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Biomedical subjects

L Gan

Publications and source records attributed to L Gan.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1993

Developmental potential of muscle cell progenitors and the myogenic factor SUM-1 in the sea urchin embryo.

During sea urchin development, esophageal muscle arises from secondary mesenchyme cells, descendants of the vegetal plate that delaminate from the coelomic epithelium at the end of gastrulation. In lithium-induced exogastrulae, where vegetal plate descendants evert rather than invaginate, myogenesis occurs normally, indicating that myocyte progenitors do not have to be near the future stomodeum for differentiation to occur. Vegetal plate descendants isolated along with the extracellular matrix at different times during gastrulation produce differentiated myocytes in culture as monitored by staining with a myosin heavy chain antibody. Vegetal isolates prepared at mid-gastrulation or later consistently produce differentiated myocytes whose form and position resembled their counterparts in the intact embryo, whereas vegetal isolates prepared a few hours earlier while capable of gut differentiation, as evidenced by the de novo synthesis of the endodermal surface marker Endo 1, did not produce differentiated myocytes. These results suggest that sometime after early gastrulation, a subset of secondary mesenchyme cells are competent to differentiate into muscle cells. RNase protection assays showed that the accumulation of sea urchin myogenic factor (SUM-1) mRNA is likely to be coincident with the earliest demonstrable commitment of myogenic precursors. Premature expression of SUM-1 coding sequences in mesenchyme blastulae resulted in the activation of muscle-specific enhancer elements, demonstrating that SUM-1 can function precociously in the early embryo. However, SUM-1 expressed in this manner did not activate the endogenous MHC gene, nor induce premature or ectopic production of muscle cells.

Amino Acid Sequence↗
PubMed1991

Sea urchin USF: a helix-loop-helix protein active in embryonic ectoderm cells.

We previously characterized a DNA-binding factor in nuclear extracts of Strongylocentrotus purpuratus embryos that bound Spec gene promoters, was ectoderm specific, and had properties similar to the vertebrate transcription factor USF. Here we describe a cDNA clone, suUSF, isolated from an S. purpuratus cDNA library, with sequence homology to human USF. Spec gene promoter fragments formed sequence-specific complexes with suUSF, and antibodies against suUSF inhibited binding activity in nuclear extracts. Reaction of USF-site containing probes with filter-bound nuclear proteins demonstrated that suUSF binding activity was enriched in ectoderm cells, and immunoblotting showed a similar ectoderm enrichment. These data demonstrated that suUSF was responsible for the ectoderm-specific activity observed in sea urchin extracts.

Amino Acid Sequence↗
PubMed1990

Hyperthermic enhancement of rhodamine 123 cytotoxicity in B16 mouse melanoma cells in vitro.

The effect of elevated temperature on cytotoxicity of rhodamine 123 (R123) was tested in vitro on B16 mouse melanoma cells. Simultaneous 1-h exposure to R123 and hyperthermia (43 degrees C for 1 h) resulted in marked enhancement of R123 cytotoxicity. Thermal enhancement of R123 cytotoxicity occurred at temperatures as low as 38 degrees C. Heat treatment (43 degrees C for 1 h) given immediately before or after R123 exposure (37 degrees C for 1 h) yielded no significant increase in cytotoxicity over that expected for strict additivity. The effects of heat on two mechanisms reported to be associated with R123 cytotoxicity were evaluated: (a) target inactivation by R123; and (b) R123 intracellular accumulation. Hyperthermia caused an increased rate of target inactivation by R123 and also caused an increased net intracellular accumulation of R123. This indicates that at least two mechanisms are responsible for the synergistic cytotoxicity of R123 and hyperthermia.

Animals↗
PubMed1990

Repetitive DNA sequences linked to the sea urchin spec genes contain transcriptional enhancer-like elements.

The 5' flanking DNA of three related Strongylocentrotus purpuratus genes, Spec1, Spec2a, and Spec2c, were analyzed with respect to structure and cis-regulatory activity. The structural features of DNA sequences upstream of the first intron were highly unusual and implicated certain regions as sites of coordinate control for gene expression. By aligning the genes with a common upstream 600-bp repetitive DNA sequence element, termed RSR, it was shown that a conserved DNA block of approximately 800 bp extended from the 3' end of the first exon to the 5' end of the RSR element. In Spec2a, the conserved sequence block was a continuous stretch of DNA, but in Spec1 and Spec2c, 2.5 to 3 kb of inserted DNA bounded by short direct repeats interrupted the conserved sequence block, thus changing the relative placement of the RSR element and other 5' flanking DNA. Deletion of XhoI fragments containing the 5' half but not the 3' half of the RSR element resulted in a significant decrease in chloroamphenicolacetyl transferase (CAT) activity when Spec-CAT reporter gene fusion plasmids were injected into Lytechinus pictus eggs. These results strongly suggested, but did not prove, that the sequences held in common among the XhoI fragments, that is, the 5' half of the RSR elements, were responsible for the decrease in CAT activity. The Spec2a gene was particularly sensitive to deletions of the XhoI fragment containing the 5' half of the RSR element. The deleted element had several enhancer-like properties when inserted back into various test plasmids: it could be positioned in locations different from the transcriptional start site; in some but not all cases, it could be made to work in the reverse orientation; and it could drive expression of the CAT gene using an SV40 promoter or cryptic promoter elements. These findings suggested that an enhancer-like element important for Spec gene expression was contained within a repetitive DNA sequence. Genomic DNA blots suggested that there are many more of these RSR elements than there are Spec genes.

Animals↗
PubMed1990

Regulatory elements from the related spec genes of Strongylocentrotus purpuratus yield different spatial patterns with a lacZ reporter gene.

The Spec1 and Spec2 genes of Strongylocentrotus purpuratus are closely associated with the differentiation of aboral ectoderm. To examine cis-regulatory elements involved in the spatial expression of the Spec genes, we fused the Escherichia coli lacZ gene containing a nuclear targeting signal to 5'flanking DNA plus 5' untranslated leader sequences from Spec1, Spec2a, and Spec2c. All three genes contain 700 bp of highly conserved DNA in their upstream regions, but in Spec1 and Spec2c large insertions interrupt the conserved regions. The Spec-lacZ reporter gene plasmids were microinjected into eggs of S. purpuratus, Lytechinus variegatus, and L. pictus, and beta-galactosidase activity was determined in situ by X-gal staining. The Spec2a-lacZ fusion gene, which contained 1516 bp of 5' flanking DNA and 18 bp of 5' untranslated leader sequence, was preferentially expressed in aboral ectoderm cells in all three species. The Spec1-lacZ fusion gene was expressed in a strikingly different fashion--preferentially in primary and secondary mesenchyme cells, occasionally in aboral ectoderm cells, and less often in oral ectoderm and endoderm cells. The staining pattern was the same in either homologous or heterologous embryos. The Spec2c-lacZ fusion gene, like Spec2a-lacZ, was preferentially expressed in aboral ectoderm, but staining of other cell types was frequently observed. To further delineate sequences required for correct spatial expression, we deleted 800 bp of 5' flanking DNA from the Spec2a-lacZ fusion gene, resulting in a delta Spec2a-lacZ fusion gene that contained only the conserved DNA region. This gene fusion showed preferential expression in aboral ectoderm cells. However, the cell type specificity was not as great as with the parental Spec2a-lacZ plasmid. These experiments implied that the conserved DNA region, associated with all Spec genes examined, was insufficient for complete aboral ectoderm specificity, and suggested that a spatial repressor element existed between -1516 and -697 bp in the 5' flanking DNA of Spec2a.

Animals↗
PubMed1989

Relationship between cellular accumulation of rhodamine 123 (R123) and cytotoxicity in B16 melanoma cells.

Rhodamine 123 (R123) is a mitochondria-specific prototype anticancer agent because its target is the energy-producing mechanism of the cell. The goal of this study was to investigate the relationship between intracellular R123 accumulation and cytotoxicity in a R123-sensitive cell line (RS) and a R123-resistant subline (RR) that we developed. Cytotoxicity after exposure to R123 (0-60 micrograms/ml) was assessed using the clonogenic assay. Intracellular R123 was extracted with acid-alcohol and measured by fluorimetry. The rate of R123 accumulation over 1 hr was significantly higher (P less than 0.0001) for RS cells (4.65 +/- 0.39 micrograms/min/10(6) cells) than for RR cells (1.29 +/- 0.24 micrograms/min/10(6) cells). R123 accumulation in RS cells was strongly correlated (r = 0.80; P less than 0.0001) with cytotoxicity. Treatment of RR cells with verapamil (100 microM) reversed R123 resistance. The resulting dose-survival curve was identical to the dose-response curve of RS cells treated with R123 alone. Cellular content of R123 in RR cells treated with verapamil increased to a level similar to that of RS cells and correlated with cytotoxicity. These data suggest that cytotoxicity of R123 in B16 cells results from increased cellular accumulation of R123.

Animals↗
PubMed1989

Preparation of clear solutions of reconstituted acetylcholinesterase. Effect of ionic strength and lipid/protein ratio.

The detergent-soluble globular dimer of acetylcholinesterase from Torpedo californica was reconstituted through dialysis into preformed egg phosphatidylcholine vesicles. The formation of the enzyme-lipid complexes depended on the ionic strength of the dialysis buffer as well as the molar lipid/protein ratio (R). The enzyme was unstable at I less than 0.05; increasing the ionic strength increased the size of the complex. A too low R value (e.g. 1000) would promote self-aggregation of the enzyme and produce heterogeneous complexes, especially at high I values. On the other hand, a too high R value (e.g. greater than 5000) favored the formation of large enzyme-lipid complexes; their solutions were too turbid for optical studies. The enzyme reconstituted at I = 0.07 and R = 4000 gave a clear solution and showed no artifacts due to light scattering. The conformation based on circular dichroism and enzymatic activity of the detergent-soluble enzyme were unchanged upon reconstitution. The reconstituted enzyme in lipid vesicles seemed to be slightly more stable against thermal denaturation than the protein in sodium cholate solution.

Acetylcholinesterase↗
PubMed1987

Conformation similarities of the globular and tailed forms of acetylcholinesterase from Torpedo californica.

The conformation of the globular dimer (G2), the tailed asymmetric dodecamer (A12, also containing some tailed octamer A8) and the globular tetramer (G4, prepared by removing the collagen-like tail from A12) of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) was studied by circular dichroism (CD) in the ultraviolet region. The G2 and G4 forms had similar conformation with about 40% alpha-helix, 35% beta-sheets and 4% beta-turns; the tailed form had a lower helicity (about 34%) and beta-form (about 25%) content probably because of the presence of the tail whose CD spectrum resembles that of an unordered form, but it had about the same amount of beta-turns as the other two forms. All three forms also had similar CD spectra in the near-ultraviolet region due to their non-peptide chromophores. The pH, thermal and urea denaturation of the three acetylcholinesterase forms was also similar to each other. The pH-dependency of both the enzymatic activity and CD intensity of the three forms showed bell-shaped curves with a plateau at pH 7-8. The activity was completely lost at pH below 5 or above 10, but the corresponding CD spectra retained 70-80% of the original magnitudes. Thermal denaturation of the three forms at pH 7.5 showed a conformational transition and loss of activity between 30 and 40 degrees C, but the CD intensity of the helical band at 222 nm was reduced by only 20-30%. Urea denaturation of the three forms began at 1 M urea; it was protein concentration- and time-dependent. Again, the activity disappeared faster than the decreasing CD intensity. Thus, the overall conformation of the three acetylcholinesterase forms appears to be relatively stable, but their active site is easily perturbed by changing the environment. The loss of activity correlated well with the disappearance of the CD band of tryptophan(s) in the near-ultraviolet region, suggesting that the Trp residue(s) might be at or near the active center of the enzyme.

Acetylcholinesterase↗
PubMedDate not supplied

A new computerized biomechanical perfusion model for ex vivo study of fluid mechanical forces in intact conduit vessels.

We have developed a new computerized biomechanical ex vivo perfusion system for intact conduit vessels in which a wide range of combinations of intraluminal pressure, fluid flow and shear stress could be set and maintained at target levels in mammalian conduit vessels under controlled metabolic conditions. Mean wall shear stress is calculated using the formula: Accuracy of the wall shear stress calculation was validated by ultrasonographic imaging of the vessel radius. In a series of simulation experiments, the hemodynamic homeostasis functions of the system were challenged by generating a wide range of vascular resistance in artificial vessels and by pharmacologically induced changes in vascular tone in intact human vessels. Despite rapid changes in vessel resistance, shear stress and pressure, or flow and pressure were maintained well at target levels. Shear- and pressure-stimulated production of the vasodilator prostaglandin E2 (PGE2) was used to validate the biological relevance of the model. PGE2 release was significantly more stimulated by high (25 dyn/cm2) compared to low (<4 dyn/cm2) shear (ANOVA, p = 0.012). High compared to low intraluminal pressure depressed the production of PGE2 (ANOVA, p = 0.019). In summary, the computerized perfusion model appears to offer new possibilities of investigating the complex interplay between fluid mechanics and the vascular wall.

Algorithms↗