show that the concentration of bound GABA can be estimated from the optical density of sections labeled by antibody immunocytochemistry. The applicability of this technique to fixed brain tissue is discussed.

Image Processing, Computer-Assisted↗