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Biomedical subjects

C Y Lee

Publications and source records attributed to C Y Lee.

At least 145 records · Page 8Linked to original sources
PubMed1998

Chromosomal mapping, tissue distribution and cDNA sequence of four-and-a-half LIM domain protein 1 (FHL1).

We have isolated and sequenced a human heart cDNA clone encoding a novel LIM-only protein. This full-length cDNA clone has a predicted open reading frame (ORF) encoding 280 amino acids. The ORF of this cDNA codes for a LIM-only protein that possesses four repeats of LIM domain and an extra zinc finger and this putative protein is named four-and-a-half LIM domain protein 1 (FHL1). FHL1 is unique when compared with other LIM-only proteins because it possesses an odd number of zinc fingers. When the FHL1 cDNA probe was used to hybridize with poly-(A) RNA of various human tissues, a very strong signal was detected in skeletal muscle, a moderate one in the heart; only weak signals were associated with the placenta, ovary, prostate, testis, small intestine, colon and spleen, and virtually no signal could be detected in brain, lung, liver, kidney, pancreas, thymus and peripheral blood leukocytes. The FHL1 gene was located to human chromosome at Xq27.2 by somatic cell hybrid mapping, fluorescent in situ hybridization (FISH) and radiation hybrid mapping.

Amino Acid Sequence↗
PubMed1998

Ca2+ is released from the nuclear tubular structure into nucleoplasm in C6 glioma cells after stimulation with phorbol ester.

It is well established that cellular Ca2+ is an important messenger that controls many nuclear functions but the source of nuclear Ca2+ is far from clear. It has long been thought that Ca2+ is translocated from the cytosol over a long distance to activate the nuclear transcription machinery. However, this model is at best an incomplete one. With the aid of confocal microscopy, we observed tubules extended deep inside the nucleus of C6 cells in agreement with previous studies (Fricker et al. (1997) J. Cell Biol. 136, 531-544). When cells were stimulated with phorbol 12-myristate 13-acetate or phorbol 12,13-diacetate, Ca2+ was released from these tubules. DiOC6(3), a vital marker for intracellular membranes, stained the tubule in the nucleus of the same cell used for Ca2+ imaging. Moreover, results from labelling the cells with rhodamine 123 further indicate that the tubule was formed by a double-membraned invagination with mitochondria inside. Studies with acridine orange showed that chromatin was excluded from the tubules. Taken together, our results demonstrate that the nuclear tubule is a structural entity responsible for the release of Ca2+ into the nucleoplasm after stimulation with phorbol ester.

Calcium↗
PubMed1998

The nucleus of HeLa cell contains tubular structures for Ca2+ signalling.

It has long been assumed that Ca2+ are translocated from the cytosol to the cell nucleus by a long distance to activate transcription machinery buried deep in the nucleoplasm. However, this model has been recently challenged. When HeLa cells were loaded with fluo-3, highly fluorescent spots of approximately 2 microns in diameter were observed in the cell nucleus while the fluo-3 signals were low in their neighbouring nucleoplasm as determined by confocal microscopy. These fluorescent spots were devoid of but usually associated with chromatin on their boundary. When cells were stimulated by ionomycin (1 microM), the fluo-3 fluorescence in these spots increased faster than that in their neighbouring nucleoplasm. In another experiment, optical sections with hot spot(s) were used to construct 3-D images to study the morphology of the hot spots. Views of reconstruction from different angles indicated that the hot spots formed a tubular structure with a connection to the nucleocytoplasmic interface. Moreover, injection of calcium green-dextran (70 kDa), a Ca(2+)-sensitive indicator conjugated with an inert molecule of large molecular size, into the cytosol leads to a formation of signals also in a tubular shape inside the nucleoplasm. This suggests that the 'channels' are real inside the nucleus and they are derived from an invagination of the double-membraned nuclear envelope. Taken together, our results indicate (1) tubular structures are found inside the cell nucleus; (2) they are extended from the cytosol into the nucleus through the invagination of the double membraned nuclear envelope; (3) molecules of molecular size up to 70 kDa could penetrate into these 'tunnels'; (4) Ca2+ can be released or transported into the cell nucleus through these tubular structures after ionomycin stimulation; and (5) the structures are usually associated with chromatin.

Aniline Compounds↗
PubMed1998

Molecular cloning and characterization of FHL2, a novel LIM domain protein preferentially expressed in human heart.

A full-length cDNA clone encoding a novel LIM-only protein was isolated and sequenced from a human fetal heart cDNA library. This full-length clone consists of 1416 base pairs and has a predicted open reading frame (ORF) encoding 279 amino acids. The ORF of this polypeptide codes for the human heart-specific four and a half LIM-only protein 2 (FHL2). It possesses an extra zinc finger that is a half LIM domain and four repeats of LIM domain. When the human FHL2 cDNA probe was used to hybridize with poly-A RNA of various human tissues, a very strong signal could be seen in heart tissues, and only moderately low signals could be detected in placenta, skeletal muscle and ovary. Virtually no signal could be detected in brain, lung, liver, kidney, pancreas, spleen, thymus, prostate, testis, small intestine, colon or peripheral blood leukocyte. FHL2 was mapped to chromosome 2q12-q13 by fluorescent in-situ hybridization (FISH).

Amino Acid Sequence↗
PubMed1998

Developmental regulation of 14-3-3 epsilon isoform in rat heart.

Human heart cDNA sequencing yielded a cDNA clone that is similar in DNA and amino acid sequences to that of mouse 14-3-3 epsilon isoform. The 6xHis-tagged H1433 epsilon recombinant protein was expressed in Escherichia coli and its size was approximately 30 kDa. From Northern blot results with human multiple tissues, human skeletal muscle was found to have the highest level of h1433 epsilon mRNA expression, whereas Northern blots of human cancer cell lines detected the highest mRNA level of h1433 epsilon in colorectal adenocarcinoma SW480. The protein expression level of h1433 epsilon and Raf-1 is found to be regulated coordinately during rat heart development, and their protein expression was highest from 14.5 to 16.5 days postcoitum.

14-3-3 Proteins↗
PubMed1998

Mapping of the human cysteine-rich intestinal protein gene CRIP1 to the human chromosomal segment 7q11.23.

We report here on the mapping of a cDNA encoding for human cysteine-rich heart protein (HCRHP), a counterpart of the murine cysteine-rich intestinal protein CRIP. By somatic cell hybrid analysis and radiation hybrid mapping, we have located the gene CRIP1 (HGMW-approved symbol) on the subcentromeric region of the q arm of human chromosome 7, flanking a deletion associated with Williams syndrome.

Animals↗
PubMed1998

The nuclear envelope of resting C6 glioma cells is able to release and uptake Ca2+ in the absence of chemical stimulation.

Many agonists evoke events in the cell nucleus through the control of Ca2+ signals. Recent studies using isolated nuclei have indicated that the nuclear envelope is a store for nuclear Ca2+. However, the release of Ca2+ directly from the nuclear envelope in living cells has never been reported. In the present study, we have investigated the changes of Ca2+ signals at the cyto-nucleoplasmic interface of rat C6 glioma cells using confocal microscopy. Digital imaging indicates that fluo-3, a Ca2+-sensitive fluorescent probe, was concentrated in or around the nuclear envelope. Our experiments also revealed that C6 cells at rest produced spontaneous Ca2+ spikes in the absence of chemical stimulation. The amplitude of the repetitive Ca2+ spikes was higher at the nuclear envelope than in the whole cell or cytosol. After image subtraction, circular rims of Ca2+ release and uptake were seen at the outer boundary of the nucleus. When the cells were treated with thapsigargin (2 muM), a specific Ca2+-ATPase inhibitor, a long-lasting Ca2+ release was observed at the nuclear envelope. Moreover, most of the released Ca2+ was directed inwardly to the nucleoplasm with little outward diffusion. Our results thus indicate: (1) that the nuclear envelope is a Ca2+ store that possesses the ability to discharge and sequestrate Ca2+; and (2) the Ca2+-releasing channels are present in the inner nuclear membrane.

Adenosine Triphosphate↗
PubMed1998

The rise of nuclear and cytosolic Ca2+ can be uncoupled in HeLa cells.

It has long been assumed that ionized Ca2+ is transmitted from the cytosol to the cell nucleus through the nuclear pore complexes to trigger many nuclear functions such as gene transcription after agonist stimulation. However, this hypothesis has been challenged recently. In the present study, we have investigated the interplay of Ca2+ in the cytosol and nucleus of HeLa cells upon histamine stimulation by using confocal microscopy. In resting cells, addition of histamine (50 microM) produced synchronous Ca2+ signals in the cytosol and nucleus with a stronger fluo-3 emission in the nucleus. Our results also demonstrate that the rise of cytosolic and nuclear free Ca2+ can be uncoupled after histamine stimulation. This assertion is supported by the following observations: (1) when heparin was injected into the cytosol, the increase in fluo-3 fluorescence in the cytosol mediated by histamine was almost eliminated while that in the nucleus still occurred; (2) when heparin was in the nucleus, the increase in the free nucleoplasmic Ca2+ concentration ([Ca2+]n) elicited by histamine was abolished while the rise of cytosolic Ca2+ level ([Ca2+]c) was seen; (3) when we injected inositol 1,4,5-trisphosphate (IP3) directly into the nucleus, [Ca2+]n increased; and (4) in the cells given cytosolic injections of BAPTA-dextran (70 kDa), histamine evoked additional Ca2+ transients in the nucleus. These results suggest that the nucleus of the HeLa cell has its own IP3-sensitive Ca2+ store and that the Ca2+ signals in the nucleus and cytosol can be uncoupled.

Biological Transport↗
PubMed1998

Chromosomal mapping of a skeletal muscle specific LIM-only protein FHL3 to the distal end of the short arm of human chromosome 1.

Four-and-a-half LIM domain proteins (FHL) possess four tandem repeats of LIM domain and an extra zinc finger. FHL family LIM proteins are unique when compared with other LIM-only proteins because they possess an odd number of zinc fingers. In this study, the tissue distribution and chromosomal mapping of skeletal muscle LIM protein FHL3 were reported. When the FHL3 cDNA probe was used to hybridize with poly-(A) RNA of various human tissues, a very strong signal was detected in skeletal muscle, and virtually no signal could be detected in heart, brain, placenta, lung, liver, kidney and pancreas. Using radiation hybrid technique, FHL3 gene was mapped to the distal end of the short arm of chromosome 1 (123.26 cR from the top of the Chr1 linkage group) and this region (near 1p34) is related to several human malignancies.

Animals↗
PubMed1998

Trichloroethylene biodegradation by phenoloxidizing cultures grown from various conditions.

The rate and extent of trichloroethylene (TCE) degradation by three cultures of phenol oxidizing bacteria grown from two chemostats and one from rotating biological contactors was investigated. Batch experiments were performed to measure the disappearance of TCE both with the resting cell alone and with the formate added. Experimental data were then compared with mathematical predictions from a model describing TCE transformation. Through nonlinear regression analysis, a best fit between the measurements and predictions was achieved when residual sum of squares reached a minimum. Based on the resultant parameters of transformation capacity and reaction rate, the suspended cells with a mean cell retention time (MCRT) of 3.8 days were the most active in degrading TCE, while the attached bacteria had the least activity. Based on the results presented herein, it is recommend that using suspended-growth reactors operated at short MCRT to produce desirable cells for cometablic transformation of TCE.

Bacteria↗
PubMed1998

Estimates of relative binding free energies for HIV protease inhibitors using different levels of approximations.

Although the free energy perturbation approach is a rigorous method for estimating the relative binding free energy between an enzyme and its inhibitors, it is computationally expensive. This paper examines the accuracy at different levels of approximations, following the series expansion of free energy derived by Aqvist et al. Level-0 calculates only the enzyme-inhibitor interaction energy at the minimum energy configuration without solvent. In Level-0MD, the inhibitor configurations are sampled by molecular dynamics. These levels assume that the second- and higher order terms in the series expansion can be neglected and that the interaction energies in the bound and unbound states are equal. Level-1 does not assume equal interaction energies in the bound and unbound states. Level-1S includes the solvent contribution but both enzyme and inhibitor are fixed. In Level-1SMD, the inhibitor configurations are sampled by molecular dynamics. Level-2SMD retains the second-order term. We chose seven HIV-1 protease inhibitors for study: A77003, A76889, A76928, A78791, A74704, JG365 and MVT101. Level-0 and Level-0MD were found to give essentially the same relative interaction energies by using the AMBER force field, suggesting that fixing atomic positions may be a good approximation in some cases. However, as expected, Level-0 or Level-0MD gave poor predictions for the relative binding free energies between hydrophobic inhibitors (e.g. A77003) and more hydrophilic inhibitors (e.g. JG365). Level-1SMD produced a much better correlation between calculated and experimental results. Inclusion of the second-order term did not improve the accuracy.

Chemical Phenomena↗
PubMed1998

Diphtheria, tetanus and whole cell pertussis vaccine combined with hepatitis B vaccines: a comparison of two doses (10 microg and 5 microg).

BACKGROUND: A combined diphtheria-tetanus-whole cell pertussis-hepatitis B (DTPwHB) vaccine might facilitate the achievement of universal vaccination of infants against hepatitis B. METHODS: A double blind, randomized, two-armed, single center study was undertaken to evaluate the immunogenicity and reactogenicity of combined tetravalent DTPwHB vaccine, with two dosages of hepatitis B component (10 microg and 5 microg). The combined vaccine was tested in the context of a simplified vaccination schedule at 1.5, 3.5 and 6 months of age, to 120 healthy infants born to hepatitis B surface antigen-negative mothers after priming with one dose of hepatitis B vaccine (10 microg) at birth. Antibodies to each antigenic component were measured from blood samples collected immediately after birth, pre- and postvaccination blood samples. RESULTS: The reactogenicity profiles were similar in the two groups. No serious adverse events were reported. One month after completion of the four-dose vaccination schedule, all subjects except one in Group 1 (10 microg) had protective titers of anti-HBs (10 mIU/ml). At this time the geometric mean titer in Group 1 (10 microg) was higher than that observed in Group 2 (5 microg), 696 vs. 488 mIU/ml (P = 0.19). One month after three doses all subjects in both groups had protective antidiphtheria titers and antitetanus titers. The vaccine response rate to the Bordetella pertussis component of the vaccine was 88.0% in Group 1 and 96.2% in Group 2 (P = 0.86). CONCLUSION: Both combined tetravalent vaccines are safe and immunogenic when administered to infants born to a hepatitis B surface antigen-negative mother, with a 10-microg dose of priming hepatitis B vaccine at birth. This combined tetravalent DTPwHB vaccine may play an important role to promote integration of HB vaccine into the Expanded Program of Immunization in hepatitis B-endemic areas.

Antibodies, Bacterial↗
PubMed1998

Assessment of progesterone-induced acrosome reaction by biotinylated monoclonal antibody probes.

PROBLEM: To develop an immunohistochemical assay for determination of acrosome-reacted human sperm and to study the effects of progesterone and cholesterol treatment on human sperm acrosome reaction. METHOD OF STUDY: Three distinct anti-sperm monoclonal antibodies were biotinylated and used as probes for assessment of acrosome reaction in a 30-min immunohistochemical assay. Progesterone and/or cholesterol were added to sperm preparation to influence the acrosome reaction in different experimental conditions. RESULTS: Percentages of acrosome-intact sperm decreased significantly during the 18-hr incubation. Acrosome reaction could be induced by progesterone as early as 2 hr after sperm incubation in human tubal fluid. The degree of progesterone-induced acrosome reaction was time dependent and the optimal effect was reached by adding 10 micrograms/ml progesterone for 30 min incubation. Progesterone-induced acrosome reaction was shown to be hormone-concentration dependent with 50% stimulation at 1 microgram/ml. Cholesterol (1 microgram/ml) was found to inhibit progesterone-induced acrosome reaction either by co-incubation with human sperm during capacitation, or by simultaneous incubation with progesterone during acrosome reaction induction. CONCLUSIONS: Assessment of human sperm acrosomal status by avidin-biotin immunohistochemical assay can be a routine in clinical laboratories for male infertility services. Cholesterol can inhibit progesterone-induced acrosome reaction, possibly by its modifications of sperm plasma membrane and/or interference of progesterone binding to its surface receptors.

Acrosome↗
PubMed1998

Factors affecting the collagen binding capacity of Staphylococcus aureus.

To determine whether the ability of Staphylococcus aureus to bind collagen involves an adhesin other than the collagen adhesin encoded by cna, we examined the collagen binding capacity (CBC) of 32 strains of S. aureus. With only two exceptions, a high CBC corresponded with the presence of cna. Both exceptions involved cna-positive strains with a low CBC. The first was a single strain (ACH5) that encoded but did not express cna. The second were the mucoid strains Smith diffuse and M, both of which encoded and expressed cna but bound only minimal amounts of collagen. Analysis of capsule mutants suggests that the reduced CBC observed in the mucoid strains was due to masking of the collagen adhesin on the cell surface and that this masking effect is restricted to heavily encapsulated strains. Differences in the CBC of the remaining cna-positive strains were correlated to variations in the level of cna transcription and were independent of the number of B domain repeats in the cna gene. In all cna-positive strains other than ACH5, cna transcription was temporally regulated, with cna mRNA levels being highest in cells taken from exponentially growing cultures and falling to almost undetectable levels as cultures entered the post-exponential growth phase. The CBC was also highest with cells taken from exponentially growing cultures. Mutation of agr resulted in a slight increase in cna transcription and a corresponding increase in CBC during the exponential growth phase but did not affect the temporal pattern of cna transcription. Mutation of sar resulted in a more dramatic increase in CBC and a delay in the post-exponential-phase repression of cna transcription. Mutation of both sar and agr had an additive effect on both CBC and cna transcription. We conclude that (i) cna encodes the primary collagen-binding adhesin in S. aureus, (ii) sar is the primary regulatory element controlling expression of cna, and (iii) the regulatory effects of sar and agr on cna transcription are independent of the interaction between sar and agr.

Adhesins, Bacterial↗
PubMed1998

Mononucleosis syndrome and coincidental human herpesvirus-7 and Epstein-Barr virus infection.

Two girls (a 5 year old and a 21 month old) experiencing mononucleosis syndrome with coincidental human herpesvirus (HHV)-7 and Epstein-Barr virus (EBV) infections are described. One patient had primary HHV-7 infection and reactivated EBV infection. The other had primary HHV-7 and EBV infections. These cases indicated that HHV-7 is capable of inducing infectious mononucleosis-like illness. Multiple herpesvirus infection in one of the patients also suggests that interaction among herpesviruses can occur in vivo. The consequence of this interaction may have clinical implications.

Child, Preschool↗
PubMed1998

Regulation of the depth of surface liquid in bovine trachea.

The luminal surface of airways is lined by a thin film of airway surface liquid (ASL). Physiological regulation of the depth of ASL has not been reported previously. In this paper, we have used low-temperature scanning electron microscopy of rapidly frozen specimens of bovine tracheal epithelium to demonstrate alterations in the depth of ASL in response to the cholinergic agonist methacholine. We first established that methacholine selectively stimulated airway glands, with maximal secretion at approximately 2 min and a return to baseline within approximately 5 min. A 2-min exposure to methacholine increased the depth of ASL from 23 to 78 microns. Thereafter, depth decreased linearly with time, reaching 32 microns at 30 min. The initial increase in depth was blocked by bumetanide, an inhibitor of active chloride secretion, whereas the slow decline back to baseline was inhibited by amiloride, a blocker of active sodium absorption. We conclude that the methacholine-induced changes in ASL depth reflect transient gland secretion followed by liquid absorption across the surface epithelium.

Animals↗