Biomedical subjects

K Park

Publications and source records attributed to K Park.

At least 19 recordsLinked to original sources

PubMed1992

Inhibition of protein phosphatases blocks myogenesis by first altering MyoD binding activity.

To examine the role of protein phosphatases in skeletal muscle differentiation, C2C12 myoblasts were treated with okadaic acid, a potent in vitro inhibitor of protein phosphatases 1 and 2A which regulate various cellular events in intact cells. We now show that okadaic acid treatment of the mouse myoblast C2C12 cell line reversibly altered the morphology of the cells and blocked differentiation. At a molecular level, it extinguished expression of the myogenic determination genes, MyoD1 and myogenin, but induced the expression of an inhibitor of differentiation, Id. Analysis of the MyoD1 promoter showed that inhibition of MyoD1 expression by okadaic acid occurs at the transcriptional level. These changes occur 10-20 h after okadaic acid treatment. However, within 1 h of treatment the ability of muscle extracts to support a specific MyoD-dependent gel mobility shift using a MyoD DNA binding site is lost. These data suggest that protein phosphatases play an important role during myogenic differentiation.

Animals↗

PubMed1992

Post-transcriptional regulation of the human transforming growth factor-beta 1 gene.

Since many lines of evidence suggest that expression of the transforming growth factor-beta 1 (TGF-beta 1) gene may be regulated post-transcriptionally, we examined the effect of the 5'-untranslated region (UTR) of this gene on TGF-beta 1 expression. For this purpose, fragments of the 840-nucleotide highly GC-rich TGF-beta 1 5'-UTR were inserted into the 5'-UTR of the structural gene for human growth hormone driven by the simian virus 40 early promoter. A portion of the 5'-UTR of TGF-beta 1 mRNA spanning the sequences from +11 to +147 was shown to inhibit growth hormone expression by as much as 22-fold. This effect was cell-specific; growth hormone production was inhibited in PC-3 human prostate adenocarcinoma and A-549 human lung adenocarcinoma cells, while no effect was seen in rat pheochromocytoma PC12 cells, which show efficient translation of endogenous TGF-beta 1 mRNA. Computer analysis showed that this region of the 5'-UTR contained a stable secondary stem-loop structure spanning sequences +49 to +76. This stem-loop region alone is sufficient to inhibit expression of the growth hormone gene, suggesting that it plays an important role in post-transcriptional regulation of TGF-beta 1 gene expression.

Animals↗

PubMed1992

Inhibition of myogenesis by okadaic acid, an inhibitor of protein phosphatases, 1 and 2A, correlates with the induction of AP1.

Recently, we demonstrated that okadaic acid, an inhibitor of protein phosphatases 1 and 2A, inhibits myogenesis by extinguishing the expression of MyoD1 and inducing the expression of Id. Since it has been reported that transformation by c-fos also inhibits myogenesis through inhibition of MyoD1 expression, we examined the effects of okadaic acid on the activation of the c-fos and jun family of proto-oncogenes in an attempt to understand the mechanism by which okadaic acid inhibits the myogenic differentiation. Treatment of C2C12 cells in growth medium with okadaic acid increased expression of the mRNAs for the c-fos family continuously and for the jun family to a lesser extent. In contrast, in differentiation medium, the induction of c-fos, c-jun, and fos B mRNAs by okadaic acid was transient, whereas fra-1, jun D, and jun B mRNAs were induced continuously, suggesting that okadaic acid regulates the expression of the c-fos and jun family through complex regulatory mechanisms depending on the state of differentiation of the cells. Transfection of c-jun and c-fos promoter-chloramphenicol acetyltransferase constructs demonstrated that the effects of okadaic acid on the induction of c-fos and c-jun are mediated through the activation of promoter elements. These results suggest that some of the targets of protein phosphatases 1 and 2A may include transcription factors capable of forming AP1 complexes and that these factors may play an important role during myogenic differentiation.

Base Sequence↗

PubMed1992

Analysis of the circular dichroism spectrum of proteins using the convex constraint algorithm: a practical guide.

Due to the time scale of circular dichroism (CD) measurements, it is theoretically possible to deconvolute such a spectrum if the pure CD spectra differ significantly from one another. In the last decade several methods have been published aiming at obtaining the conformational weights, or percentages (which are the coefficients for a linear combination) of the so-called typical secondary structural elements making up the three-dimensional structure of proteins. Two methods that can be used to determine the secondary structures of proteins are described here. The first method, called LINCOMB, is a simple algorithm based on a least-squares fit with a set of reference spectra representing the known secondary structures and yielding an estimation of weights attributed to alpha-helix, beta-pleated sheet (mainly antiparallel), beta-turns, unordered form, and aromatic/disulfide (or nonpeptide) contributions of the protein being analyzed. This method requires a "template" or reference curve set, which was obtained from the second method. The second method, "convex constraint analysis," is a general deconvolution method for a CD spectra set of any variety of conformational type. The algorithm, based on a set of three constraints, is able to deconvolute a set of CD curves to its common "pure"-component curves and conformational weights. To analyze a single CD spectrum with this method, the spectrum is appended to the data set used as a reference data set. As a way to determine the reliability of the algorithm and provide a guideline to its usage, some applications are presented.

Algorithms↗

PubMed1992

Synthesis of photoreactive poly(ethylene glycol) and its application to the prevention of surface-induced platelet activation.

Photoreactive poly(ethylene glycol) (PEG) was synthesized by reacting 4-fluoro-3-nitrophenyl azide (FNPA) with sodium salt of PEG. The synthesized 4-azido-2-nitrophenyl PEG (ANP-PEG) was characterized by 1H-NMR, IR, and UV spectroscopy. ANP-PEG was grafted to dimethyldichlorosilane-coated glass (DDS-glass) by photolysis without any premodification of the surface. The effects of various grafting factors, such as the polymer adsorption time, concentration of ANP-PEG, and UV irradiation time, on the PEG grafting efficiency were examined. The PEG-grafted DDS-glass was characterized by measuring surface free energies, surface-induced platelet activation, and the relative amount of PEG grafted on the surface using electron spectroscopy for chemical analysis (ESCA). Platelet adhesion and activation was analyzed by measuring the number and spread area of adherent platelets. The results showed that ANP-PEG had to be adsorbed onto DDS-glass for at least 12 h before photolysis for the maximum grafting efficiency. No platelets could adhere to the PEG-grafted DDS-glass, if the bulk concentration of ANP-PEG in the adsorption solution was between 1 mg/mL and 10 mg/mL. Above 10 mg/mL, platelet activation gradually increased and reached the maximum at 30 mg/mL. Our data indicate that the grafting of ANP-PEG requires careful control of the grafting conditions and that the grafted PEG can prevent surface-induced platelet activation.

Biocompatible Materials↗

PubMed1992

Differentiation between transmembrane helices and peripheral helices by the deconvolution of circular dichroism spectra of membrane proteins.

The interpretation of the circular dichroism (CD) spectra of proteins to date requires additional secondary structural information of the proteins to be analyzed, such as X-ray or NMR data. Therefore, these methods are inappropriate for a CD database whose secondary structures are unknown, as in the case of the membrane proteins. The convex constraint analysis algorithm (Perczel, A., Hollósi, M., Tusnády, G., & Fasman, G. D., 1991, Protein Eng. 4, 669-679), on the other hand, operates only on a collection of spectral data to extract the common spectral components with their spectral weights. The linear combinations of these derived "pure" CD curves can reconstruct the original data set with great accuracy. For a membrane protein data set, the five-component spectra so obtained from the deconvolution consisted of two different types of alpha helices (the alpha helix in the soluble domain and the alpha T helix, for the transmembrane alpha helix), a beta-pleated sheet, a class C-like spectrum related to beta turns, and a spectrum correlated with the unordered conformation. The deconvoluted CD spectrum for the alpha T helix was characterized by a positive red-shifted band in the range 195-200 nm (+95,000 deg cm2 dmol-1), with the intensity of the negative band at 208 nm being slightly less negative than that of the 222-nm band (-50,000 and -60,000 deg cm2 dmol-1, respectively) in comparison with the regular alpha helix, with a positive band at 190 nm and two negative bands at 208 and 222 nm with magnitudes of +70,000, -30,000, and -30,000 deg cm2 dmol-1, respectively.

Adenosine Triphosphatases↗

PubMed1992

Deconvolution of the circular dichroism spectra of proteins: the circular dichroism spectra of the antiparallel beta-sheet in proteins.

A recently developed algorithm, called Convex Constraint Analysis (CCA), was successfully applied to determine the circular dichroism (CD) spectra of the pure beta-pleated sheet in globular proteins. On the basis of X-ray diffraction determined secondary structures, the original data set used (Perczel, A., Hollosi, M., Tusnady, G. Fasman, G.D. Convex constraint analysis: A natural deconvolution of circular dichroism curves of proteins, Prot. Eng., 4:669-679, 1991), was improved by the addition of proteins with high beta-pleated sheet content. The analysis yielded CD curves of the pure components of the main secondary structural elements (alpha-helix, antiparallel beta-pleated sheet, beta-turns, and unordered conformation), as well as a curve attributed to the "aromatic contribution" in the wavelength range of 195-240 nm. Upon deconvolution the curves obtained were assigned to various secondary structures. The calculated weights (percentages determining the contributions of each pure component curve in the measured CD spectra of a given protein) were correlated with the X-ray diffraction determined percentages in an assignment procedure and were evaluated. The Pearson product correlation coefficients (R) are significant for all five components. The new pure component curves, which were obtained through deconvolution of the protein CD spectra alone, are promising candidates for determining the percentages of the secondary structural components in globular proteins without the necessity of adopting an X-ray database. The CD spectrum of the CheY protein was interesting because it has the characteristic shape associated with the alpha-helical structure, but upon analysis yielded a considerable amount of beta-sheet in agreement with the X-ray structure.

Circular Dichroism↗

PubMed1992

Use of ultrasound imaging and fluoroscopic imaging to study gastric retention of enzyme-digestible hydrogels.

Ultrasound and fluoroscopic imaging techniques were used to monitor the gastric retention of enzyme-digestible hydrogels in the canine stomach. When water was present in the stomach, ultrasound imaging was very effective in monitoring the position of the hydrogel in the stomach, solvent penetration into the gel, and the gastric tissue-gel interactions during peristalsis. Rubbery or fully swollen hydrogels appeared as sonolucent objects with ultrasound imaging. Partially swollen hydrogels displayed a sonolucent outer layer due to solvent penetration and a centrally located bright echo resulting from the acoustic impedance mismatch at the glassy/rubbery interface. The degree of gastric tissue-gel interactions during peristalsis was inversely related to the extent of lumenal distention with water. The effectiveness of peristaltic contractions in driving the hydrogel toward the pyloric sphincter increased as the water was emptied from the stomach. In the absence of water, imaging of the gel with ultrasound became difficult. For this reason, gels were loaded with diatrizoate meglumine/sodium diatrizoate to visualize in real-time using fluoroscopic imaging. Fluoroscopic imaging allowed only indirect assessment of the hydrogel movement during peristalsis and the degree of hydrogel swelling. The gastric retention of the hydrogel under fasted conditions was influenced by the degree of gel deformation in response to peristaltic contractions. Hydrogels with a low degree of deformation during peristalsis showed long gastric retention times. The utilization of ultrasound imaging and fluoroscopic imaging for monitoring dynamic events in the stomach provided information on hydrogel properties which are important to gastric retention. The use of these imaging techniques in the development of long-term oral drug delivery systems is described.

Administration, Oral↗

PubMed1992

Prevention of protein adsorption and platelet adhesion on surfaces by PEO/PPO/PEO triblock copolymers.

Fibrinogen adsorption and platelet adhesion on to dimethyldichlorosilane-treated glass and low-density polyethylene were examined. The surfaces were treated with poly(ethylene glycol) and poly(ethylene oxide)/poly(propylene oxide)/poly(ethylene oxide) triblock copolymers (Pluronics). Poly(ethylene glycol) could not prevent platelet adhesion and activation, even when the bulk concentration for adsorption was increased to 10 mg/ml. Pluronics containing 30 propylene oxide residues could not prevent platelet adhesion and activation, although the number of ethylene oxide residues varied up to 76. However, Pluronics containing 56 propylene oxide residues inhibited platelet adhesion and activation, even though the number of ethylene oxide residues was as small as 19. Fibrinogen adsorption on the Pluronic-coated surfaces was reduced by more than 95% compared to the adsorption on control surfaces. The ability of Pluronics to prevent platelet adhesion and activation was mainly dependent on the number of propylene oxide residues, rather than the number of ethylene oxide residues. The large number of propylene oxide residues was expected to result in tight interaction with hydrophobic dimethyldichlorosilane-treated glass and low-density polyethylene surfaces and thus the tight anchoring of Pluronics to the surfaces. The presence of 19 ethylene oxide residues in the hydrophilic poly(ethylene oxide) chains was sufficient to repel fibrinogen and platelets by the mechanism of steric repulsion.

Adsorption↗

PubMed1992

Functional antagonism between YY1 and the serum response factor.

The rapid, transient induction of the c-fos proto-oncogene by serum growth factors is mediated by the serum response element (SRE). The SRE shares homology with the muscle regulatory element (MRE) of the skeletal alpha-actin promoter. It is not known how these elements respond to proliferative and cell-type-specific signals, but the response appears to involve the binding of the serum response factor (SRF) and other proteins. Here, we report that YY1, a multifunctional transcription factor, binds to SRE and MRE sequences in vitro. The methylation interference footprint of YY1 overlaps with that of the SRF, and YY1 competes with the SRF for binding to these DNA elements. Overexpression of YY1 repressed serum-inducible and basal expression from the c-fos promoter and repressed basal expression from the skeletal alpha-actin promoter. YY1 also repressed expression from the individual SRE and MRE sequences upstream from a TATA element. Unlike that of YY1, SRF overexpression alone did not influence the transcriptional activity of the target sequence, but SRF overexpression could reverse YY1-mediated trans repression. These data suggest that YY1 and the SRF have antagonistic functions in vivo.

Animals↗

PubMed1992

Study on the prevention of surface-induced platelet activation by albumin coating.

To understand how albumin on the surface inhibits surface-induced platelet activation, we adsorbed albumin on dimethyldichlorosilane-coated glass (DDS-glass) and modified the adsorbed albumin by three different methods. The adsorbed albumin was crosslinked with glutaraldehyde, dried and rehydrated, or digested with trypsin. Surface albumin concentration did not change by crosslinking; however, it decreased by about 15% by a simple dry-and-rehydration process. Trypsin digestion reduced the surface albumin concentration by 50%. Platelets were found to adhere and activate on albumin coated DDS-glass, if the adsorbed albumin was modified. The extent of platelet activation was quantified with two numeric parameters, the spread area and circularity. Fibrinogen adsorption to the dried or digested albumin layer resulted in enhancement of platelet activation, while adsorption of more albumin inhibited platelet activation. The results suggest that albumin can inhibit platelet activation as long as it covers the surface completely and remains flexible on the surface. This study indicates that steric repulsion is one of the mechanisms of surface passivation by albumin.

Adsorption↗

PubMed1992

Development of the mucociliary system in the eustachian tube and middle ear: murine model.

In order to describe the developmental anatomy of the murine eustachian tube and its related structures, seventy six mice of ages ranging from gestational day 11 to postnatal day 21 were investigated through the light and electron microscopic observations. Development of the ciliated cells was seen concurrently in both the eustachian tube and middle ear on the 16 th gestation day, one day earlier than the epithelial secretory cells appeared in both the eustachian tube and middle ear. The number of ciliated cells and secretory cells increased rapidly after birth. Tubal glands were well identified with evidence of secretory activity around the time of birth. Thus, the findings of this study indicate that the mucociliary defense system starts to develop during the fetal stage and is well established immediately after birth.

Animals↗

PubMed1992

Luminal development of the eustachian tube and middle ear: murine model.

The purpose of this study was to describe the luminal development of the murine eustachian tube and middle ear. Thirty specimens, aging from gestational day 11 to postnatal day 21, were investigated through the light microscopic observations. The present study also used digitizer, computer, and serially sectioned temporal bone specimens for three-dimensional reconstruction to measure the volume of the eustachian tube and middle ear cavity at different gestational and postnatal ages. The first pharyngeal pouch elongated during gestational day 12 to form the tubotympanic recess. Between gestational day 13 and 14 this tubotympanic recess extended to the middle ear area. A rapid increment in the volume of the tubotympanic recess was noted between gestational day 15 and 16. At this age, a definite division of the tubotympanic recess into the eustachian tube and middle ear cavity was observed. During the postnatal period, the maximum change of the middle ear volume was noted on postnatal day 11 when the mesenchymal tissue in the middle ear cavity disappeared completely.

Animals↗

PubMed1992

Single-center experience of unrelated living-donor renal transplantation in the cyclosporine era.

Renal transplantation from the unrelated living-donor might be an alternative when the cadaveric or related donor is not available, because graft and patient survival rates are superior to those of kidneys from cadaveric donors and even comparable to those from related donors. Among the many factors that might contribute to these excellent results, we believe that the good quality of kidneys (lacking preservation or ischemic injury) is the most important one. In addition, our criteria for patient selection requiring well-matched HLA typing might partially contribute to our success. For the successful renal transplantation program using unrelated-living donors, high ethical standards, accumulated experience from living-related donor transplantation, and dedicated professionalism are strongly recommended.

ABO Blood-Group System↗

PubMed1992