Biomedical subjects

L Gan

Publications and source records attributed to L Gan.

At least 19 recordsLinked to original sources

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↗

PubMed1997

Essential role of POU-domain factor Brn-3c in auditory and vestibular hair cell development.

The Brn-3 subfamily of POU-domain transcription factor genes consists of three highly homologous members-Brn-3a, Brn-3b, and Brn-3c-that are expressed in sensory neurons and in a small number of brainstem nuclei. This paper describes the role of Brn-3c in auditory and vestibular system development. In the inner ear, the Brn-3c protein is found only in auditory and vestibular hair cells, and the Brn-3a and Brn-3b proteins are found only in subsets of spiral and vestibular ganglion neurons. Mice carrying a targeted deletion of the Brn-3c gene are deaf and have impaired balance. These defects reflect a complete loss of auditory and vestibular hair cells during the late embryonic and early postnatal period and a secondary loss of spiral and vestibular ganglion neurons. Together with earlier work demonstrating a loss of trigeminal ganglion neurons and retinal ganglion cells in mice carrying targeted disruptions in the Brn-3a and Brn-3b genes, respectively, the Brn-3c phenotype reported here demonstrates that each of the Brn-3 genes plays distinctive roles in the somatosensory, visual, and auditory/vestibular systems.

Animals↗

PubMed1997

The molecular evolution of the vertebrate trypsinogens.

We expand the already large number of known trypsinogen nucleotide and amino acid sequences by presenting additional trypsinogen sequences from the tunicate (Boltenia villosa), the lamprey (Petromyzon marinus), the pufferfish (Fugu rubripes), and the frog (Xenopus laevis). The current array of known trypsinogen sequences now spans the entire vertebrate phylogeny. Phylogenetic analysis is made difficult by the presence of multiple isozymes within species and rates of evolution that vary highly between both species and isozymes. We nevertheless present a Fitch-Margoliash phylogeny constructed from pairwise distances. We employ this phylogeny as a vehicle for speculation on the evolution of the trypsinogen gene family as well as the general modes of evolution of multigene families. Unique attributes of the lamprey and tunicate trypsinogens are noted.

Amino Acid Sequence↗

PubMed1997

A highly conserved apoptotic suppressor gene is located near the chicken T-cell receptor alpha chain constant region.

A cosmid clone containing the chicken T-cell receptor alpha chain constant region (TCRAC) was sequenced. The cosmid contains the TCRAC gene, six putative joining gene segments (TCRAJ), and surprisingly, a chicken homologue for the human apoptotic suppressor gene, defender against cell death (DAD1). The DAD1 gene is 6.3 kilobases downstream of the TCRAC gene and has an inverted transcription orientation with respect to the TCRAC gene. The cDNA for the chicken DAD1 gene is 597 base pairs in length and encodes a highly conserved hydrophobic protein. The proximal location of DAD1 to the TCRAC locus has also been confirmed in both humans and mouse. The location of the DAD1 gene suggests that DAD1 may play an important role in T-cell related apoptotic activities.

Amino Acid Sequence↗

PubMed1996

Targeted deletion of the mouse POU domain gene Brn-3a causes selective loss of neurons in the brainstem and trigeminal ganglion, uncoordinated limb movement, and impaired suckling.

The Brn-3 subfamily of POU domain genes are expressed in sensory neurons and in select brainstem nuclei. Earlier work has shown that targeted deletion of the Brn-3b and Brn-3c genes produce, respectively, defects in the retina and in the inner ear. We show herein that targeted deletion of the Brn-3a gene results in defective suckling and in uncoordinated limb and trunk movements, leading to early postnatal death. Brn-3a (-/-) mice show a loss of neurons in the trigeminal ganglia, the medial habenula, the red nucleus, and the caudal region of the inferior olivary nucleus but not in the retina and dorsal root ganglia. In the trigeminal and dorsal root ganglia, but not in the retina, there is a marked decrease in the frequency of neurons expressing Brn-3b and Brn-3c, suggesting that Brn-3a positively regulates Brn-3b and Brn-3c expression in somatosensory neurons. Thus, Brn-3a exerts its major developmental effects in somatosensory neurons and in brainstem nuclei involved in motor control. The pheno-types of Brn-3a, Brn-3b, and Brn-3c mutant mice indicate that individual Brn-3 genes have evolved to control development in the auditory, visual, or somatosensory systems and that despite differences between these systems in transduction mechanisms, sensory organ structures, and central information processing, there may be fundamental homologies in the genetic regulatory events that control their development.

Animals↗

PubMed1996

POU domain factor Brn-3b is required for the development of a large set of retinal ganglion cells.

The three members of the Brn-3 family of POU domain transcription factors are found in highly restricted sets of central nervous system neurons. Within the retina, these factors are present only within subsets of ganglion cells. We show here that in the developing mouse retina, Brn-3b protein is first observed in presumptive ganglion cell precursors as they begin to migrate from the zone of dividing neuroblasts to the future ganglion cell layer, and that targeted disruption of the Brn-3b gene leads in the homozygous state to a selective loss of 70% of retinal ganglion cells. In Brn-3b (-/-) mice other neurons within the retina and brain are minimally or not at all affected. These experiments indicate that Brn-3b plays an essential role in the development of specific ganglion cell types.

Animals↗

PubMed1996

Cloning and characterization of the promoter for a potassium channel expressed in high frequency firing neurons.

The Kv3.1 potassium channel is expressed in neurons that generate trains of high frequency action potentials in response to synaptic inputs. To understand the mechanisms underlying the regulation and restricted expression pattern of the Kv3.1 gene, we have cloned and characterized its promoter. We first isolated a 5.3-kilobase pair fragment of the Kv3.1 5'-flanking region. When linked to the chloramphenicol acetyltransferase reporter gene, this fragment was found to be active in the undifferentiated PC12 cell line, a neuron-like cell line, but not in a fibroblast cell line. By carrying out a series of deletion analyses in undifferentiated PC12 cells, we have localized the essential promoter region to a highly GC-rich region containing four Sp-1 binding sites. Similar deletion analysis in NIH3T3 cells suggests that multiple silencing elements and enhancing element(s) are involved in the cell type-specific expression of this gene. Further regulatory elements, including one cyclic AMP/calcium response element (CRE) and one Ap-1 element were found in the upstream region of the promoter. Using a stable undifferentiated PC12 cell line transfected with the Kv3.1 5'-flanking region, we determined that promoter activity is enhanced by a cAMP analog and a calcium ionophore. Deletion of the CRE-like element at position -252 eliminated the enhancement of promoter activity by cAMP, and mobility shift assays confirmed that the Kv3.1 CRE sequence binds both a nuclear factor in undifferentiated PC12 cells and recombinant CRE binding protein. Our results suggest that the transcription of the Kv3.1 channel may be regulated by neurotransmitters that elevate cAMP levels in neurons.

3T3 Cells↗

PubMed1996

Altering cell fates in sea urchin embryos by overexpressing SpOtx, an orthodenticle-related protein.

While many general features of cell fate specification in the sea urchin embryo are understood, specific factors associated with these events remain unidentified. SpOtx, an orthodenticle-related protein, has been implicated as a transcriptional activator of the aboral ectoderm-specific Spec2a gene. Here, we present evidence that SpOtx has the potential to alter cell fates. SpOtx was found in the cytoplasm of early cleavage stage embryos and was translocated into nuclei between the 60- and 120-cell stage, coincident with Spec gene activation. Eggs injected with SpOtx mRNA developed into epithelial balls of aboral ectoderm suggesting that SpOtx redirected nonaboral ectoderm cells to an aboral ectoderm fate. At least three distinct domains on SpOtx, the homeobox and regions in the N-terminal and C-terminal halves of the protein, were required for the morphological alterations. These same N-terminal and C-terminal regions were shown to be transactivation domains in a yeast transactivation assay, indicating that the biological effects of overexpressing SpOtx were due to its action as a transcription factor. Our results suggest that SpOtx is involved in aboral ectoderm differentiation by activating aboral ectoderm-specific genes and that modulating its expression can lead to changes in cell fate.

Animals↗

PubMed1995

Isolation and characterization of the chicken trypsinogen gene family.

Based on genomic Southern hybridizations and cDNA sequence analyses, the chicken trypsinogen gene family can be divided into two multi-member subfamilies, a six-member trypsinogen I subfamily which encodes the cationic trypsin isoenzymes and a three-member trypsinogen II subfamily which encodes the anionic trypsin isoenzymes. The chicken cDNA and genomic clones containing these two subfamilies were isolated and characterized by DNA sequence analysis. The results indicated that the chicken trypsinogen genes encoded a signal peptide of 15 to 16 amino acid residues, an activation peptide of 9 to 10 residues and a trypsin of 223 amino acid residues. The chicken trypsinogens contain all the common catalytic and structural features for trypsins, including the catalytic triad His, Asp and Ser and the six disulphide bonds. The trypsinogen I and II subfamilies share approximately 70% sequence identity at the nucleotide and amino acid level. The sequence comparison among chicken trypsinogen subfamily members and trypsin sequences from other species suggested that the chicken trypsinogen genes may have evolved in coincidental or concerted fashion.

Amino Acid Sequence↗

PubMed1995

A microtiter plate-based high-throughput DNA purification method.

A fast, reliable, inexpensive, and high-throughput method to purify DNA has been developed. It is based on DNA amplification by polymerase chain reaction utilizing a mini spin-column made from a 96-well membrane-bottomed assay plate. With this method, 50% of the DNA is recovered routinely using Sephacryl-500HR as filtration media. Purified DNA can then be used in various enzymatic manipulations, such as sequencing and hybridization. This provides an economical alternative protocol for routine large-scale purification of DNA templates for sequencing or other enzymatic manipulations.

Base Sequence↗

PubMed1995

An orthodenticle-related protein from Strongylocentrotus purpuratus.

Orthodenticle-related proteins function as regulators of head formation and other developmental events in flies and mice. Here, we characterize a cDNA clone encoding an orthodenticle-related protein from the sea urchin Strongylocentrotus purpuratus. The cDNA, termed SpOtx, has a highly conserved orthodenticle homeobox but otherwise diverges in sequence from its fly and mouse counterparts. Orthodenticle-related proteins bind with high affinity to DNA containing the sequence motif TAATCC/T. The S. purpuratus aboral ectoderm-specific Spec2a gene has several TAATCC/T sites in its control region, and we provide evidence, using bandshift analysis, that Spec2a may be target gene for SpOtx. Two SpOtx transcripts accumulate during embryogenesis, an early transcript whose level peaks at blastula stage and a late transcript accumulating to highest concentrations at gastrula stage. SpOtx transcripts were found initially in all cells of the cleaving embryo, but they gradually became restricted to oral ectoderm and endoderm cells. In contrast, SpOtx protein was found in nuclei of all cells at both blastula and pluteus stages. Our results suggest that SpOtx plays a role in the activation of the Spec2a gene and most likely has additional functions in the developing sea urchin embryo.

Amino Acid Sequence↗

PubMed1995

Proliferating cell nuclear antigen: contradictory results regarding its presence in the lens.

BACKGROUND: Cell division of the normal lens is believed to occur in the germinative zone of the lens epithelium only. The expression of proliferating cell nuclear antigen (PCNA) at least in its insoluble form, is restricted to cells shortly after and during DNA synthesis. METHOD: To elucidate the location of the previously reported notable amounts of PCNA in lens epithelium, 6-micron sections of 10 New Zealand rabbit corneas were stained with specific antibodies. RESULTS: No PCNA-positive cells were detected in the lens epithelium, neither in the central-zone nor in the germinative zone. CONCLUSION: As the mitotic activity of lens epithelium is physiologically low (below 0.03% in adult rabbits), the likelihood of detecting mitotic figures in histologic sections is very limited. We hence conclude that PCNA may be expressed in the epithelia of the lens but is only occasionally visible in histological sections. Our most recent studies have confirmed this. The fixation process and the use of specific antibodies are decisive factors that may, when used correctly, allow specific localization PCNA that is involved in cell replication of lens epithelium. However, it has to be pointed out that the mitotic index of lens epithelium is age- and species-related, and hence so is the expression of PCNA, which makes comparison with other studies more difficult.

Animals↗

PubMed1995

Proliferating cell nuclear antigen colocalization with corneal epithelial stem cells and involvement in physiological cell turnover.

Corneal integrity is dependent on a constant turnover of epithelial cells. According to the current hypothesis essential contributors to this process, the so-called stem-cells, are localized in the limbal area of the cornea. However, histological identification of stem cells of the cornea is thus far not possible. In the present study we have used specific antibodies against proliferating cell nuclear antigen (PCNA). The nuclear protein is expressed in cells during replication. Prior to establishment of staining indices the optimal fixation procedure was found to be of major importance for the results and standardized for fixation in 4% formaldehyde. This procedure also gave the best preservation of the morphology. In the corneas of ten New Zealand rabbits the staining index for PCNA was found to be on average 23% (SD +/- 13%) for the basal layer of the limbal epithelium. In the mid-peripheral and central corneal epithelium only occasional staining of PCNA was detected with a 2% staining index (SD +/- 2%). The agglomeration of PCNA-positive cells in the basal layer of the limbal epithelium together with their colocalization with the known location of stem cells strongly suggests that PCNA could serve as a reliable indicator for the proximity of proliferating corneal epithelial stem cells in histological sections, which would be of significant clinical importance.

Animals↗

PubMed1995

Electrophysiological and pharmacological characterization of a mammalian Shaw channel expressed in NIH 3T3 fibroblasts.

1. The Shaw-like voltage-activated potassium channel Kv3.1 is expressed in neurons that generate rapid trains of action potentials. By expressing this channel in a mammalian cell line and by simulating its activation, we tested the potential role of this channel in action potential repolarization. 2. NIH 3T3 fibroblasts were stably transfected with Kv3.1 DNA. Currents recorded in these cells had a threshold of activation at approximately -10 mV, showed little inactivation, and were very sensitive to blockade by 4-aminopyridine and tetraethylammonium. 3. Kv3.1 currents activated rapidly at the onset of depolarizing voltage pulses. After an initial rapid phase of activation, which could be fit by an n4 Hodgkin-Huxley model, Kv3.1 currents expressed in fibroblasts had a second, slower phase of activation, and, in some cells, a slower phase of partial inactivation, both of which could be fit with modified n4p models. 4. Cell-attached single-channel recordings indicated that the Kv3.1 channel displays two gating behaviors, a short-open-time pattern, which occurs only at the onset of depolarization, and a long-open-time pattern, which predominates during prolonged depolarizations. 5. The amplitude of Kv3.1 currents, and the probability of channel openings, was reduced by a phorbol ester activator of protein kinase C, and the action of this agent was blocked by preincubation with the protein kinase inhibitor H7 (1-[5-isoquinolinesulfonyl]-2-methyl piperazine). In contrast, the effects of dioctanoyl glycerol, which also attenuated the currents, could not be completely blocked by H7, suggesting that diacylglycerols may act on the channel by a kinase-independent pathway. 6. Incorporation of a current with the kinetics and voltage dependence of Kv3.1 currents into a model cell with a sustained inward current showed that, in contrast to other delayed-rectifier currents such as the Shaker-like Kv1.1 and Kv1.6 channels, the level of expression of Kv3.1 currents could be varied over a wide range without attenuation of action potential height. Our results suggest that the Kv3.1 channel may provide rapidly firing neurons with a high safety factor for impulse propagation.

Action Potentials↗

PubMed1994

Multiple Otx binding sites required for expression of the Strongylocentrotus purpuratus Spec2a gene.

The control region of the aboral ectoderm-specific Spec2a gene of Strongylocentrotus purpuratus contains a 188-bp enhancer element, the RSR enhancer, required for temporal activation and aboral ectoderm/mesenchyme cell expression. Within the enhancer is a positive cis-regulatory element with the core consensus sequence TAATCC, which is capable of binding the sea urchin orthodenticle-related homeobox protein SpOtx. In this report, we extend our analysis of the RSR enhancer by dissecting it into smaller pieces and testing these pieces in an enhancer activation assay. The 188-bp enhancer region could not be divided without partial loss of activity, and two of the three pieces tested exhibited some activity. Using site-directed mutagenesis, we showed that three Otx consensus binding sites were responsible for the activity of the enhancer, acting in a non-cooperative manner to yield full activity. Mutagenizing the three Otx sites and a fourth one just upstream abolished all activity in the context of the complete Spec2a control region. Bandshift analysis revealed that the Otx sites were able to bind SpOtx, suggesting that this transcription factor mediates positive control at these sites. Non-SpOtx binding sites overlapping two of the Otx sites may also play a role in Spec2a expression. Using a lacZ reporter gene, we showed that a 76-bp DNA fragment containing two of the Otx sites was sufficient for aboral ectoderm/mesenchyme cell expression. These results suggest that the RSR enhancer plus an upstream DNA element required for mesenchyme cell repression are necessary and sufficient for the proper temporal activation and aboral ectoderm expression of the Spec2a gene and that the Otx elements play a positive role in this process.

Animals↗

PubMed1993

A positive cis-regulatory element with a bicoid target site lies within the sea urchin Spec2a enhancer.

Activation of the aboral ectoderm-specific Spec2a gene in blastula-stage sea urchin embryos requires an upstream regulatory region that is part of a repetitive sequence element (RSR) associated with all Spec1/Spec2 genes. Deletions from the 5' end of Spec2a-flanking DNA, monitored for activity using the sea urchin embryo gene-transfer expression system, indicated that this regulatory region has multiple DNA elements and that no positive element lies upstream of the RSR. We mapped the regulatory region using Spec2a fragments containing RSR sequences fused to an SV40 minimal promoter. The region between base pairs -443 and -631, defined as the RSR enhancer, was essential for maximal activity and conferred preferential aboral ectoderm expression to a lacZ reporter gene. Expression was not fully restricted to aboral ectoderm, however, suggesting that negative spatial elements are also associated with the proper activation of Spec2a. DNaseI footprinting and band-shift analysis of the RSR enhancer identified an A/T-rich DNA element, the A/T palindrome. This element binds a single 45-kDa nuclear protein, the A/T palindrome binding protein (A/TBP), whose specificity suggests a possible relationship with the bicoid-class homeodomain proteins. Mutations of the A/T palindrome are incapable of binding the 45-kDa protein and lower promoter activity by eight-fold. DNA-binding activity for A/TBP is low in unfertilized eggs, increases by the 16-cell stage and continues rising in blastulae. These data suggest that A/TBP plays a major role in the activation of the Spec2a gene in aboral ectoderm cells.

Animals↗

PubMed1993

Transfection of human mesothelial cells mediated by different asbestos fiber types.

Several different asbestos fiber types mediate transfection of human mesothelial cells by exogenous DNA. We have employed the human MeT-5A mesothelial cell line, which allows the use of DNA replication as an assay for entry of DNA when plasmids bearing the SV40 origin of replication are used for transfection. We find that Canadian chrysotile, Calidria chrysotile, amosite, and crocidolite are each capable of introducing plasmid pSVod DNA into MeT-5A cells followed by subsequent replication of a fraction of the plasmid DNA. A significant fraction of the input plasmid DNA associated with the cells in the presence of asbestos is fragmented, and this fragmentation is particularly evident with crocidolite. Each of the fiber types is highly cytotoxic for the MeT-5A cells, and these cells actively accumulate the added fibers from the surrounding environment as visualized by phase-contrast microscopy. MeT-5A cells were transfected at higher efficiency with calcium phosphate than were several other primate cell lines. Calcium phosphate, however, did not induce fragmentation of the input plasmid DNA. Compared with several different mineral agents, including glass fibers, kaolin, and talc, Calidria chrysotile fibers were most effective at mediating transfection of the MeT-5A cells. Results provide a mechanism by which transfection can contribute to mutagenicity of asbestos fibers and indicate that this mechanism can operate in human mesothelial cells.

Asbestos↗