Search PubMed⌕ Search

Biomedical subjects

K M Pang

Publications and source records attributed to K M Pang.

At least 19 recordsLinked to original sources
PubMed2001

Regulated expression of myosin II heavy chain and RacB using an inducible tRNA suppressor gene.

An inducible expression system that indirectly regulates gene expression through the use of an inducible suppressor tRNA has been used to express both endogenous and exogenous genes in Dictyostelium. The tetracycline repressor and tRNA suppressor (Glu) are expressed from a single G418 selectable vector, while a gene engineered to contain a stop codon is expressed from a separate hygromycin selectable vector. beta-Galactosidase could be induced over 300 fold with this system, and the extent of induction could be varied depending upon the amount of tetracycline added. It took 3 days to fully induce expression, and about 3 days for expression to decrease to baseline after removal of the tetracycline. Dictyostelium myosin II heavy chain could also be expressed in an inducible manner, although the induction ratio was not as high as beta-galactosidase and the maximum expression level was not as high as wild-type levels. A significant accumulation of the truncated peptide indicates that complete suppression of the stop codon was not achieved. Partial phenotypic reversion was observed in null mutants inducibly expressing myosin II. RacB could also be inducibly expressed, whereas the protein could not be expressed from a constitutive promoter, presumably because expression at high levels is lethal. Therefore, the inducible tRNA system can be used to control expression of endogenous Dictyostelium genes.

Animals↗
PubMed2000

Actin filaments are severed by both native and recombinant dictyostelium cofilin but to different extents.

Cofilin has been reported to depolymerize F-actin alternately by either severing filaments to increase the number of depolymerizing ends or by increasing the off-rate of monomers from F-actin without increasing the number of filament ends. We have compared directly the ability of native and recombinant cofilins from Dictyostelium to sever F-actin. Our results demonstrate that native cofilin has a higher level of severing activity than recombinant cofilin. Significantly, the measurement of cofilin's severing activity by two independent methods, direct visualization with an improved light microscope assay and by scoring of the number of pointed ends by DNase I binding, clearly shows that both native and recombinant cofilins sever F-actin but to different extents. The severing activity in preparations of recombinant cofilin is variable depending on the method of preparation and, in some cases, is difficult to detect by microscopy assays. This latter point is particularly significant because it may lead to the conclusion that cofilin severs weakly or not at all depending on its method of isolation.

Actin Depolymerizing Factors↗
PubMed2000

Overexpression of beta-catenin induces apoptosis independent of its transactivation function with LEF-1 or the involvement of major G1 cell cycle regulators.

beta-Catenin promotes epithelial architecture by forming cell surface complexes with E-cadherin and also interacts with TCF/LEF-1 in the nucleus to control gene expression. By DNA transfection, we overexpressed beta-catenin and/or LEF-1 in NIH 3T3 fibroblasts, corneal fibroblasts, corneal epithelia, uveal melanoma cells, and several carcinoma cell lines. In all cases (with or without LEF-1), the abundant exogenous beta-catenin localizes to the nucleus and forms distinct nuclear aggregates that are not associated with DNA. Surprisingly, we found that with time (5-8 d after transfection) cells overexpressing beta-catenin all undergo apoptosis. LEF-1 does not need to be present. Moreover, LEF-1 overexpression in the absence of exogenous beta-catenin does not induce apoptosis, even though some endogenous beta-catenin moves with the exogenous LEF-1 into the nucleus. TOPFLASH/FOPFLASH reporter assays showed that full-length beta-catenin is able to induce LEF-1-dependent transactivation, whereas Arm beta-catenin totally abolishes the transactivating function. However, Arm beta-catenin, containing deletions of known LEF-1-transactivating domains, has the same apoptotic effects as full-length beta-catenin. Overexpressed beta-catenin also induces apoptosis in cells transfected with nuclear localization signal-deleted LEF-1 that localizes only in the cytoplasm. Thus, the apoptotic effects of overexpressed exogenous beta-catenin do not rely on its transactivating function with nuclear LEF-1. Overexpressed delta-catenin, containing 10 Arm repeats, induces only minor apoptosis, suggesting that the major apoptotic effect may be due to domains specific to beta-catenin as well as to Arm repeats. The absence of p53, Rb, cyclin D1, or E2F1 does not affect the apoptotic effect of overexpressed beta-catenin, but Bcl-x(L) reduces it. We hypothesize that in vivo apoptosis of cells overexpressing beta-catenin might be a physiological mechanism to eliminate them from the population.

3T3 Cells↗
PubMed1999

Variables controlling the expression level of exogenous genes in Dictyostelium.

Ectopic expression of genes from recombinant plasmids is commonly used to study gene function. In Dictyostelium, three drug resistance cassettes are commonly used as selectable markers in vectors. We report here a comparative study of the expression of green fluorescent protein (GFP) gene from vectors containing each of the drug-resistant cassettes. The expression was highest in cells transformed with the vectors containing the neomycin-resistant cassette (pDNeoGFP), followed by the hygromycin-resistant cassette (pDHygGFP) and the blasticidin-resistant cassette (pDBsrGFP). The level of GFP expression was directly related to the copy number of the vector in transformants. In turn, the copy number of the vector depended on the drug resistance cassette as well as the concentration of the drug used in selection. In general, cells with higher copy numbers could be selected by a higher drug concentration. The expression of GFP was also affected by the method of transformation. For pDHygGFP, expression of GFP was much higher in cells transformed by electroporation than those transformed by calcium phosphate coprecipitation. However, only a slight difference was observed for pDNeoGFP or pDBsrGFP.

Animals↗
PubMed1998

Use of a fusion protein between GFP and an actin-binding domain to visualize transient filamentous-actin structures.

Many important processes in eukaryotic cells involve changes in the quantity, location and the organization of actin filaments [1] [2] [3]. We have been able to visualize these changes in live cells using a fusion protein (GFP-ABD) comprising the green fluorescent protein (GFP) of Aequorea victoria and the 25 kDa highly conserved actin-binding domain (ABD) from the amino terminus of the actin cross-linking protein ABP-120 [4]. In live cells of the soil amoeba Dictyostelium that were expressing GFP-ABD, the three-dimensional architecture of the actin cortex was clearly visualized. The pattern of GFP-ABD fluorescence in these cells coincided with that of rhodamine-phalloidin, indicating that GFP-ABD specifically binds filamentous (F) actin. On the ventral surface of non-polarized vegetative cells, a broad ring of F actin periodically assembled and contracted, whereas in polarized cells there were transient punctate F-actin structures; cells cycled between the polarized and non-polarized morphologies. During the formation of pseudopods, an increase in fluorescence intensity coincided with the initial outward deformation of the membrane. This is consistent with the models of pseudopod extension that predict an increase in the local density of actin filaments. In conclusion, GFP-ABD specifically binds F actin and allows the visualization of F-actin dynamics and cellular behavior simultaneously.

Actins↗
PubMed1996

Bacterial morphotypes of 3-day old plaque in Chinese. A pilot study.

The aim of the this study was to estimate the % distribution of bacterial morphotypes of supragingival plaque in Chinese subjects after 3 days of no oral hygiene. 36 dental students, hygienists or dental surgery assistants (mean age: 24.3 years) were recruited. Prophylaxis and oral hygiene reinforcement were given 1 x a week for 3 weeks to obtain gingival health and a 3-day period without any plaque control started afterwards. At the end of the 3-day period, the plaque along the gingival margin of the upper right central incisor was removed and direct Gram stained smears were prepared for light microscopy. Bacteria were classified as gram-positive or gram-negative cocci, rods, filaments, fusiform organisms, spirilla or spirochetes. A differential count of 200 organisms from 3-6 microscopic fields was performed and the results expressed as a % of the organisms counted. Results from the 36 direct smears showed approximately 83% (range: 52%-96%) of the organisms to be gram-negative while gram-positive bacteria comprised only a minority of about 12% (range: 0.7%-43%). This result is remarkably different from previous reports from the West where gram-positive bacteria were the predominant bacterial types noted in early supragingival plaque. Further longitudinal studies involving both microscopy and bacteriological culture will be necessary to confirm this finding.

Adult↗
PubMed1996

Resin permeation into acid-conditioned, moist, and dry dentin: a paradigm using water-free adhesive primers.

Preservation of the morphological integrity of demineralized dentin collagen in its hydrated state may account for the success observed in wet-bonding procedures. This study investigated the micromorphological differences between moist- and dry-bonding techniques with the use of: (a) Aelitebond, an alcohol-based, water-free, single-component dentin adhesive primer system; and (b) a water-free, acetone-based experimental primer similar to the acetone-based, water-containing All-Bond 2, a two-component primer system. In the wet groups, acid-conditioned dentin surfaces were blotted so that they remained visibly moist prior to bonding. In the dry groups, dentin surfaces were air-dried for 30 sec. Following the bonding procedures, dentin discs in each group were laminated together by means of a chemical-cure resin and processed for scanning electron microscope (SEM) and transmission electron microscope (TEM) examination. Conditioning with 10% H3PO4 for 20 sec. produced complete demineralization of the outer dentin. In the wet groups, banded collagen and interfibrillar spaces could be observed at the surface of the acid-conditioned dentin. Complete wetting of the loosely arranged collagen fibrils by the resin resulted in the formation of a hybrid layer. In the dry groups, only a very thin hybrid layer was observed on the dentin surface, along the walls of the tubules, and along the course of their lateral branches. The absence of banded collagen and interfibrillar spaces within these areas suggested the existence of a collapsed dentin matrix along various liquid-vapor boundaries that restricted resin permeation into the subsurface intertubular matrix, producing an incompletely infiltrated "hybridoid region".

Dental Bonding↗
PubMed1996

Quantitative contribution of the collagen network in dentin hybridization.

PURPOSE: To determine the quantitative contribution of dentin hybridization to bonded assembly strength and demonstrate the micromorphology of the interface with and without collagen present. MATERIALS AND METHODS: Four groups of 10 molar teeth were finished to a 320 grit dentin smear layer. Two groups served as controls and two experimental groups were subjected to collagenase digestion of the collagen exposed by acid conditioning. All-Bond 2 and Amalgambond were used to bond Bisfil and Epic resin composite, respectively. Stored in water at 37 degrees C for 24 hours the assemblies were tested in a shear mode at a crosshead speed of 5 mm/minute. Means and standard deviations were subjected to analysis for statistical significance. Twenty four teeth in four groups were examined by scanning (SEM) and transmission electron microscopy (TEM) for the relationship between resin and conditioned dentin with and without the collagen network. RESULTS: All-Bond 2 and Amalgambond controls were 28.41 +/- 3.9 and 19.04 +/- 5.96 MPa, collagenase-treated groups scored 26.43 +/- 2.90 and 19.70 +/- 4.25 MPa respectively. No significant difference existed between the control and experimental groups. SEM showed an intertubular collagen network with patent tubules and a pronounced porous, irregular dentin topography following collagen digestion. A distinct hybrid zone and tubular penetration was observed but the collagenase-treated specimens showed only resin in the tubules and their lateral extensions. TEM confirmed the absence of a distinct hybrid zone in the collagenase groups with a tight, gap-free junction between the resin and the undemineralized dentin. An electron dense zone (< 50 nm) at the leading edge of conditioning was observed for All-Bond 2 and Amalgambond groups. It was concluded that the resin-reinforced or hybridized, collagenous network does not detract from, nor contribute any significant quantitative value per se to dentin bonding with the systems tested.

Acid Etching, Dental↗
PubMed1995

Comparison of three methods of critical evaluation of microleakage along restorative interfaces.

This in vitro study compared microleakage along the dentin-restorative interface by use of a conventional single-surface and two stereoscopical protocols. The extent of microleakage in class V cylindric cavities that were restored with Scotchbond 2/P-50 was assessed after thermocycling and silver staining were performed. Specimens were randomized into three groups of 10 teeth for the three different protocols to be investigated. The categories were group 1, a single longitudinal section through the midline of the restoration, group 2, a multiple-surface protocol based on computer reconstruction of digitized images from multiple serial abrasions, and group 3, a clearing protocol based on immersion of demineralized specimens in methyl salicylate. The severity of microleakage for all groups was recorded on an ordinal scale and based on linear leakage length. The median leakage score for groups 1 through 3 was 2, 4, and 4, respectively. When comparisons were performed with nonparametric statistical analysis, both the clearing and the multiple-surface protocols recorded significantly more severe leakage than those detected from a conventional, single longitudinal, midline sectioning technique, (x2 = 8.595, p < 0.02). Furthermore microleakage patterns generated from the two stereoscopical protocols revealed extensive microleakage that advanced unilaterally or bilaterally through the dentin-restorative interface along the embrasure regions into the axial wall of the restorations. No significant difference was noted between the two stereoscopical protocols (Q = 0.260, p > 0.05), which suggested that the clearing protocol, which is less labor-intensive than the multiple-surface protocol, may be recommended for future comparative microleakage studies that involve the dentin-restorative interface.

Analog-Digital Conversion↗
PubMed1995

Variability in microleakage observed in a total-etch wet-bonding technique under different handling conditions.

Using a stereoscopic clearing protocol and scanning electron microscopy, we investigated the extent and nature of microleakage in a total-etch, current-generation dentin adhesive by a wet-bonding technique under different handling conditions. The hypotheses were that inadequate light curing of the primer or incomplete drying of the primer solvent might adversely affect the sealing ability of an acetone-containing adhesive system. The study consisted of three experimental groups: (I) a control group with an adequate light source and with the primer solvent completely dried; (II) an "inadequate-light" group; and (III) an "incomplete evaporation of primer solvent" group. The extent of microleakage after silver staining and clearing of the specimens was scored based on a modified five-point scale. Nonparametric statistical analysis (Kruskal-Wallis ANOVA) followed by a multiple comparison test (Dunn test) indicated significant differences among the three groups (p < 0.05). SEM examination of the restorative interface revealed that microleakage appeared to be initiated from the bonding resin-hybrid layer interface in all three groups, representing the weak link in the adhesive system. In addition, microleakage was characterized by 5 zones, each delineating a stage in a continuous array of progressively deleterious microleakage patterns variously distributed among the three groups. It was suggested that, while the bonding resin-hybrid layer interface represented the intrinsic weakness in an already much improved dentin adhesive, extrinsic factors such as the adequacy of the curing light and, more importantly, complete removal of the primer solvents can and should be avoided to preserve the structural integrity of the marginal seal.

Acid Etching, Dental↗
PubMed1995

A method for microleakage evaluation along the dentin/restorative interface.

PURPOSE: To present a simple non-abrasive protocol in conjunction with the silver-staining method, enabling unobstructed visualization of well-defined microleakage patterns within the demineralized dentin-restorative interface. MATERIALS AND METHODS: Class V preparations were made with an occlusal margin on bevelled enamel and a gingival margin on dentin/cementum. After restoration with All Bond 2/P-50 and Scotchbond 2/Silux, storage in water at 37 degrees C for 7 days and thermocycling 300 times between 5 degrees and 55 degrees C, the specimens were placed in silver nitrate solution. After silver fixation, demineralization and clearing in methyl salicylate, silver stain location was scored ordinally. In addition, a three dimensional portrait of leakage pathways was illustrated using photographic and computer digitization. RESULTS: The Wilcoxon signed-rank test showed that the extent of silver penetration before and after acid demineralization was not significantly different (P > 0.05). This study also confirmed that microleakage is not uniform along the circumference of a restorative margin. Although the protocol is insensitive to microleakage occurring solely within enamel, the predominant concern over that occurring within dentin, in particular along the cervical restorative border, renders it a serviceable alternative for future investigation of the integrity of the marginal seal from clinically restored in vivo specimens.

Acid Etching, Dental↗
PubMed1994

Structural evidence of a sealed tissue interface with a total-etch wet-bonding technique in vivo.

The resin-dentin interface of in vivo specimens restored with the All-Bond 2 system by use of a total-etch wet-bonding technique on vital deep human dentin was investigated by: (a) SEM examination of cryofractured specimens; (b) SEM examination of acid-rinsed, highly polished, embedded specimens; and (c) TEM examination of demineralized ultrathin sections. Ultrastructural features that were pertinent to the formation of an effective clinical seal were characterized. It is suggested that the establishment of an effective seal of the patent dentinal tubules following total etching is accomplished by: (1) the formation of an outer zone of a solid resin plug surrounded by a circumferential cuff of resin-impregnated dentin; and (2) the formation of an inner zone of a hollow resin sheath with resin globules along the internal walls of the tubules, closely adapting to the odontoblast process. The structural appearance and functional implications of these two zones were strikingly similar, with the sealing of exposed dentinal tubules in arrested carious lesions or cervical abfraction lesions. It appeared that there is a common denominator in physiologic isolation defense mechanisms and the total-etch, wet-bonding technique in the All-Bond 2 system in preserving the biological and morphological integrity of the pulpo-dentinal complex.

Acid Etching, Dental↗
PubMed1994

Age estimation by Chinese permanent teeth with image analysis.

A total of 567 human teeth except third molars were studied to estimate age, using a combined method of multiple regression analysis of data from a scoring system, direct measurements with image analyser, and logarithmic transformation. As a result, the standard errors of estimate were 4.8-5.4 years in the working sample, and 2.9-5.4 years in the control sample respectively, by using a single tooth. By testing the formulae on forensic autopsy cases in Guang Dong and Guang Xi, (People's Republic of China), it is demonstrated that the standard error of estimate is 3.7 and 2.6 years respectively for any single tooth (incisor, canine, premolar or molar) except the third molar. Age determination from multiple teeth of the same individual has also been studied. As a result, according to the different age grouping, the standard errors of estimate ranged from 0.8 to 2.6 years (2 teeth from the same individual) and 0.6 to 2.3 years (3 teeth from the same individual). It is concluded that the method presented here is helpful in improving the accuracy of age determination from teeth.

Adolescent↗
PubMed1994

The microvasculature of human infant oral mucosa using vascular corrosion casts and india ink injection. II. Palate and lip.

The microvasculature of human hard and soft palate and lip originating from four infant males and six females, aged 6 months to 2 years was studied by scanning electron microscopy of vascular corrosion casts and light microscopy of India ink injected specimens. The capillary loops of the hard palate mucosa and vermilion border of the lips were found to be tall, numerous and consisted of primary, secondary and tertiary loops. Those of the soft palatal and labial mucosa were short, few in number and demonstrated a simple hair-pin shape originating directly from the subpapillary vascular network. It was concluded that the configuration of capillary loops is not only determined by the shape of the connective tissue papillae in the lamina propria but also influenced by the functional demands characteristic of the different areas of the oral mucosa.

Carbon↗
PubMed1993

The fungicidal effect of human lactoferrin on Candida albicans and Candida krusei.

Five oral isolates each of Candida albicans and Candida krusei were studied for their sensitivity to the fungicidal effect of human lactoferrin. Significant inter- and intraspecies variations were observed and with most isolates the sensitivity of C. krusei to lactoferrin was greater than that of C. albicans. Fungicidal activity of lactoferrin was dose-dependent and observable only with the iron-free form of the molecule (apo-lactoferrin). Iron-saturated lactoferrin was ineffective against all isolates. Supernatant protein assays and scanning electron microscopy indicated cell surface alterations--leakage of proteins and formation of surface blebs--only in those Candida isolates that were sensitive to apo-lactoferrin. As lactoferrin is a common, non-immune, mucosal defence protein, its varying mode of action against C. albicans and C. krusei may be related to their different oral carriage rates.

Analysis of Variance↗