Biomedical subjects

L Gan

Publications and source records attributed to L Gan.

At least 73 records · Page 4Linked to original sources

PubMed1999

POU domain factor Brn-3b is essential for retinal ganglion cell differentiation and survival but not for initial cell fate specification.

While the mammalian retina is well understood at the anatomical and physiological levels, little is known about the mechanisms that give rise to the retina's highly ordered pattern or its diverse neuronal cell types. Previous investigations have shown that gene disruption of the POU-IV class transcription factor Brn-3b (Brn-3.2) resulted in the loss of most retinal ganglion cells in retinas of postnatal mice. Here, we used lacZ and human placental alkaline phosphatase genes knocked into the brn-3b locus to follow the fate of brn-3b-mutant cells in the developing retina. We found that Brn-3b was not required for the initial commitment of retinal ganglion cell fate or for the migration of ganglion cells to the ganglion cell layer. However, Brn-3b was essential for the normal differentiation of retinal ganglion cells; without it, the cells underwent enhanced apoptosis. Retinal ganglion cells lacking brn-3b extended processes at the appropriate time in development, but these processes were disorganized, resulting in a thinner optic nerve. Explanted retinas from brn-3b-null embryos also extended processes when cultured in vitro, but the processes were shorter and less bundled than in wild-type retinas. Ultrastructural and marker analyses showed that the processes of mutant ganglion cells had dendritic rather than axonal features, suggesting that mutant cells formed dendrites in place of axons. These results suggest that Brn-3b regulates the activity of genes whose products play essential roles in the formation of retinal ganglion cell axons.

Alkaline Phosphatase↗

PubMed1999

A hypomorphic myogenin allele reveals distinct myogenin expression levels required for viability, skeletal muscle development, and sternum formation.

The myogenic basic helix-loop-helix transcription factor myogenin plays an essential role in the differentiation of skeletal muscle and, secondarily, in rib and sternum formation during mouse development. However, virtually nothing is known about the quantitative requirements for myogenin in these processes. Here, we describe the generation of mice carrying a hypomorphic allele of myogenin, which expresses myogenin transcripts at approximately one-fourth the level of the wild-type myogenin allele. The hypomorphic allele in combination with wild-type and myogenin-null alleles was used to create an allelic series. Embryos representing the complete range of genotypes from homozygous wild type to homozygous null were analyzed for their viability, ability to form normal ribs and sternum, and extent of skeletal muscle differentiation. Embryos carrying the hypomorphic myogenin allele over a wild-type allele were normal. In embryos bearing homozygous hypomorphic alleles, the sternum developed normally and extensive skeletal muscle differentiation occurred. However, muscle hypoplasia and reduced muscle-specific gene expression were apparent in these embryos, and the mice were not viable as neonates. When the hypomorphic allele was placed over a myogenin-null allele, the resulting embryos had sternum defects resembling homozygous myogenin-null embryos, and there was severe muscle hypoplasia. Our results demonstrate that skeletal muscle formation is highly sensitive to the absolute levels of myogenin and that correct sternum formation, skeletal muscle differentiation, and viability each require distinct threshold levels of myogenin.

Alleles↗

PubMed1999

Monitoring gene expression profile changes in ovarian carcinomas using cDNA microarray.

The development of cancer is the result of a series of molecular changes occurring in the cell. These events lead to changes in the expression level of numerous genes that result in different phenotypic characteristics of tumors. In this report we describe the assembly and utilization of a 5766 member cDNA microarray to study the differences in gene expression between normal and neoplastic human ovarian tissues. Several genes that may have biological relevance in the process of ovarian carcinogenesis have been identified through this approach. Analyzing the results of microarray hybridizations may provides new leads for tumor diagnosis and intervention.

Cloning, Molecular↗

PubMed1999

Molecular cloning and characterization of prostase, an androgen-regulated serine protease with prostate-restricted expression.

The identification of genes with selective expression in specific organs or cell types provides an entry point for understanding biological processes that occur uniquely within a particular tissue. Using a subtraction approach designed to identify genes preferentially expressed in specific tissues, we have identified prostase, a human serine protease with prostate-restricted expression. The prostase cDNA encodes a putative 254-aa polypeptide with a conserved serine protease catalytic triad and an amino-terminal pre-propeptide sequence, indicating a potential secretory function. The genomic sequence comprises five exons and four introns and contains multiple copies of a chromosome 19q-specific minisatellite repeat. Northern analysis indicates that prostase mRNA is expressed in hormonally responsive normal and neoplastic prostate epithelial tissues, but not in prostate stromal constituents. Prostase shares 35% amino acid identity with prostate-specific antigen (PSA) and 78% identity with the porcine enamel matrix serine proteinase 1, an enzyme involved in enamel matrix degradation and with a putative role in the disruption of intercellular junctions. Radiation-hybrid-panel mapping localized prostase to chromosome 19q13, a region containing several other serine proteases, including protease M, pancreatic/renal kallikrein hK1, and the prostate-specific kallikreins hK2 and hK3 (PSA). The sequence homology between prostase and other well-characterized serine proteases suggests several potential functional roles for the prostase protein that include the degradation of extracellular matrix and the activation of PSA and other proteases.

Amino Acid Sequence↗

PubMed1999

Cell type-specific expression of the Kv3.1 gene is mediated by a negative element in the 5' untranslated region of the Kv3.1 promoter.

The Kv3.1 potassium channel gene is restrictively expressed in the CNS, and its expression level is especially high in neurons that are able to follow synaptic inputs at high frequencies. To understand the transcriptional mechanisms controlling Kv3.1 expression, we have conducted a functional analysis of the Kv3.1 promoter in various cell lines of different tissue origins and in transgenic mice. Our results suggest that an upstream regulatory fragment coupled with the 5' untranslated region (UTR) is able to confer tissue-specific expression in both cell lines and in transgenic mice. Deletion analysis of the regulatory region carried out in cell lines reveals that a strong negatively acting element, uniquely residing in the 5' UTR (+350 to +158), appears able to confer cell type specificity on both the Kv3.1 promoter and the thymidine kinase promoter in transient transfection assays. A weak cell type-specific enhancer in the proximal region of the promoter (-123 to -71) also contributes to cell type-specific expression of the Kv3.1 gene.

3T3 Cells↗

PubMed1999

In vitro evidence for both the nucleus and cytoplasm as subcellular sites of pathogenesis in Huntington's disease.

A unifying feature of the CAG expansion diseases is the formation of intracellular aggregates composed of the mutant polyglutamine-expanded protein. Despite the presence of aggregates in affected patients, the precise relationship between aggregates and disease pathogenesis is unresolved. Results from in vivo and in vitro studies of mutant huntingtin have lead to the hypothesis that nuclear localization of aggregates is critical for the pathology of Huntington's disease (HD). We tested this hypothesis using a 293T cell culture model system that compared the frequency and toxicity of cytoplasmic and nuclear huntingtin aggregates. We first assessed the mode of nuclear transport of N-terminal fragments of huntingtin, and show that the predicted endogenous NLS is not functional, providing data in support of passive nuclear transport. This result suggests that proteolysis is a necessary step for nuclear entry of huntingtin. Additionally, insertion of nuclear import or export sequences into huntingtin fragments containing 548 or 151 amino acids was used to reverse the normal localization of these proteins. Changing the subcellular localization of the fragments did not influence their total aggregate frequency. There were also no significant differences in toxicity associated with the presence of nuclear compared with cytoplasmic aggregates. The findings of nuclear and cytoplasmic aggregates in affected brains, together with these in vitro data, support the nucleus and cytosol as subcellular sites for pathogenesis in HD.

Amino Acid Sequence↗

PubMed1999

Effect of leukocytes on corneal cellular proliferation and wound healing.

PURPOSE: To establish whether fucoidin, by blocking the adhesion of leukocytes on the limbal vascular endothelium, prevents extravasation of the cells from the blood stream into the limbal stroma and the wounded area after corneal injury. Successful leukocyte blocking enabled investigation of the influence of leukocytes on corneal cellular proliferation after corneal wounding. METHODS: Thirty-two New Zealand White rabbits were used. Photorefractive keratectomy (PRK) and a standardized alkali corneal wound were used as models in two sets of experiments. In half of the injured rabbits fucoidin was used to prevent leukocytes from leaving the local vessels. The efficiency of the blocking technique was evaluated by counting the number of leukocytes in the limbal and wounded corneal areas. Proliferating cell nuclear antigen (PCNA) was used as a marker for proliferative activity. RESULTS: The infiltration of leukocytes into the limbus and the cornea after PRK and alkali injuries can be blocked by fucoidin. The healing rate of corneal epithelium after alkali burn was retarded in the absence of leukocytes. PCNA expression was enhanced in the presence of leukocytes. Fucoidin per se had no influence on corneal cell proliferation and wound healing. CONCLUSIONS: Polymorphonuclear leukocytes (PMNs) can be prevented from entering the cornea in vivo by fucoidin after PRK and after alkali burn. The corneal epithelial healing rate is delayed in the absence of PMNs in vivo, and PCNA expression increases in the presence of leukocytes.

Administration, Topical↗

PubMed1998

Expression of a gene trap reporter construct in a subset of cells in embryonic sites of hematopoiesis: evidence for alternative rRNA production in hematopoietic cells.

Three mouse lines were generated from independent gene trap events in embryonic stem cells. These lines express a betageo reporter gene in a subset of cells at sites of embryonic hematopoiesis. The 5' breakpoints of all three lines were found to lie in 45S ribosomal RNA transcription units. Expression was apparently linked to metabolic activity in these cells, since the kinetics of expression during embryogenesis matched that of cycling cells with colony forming unit spleen (CFU-S) potential. Expression was not seen in adult tissues unless the animals were treated with hydroxyurea, inducing synchronous entry of quiescent CFU-S into the cell cycle. Our results suggest that there is a subset of hematopoietic stem cells, which when actively proliferating, express the SAbetageo reporter construct from RNA polymerase I transcription units.

Animals↗

PubMed1998

New cyclooxygenase-2/5-lipoxygenase inhibitors. 3. 7-tert-butyl-2, 3-dihydro-3,3-dimethylbenzofuran derivatives as gastrointestinal safe antiinflammatory and analgesic agents: variations at the 5 position.

We report an expansion of the scope of our initial discovery that 5-keto-substituted 7-tert-butyl-2,3-dihydro-3,3-dimethylbenzofurans (DHDMBFs) are antiinflammatory and analgesic agents. Several other functional groups have been introduced at the 5 position: amides, amidines, ureas, guanidines, amines, heterocycles, heteroaromatics, and heteroaryl ethenyl substituents in the 5 position all provide active compounds. These compounds are dual cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) inhibitors. They inhibit both COX-1 and COX-2 with up to 33-fold selectivity for COX-2.

Analgesics↗

PubMed1998

Langmuir-Blodgett Film Formation of a Fullerene Dicarboxylic Acid Derivative C60(HOOCCHNHCHCOOH) and its Photocurrent Generation.

The Langmuir films of a fullerene dicarboxylic acid derivative I, C60(HOOCCHNH2CHCOOH)CF3SO3H (FDA), at the air/water interface have been investigated. FDA formed a stable monolayer on pure water when dilute solution (1.04 x 10(-5) mol/L) was used as the spreading solution. The monolayer formation on various subphases was investigated, including solutions containing different monovalent (Na+, Ag+) and divalent (Ca2+, Cd2+, Cu2+) cations. The introduction of these cations makes the monolayer more stable. The limiting molecular area is also dependent on the pH of the subphase. The surface pressure versus area isotherms at different temperature clearly indicate that FDA molecules easily aggregate at high temperature. The monolayer and multilayer LB films of FDA were deposited on quartz and glass substrates from Ca2+, Cd2+, Cu2+, or Cu2+/Phen solution subphase, and their UV-vis and XPS spectra were obtained. The data showed that for Cd2+ and Cu2+/Phen subphase the cation can be transferred onto the substrates together with FDA but not the other two. The photoelectrochemical behavior of FDA on an ITO electrode was investigated, which showed that the photocurrent is cathodic. Oxygen and negative bias are beneficial factors for the photocurrent generation. The external quantum yield is ca. 4%. Copyright 1998 Academic Press.

Journal Article↗

PubMed1998

Expression and functional analysis of mouse EXT1, a homolog of the human multiple exostoses type 1 gene.

Hereditary multiple exostoses (EXT) is a genetically heterogeneous, autosomal dominant skeletal disorder. The gene for EXT1 maps to human chromosome 8q24.1 and encodes an evolutionary conserved protein that is a member of a multigene family. The mouse homolog of human EXT1 protein is 99% similar to its human counterpart. Here, we present the expression profiles of the mouse EXT1 gene. EXT1 mRNA is initially expressed at 6.5 days post-coitum (d.p.c.), which coincides with gastrulation of the mouse embryo. Whole mount in situ hybridization with 10.5 to 12.5 d.p.c. mouse embryos showed a high level of expression of EXT1 mRNA in developing limb buds. Epitope tagging experiments revealed the endoplasmic reticulum localization of EXT1 protein. This localization was consistent with a hydrophobic stretch of amino acids present at the N-terminal end of the EXT1 protein. These results provide novel information on the function of EXT1 and the etiology of hereditary multiple exostoses.

3T3 Cells↗

PubMed1998

lunatic fringe is an essential mediator of somite segmentation and patterning.

The gene lunatic fringe encodes a secreted factor with significant sequence similarity to the Drosophila gene fringe. fringe has been proposed to function as a boundary-specific signalling molecule in the wing imaginal disc, where it is required to localize signalling activity by the protein Notch to the presumptive wing margin. By targeted disruption in mouse embryos, we show here that lunatic fringe is likewise required for boundary formation. lunatic fringe mutants fail to form boundaries between individual somites, the initial segmental unit of the vertebrate trunk. In addition, the normal alternating rostral-caudal pattern of the somitic mesoderm is disrupted, suggesting that intersomitic boundary formation and rostral-caudal patterning of somites are mechanistically linked by a process that requires lunatic fringe activity. As a result, the derivatives of the somitic mesoderm, especially the axial skeleton, are severely disorganized in lunatic fringe mutants. Taken together, our results demonstrate an essential function for a vertebrate fringe homologue and suggest a model in which lunatic fringe modulates Notch signalling in the segmental plate to regulate somitogenesis and rostral-caudal patterning of somites simultaneously.

Animals↗

PubMed1998

Cloning and comparative sequence analysis of TP53 in Xiphophorus fish hybrid melanoma models.

We have cloned and sequenced the p53-encoding cDNA of green swordtail (X. helleri) and southern platyfish (X. maculatus). These two fish species are often used to produce hybrids that develop melanomas after genetic crossing. Computer translation of derived cDNA sequences revealed that p53 polypeptides from these two species are virtually identical, exhibiting only two conservative amino acid substitutions. TP53 mRNA expression was detected in virtually all tissues tested. Comparison of these fish p53 polypeptide sequences with those of other vertebrates, including other fishes, amphibians, and mammals, revealed that conservation is especially high in several previously defined protein domains. In addition, sequencing of the 3' TP53 genomic region of X. maculatus reveals similarity to the human TP53 locus in overall organization. Knowledge of the Xiphophorus TP53 sequences will allow assessment of mutational alterations within tumors generated from numerous fish genetic crosses.

Amino Acid Sequence↗

PubMed1998

Contribution of the Kv3.1 potassium channel to high-frequency firing in mouse auditory neurones.

1. Using a combination of patch-clamp, in situ hybridization and computer simulation techniques, we have analysed the contribution of potassium channels to the ability of a subset of mouse auditory neurones to fire at high frequencies. 2. Voltage-clamp recordings from the principal neurones of the medial nucleus of the trapezoid body (MNTB) revealed a low-threshold dendrotoxin (DTX)-sensitive current (ILT) and a high-threshold DTX-insensitive current (IHT). 3. IHT displayed rapid activation and deactivation kinetics, and was selectively blocked by a low concentration of tetraethylammonium (TEA; 1 mM). 4. The physiological and pharmacological properties of IHT very closely matched those of the Shaw family potassium channel Kv3.1 stably expressed in a CHO cell line. 5. An mRNA probe corresponding to the C-terminus of the Kv3.1 channel strongly labelled MNTB neurones, suggesting that this channel is expressed in these neurones. 6. TEA did not alter the ability of MNTB neurones to follow stimulation up to 200 Hz, but specifically reduced their ability to follow higher frequency impulses. 7. A computer simulation, using a model cell in which an outward current with the kinetics and voltage dependence of the Kv3.1 channel was incorporated, also confirmed that the Kv3.1- like current is essential for cells to respond to a sustained train of high-frequency stimuli. 8. We conclude that in mouse MNTB neurones the Kv3.1 channel contributes to the ability of these cells to lock their firing to high-frequency inputs.

Animals↗

PubMed1998

New cyclooxygenase-2/5-lipoxygenase inhibitors. 1. 7-tert-buty1-2,3-dihydro-3,3-dimethylbenzofuran derivatives as gastrointestinal safe antiinflammatory and analgesic agents: discovery and variation of the 5-keto substituent.

A series of 5-keto-substituted 7-tert-buty1-2,3-dihydro-3,3- dimethylbenzofurans (DHDMBFs) were prepared and evaluated as potential nonsteroidal antiinflammatory and analgesic agents. Interest in this class of compounds arose when a DHDMBF was found to be an active metabolite of the di-tert-butylphenol antiinflammatory agent tebufelone. We have now found that a variety of 5-keto-substituted DHDMBFs have good in vivo antiinflammatory and analgesic activity after oral administration. These compounds inhibit both cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) in vitro. The cyclooxygenase inhibition was found to be selective for the cyclooxygenase-2 isoform, and this combination of COX-2/5-LOX inhibition may be responsible for the gastrointestinal safety of compounds such as 30.

Animals↗

PubMed1998

New cyclooxygenase-2/5-lipoxygenase inhibitors. 2. 7-tert-butyl-2,3-dihydro-3,3-dimethylbenzofuran derivatives as gastrointestinal safe antiinflammatory and analgesic agents: variations of the dihydrobenzofuran ring.

A series of 5-keto-substituted 7-tert-buty1-2,3-dihydro-3,3- dimethylbenzofurans (DHDMBFs) were found to be nonsteroidal antiinflammatory and analgesic agents. These compounds are inhibitors of 5-lipoxygenase (5-LOX) and cyclooxygenase (COX) with selectivity for the COX-2 isoform. A series of analogues were prepared to investigate the scope of this lead. Five ketone side chains from active DHDMBFs were used to investigate the effects of changes in the DHDMBF "core": the size and identity of the heterocycle and the substituent requirements of the heterocycle and phenyl ring. Biological testing showed that a variety of structural changes can be accommodated, but no structure was clearly superior to the DHDMBF structure.

Analgesics↗

PubMed1998

Activation of Kv3.1 channels in neuronal spine-like structures may induce local potassium ion depletion.

Spines are specialized neuronal membrane structures, often localized at sites where synaptic information is relayed from one cell to another in the central nervous system. By electron immunomicroscopy we have found that the mammalian Shaw family potassium channel Kv3.1 is localized on spine-like protrusions, adjacent to postsynaptic membranes of bushy cells in the cochlear nucleus. As direct characterization of the electrophysiological behavior of ion channels in such structures is difficult, we have used Kv3. 1-transfected CHO cells to create artificial spine-like membrane compartments. Membrane patches were sucked into microelectrodes to form small, cell-attached vesicles with dimensions comparable to those of the neuronal structures. Currents mediated by the Kv3.1 channel in these vesicles undergo rapid and complete inactivation, in contrast to their noninactivating behavior in whole-cell recordings. This apparent inactivation is caused by the rapid depletion of K+ from the vesicle and the slow refilling of K+ into the vesicle compartment from the bulk cytoplasm. Our data provide evidence that compartmentalized ionic transients can be generated in spine-like membrane structures and support the view that the localization of ion channels in spine-like structures may influence responses to synaptic stimulation.

Animals↗

PubMed1998

Hevin, an antiadhesive extracellular matrix protein, is down-regulated in metastatic prostate adenocarcinoma.

Hevin, a gene closely related to the extracellular matrix protein SPARC, is an acidic cysteine-rich glycoprotein shown to be important for the adhesion and trafficking of cells through the endothelium. Through the use of differential display and differential EST analysis, we identified Hevin as a gene whose transcription is down-regulated in transformed prostate epithelial cell lines and metastatic prostate adenocarcinoma. These results were confirmed by comparing expression levels between normal and neoplastic human prostate tissues using Northern analysis. In situ hybridization with an 35S-labeled antisense riboprobe demonstrated the loss of Hevin expression in metastatic prostate carcinoma. The expression pattern of Hevin in transformed and metastatic epithelium may provide further insights into the complex cell adhesion events involved in the metastatic progression of prostate carcinoma.

Adenocarcinoma↗