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Biomedical subjects

L B Kong

Publications and source records attributed to L B Kong.

12 recordsLinked to original sources
PubMed2005

Establishment of stable HeLa cell lines expressing enzymatically active hepatitis C virus RNA polymerase.

The hepatitis C virus RNA polymerase (NS5B) is strictly required for viral replication and thus represents an attractive target for antiviral drug development. In this study, stable HeLa cell lines with an integrated NS5B gene were selected by G418 and then confirmed by genome PCR. Subsequently, transcription and expression of the integrated NS5B genes were demonstrated by RT-PCR and Western blot analysis. Further analysis demonstrated enzymatic activity of the expressed NS5B polymerase. The stable HeLa cell lines should be useful for the identification of NS5B inhibitors and for studying the mechanisms of HCV replication.

Gene Expression↗
PubMed2003

Colloidal characterization and electrophoretic deposition of hydroxyapatite on titanium substrate.

Hydroxyapatite (HA) powders were prepared by a modified chemical co-precipitation method and electrophoretically deposited onto a titanium tubular substrate. The zeta potential, electromobility and the particle size of the HA suspension was characterized at various pH values and the most stable and dispersed suspension condition was identified. Electrophoretic deposition of the HA particles on the titanium substrate was then carried out at this optimum suspension condition. Studies on deposition rate and examination on the microstructure of the sintered deposit were performed. The stoichiometry of the HA before and after sintering were also confirmed. The deposition experimental data obtained in the present work was also compared with theoretical model proposed in the literature. Lastly, the adhesion strength of the coating was also quantified using shear strength tests.

Journal Article↗
PubMed2001

The Telomerase/vault-associated protein TEP1 is required for vault RNA stability and its association with the vault particle.

Vaults and telomerase are ribonucleoprotein (RNP) particles that share a common protein subunit, TEP1. Although its role in either complex has not yet been defined, TEP1 has been shown to interact with the mouse telomerase RNA and with several of the human vault RNAs in a yeast three-hybrid assay. An mTep1(-/-) mouse was previously generated which resulted in no apparent change in telomere length or telomerase activity in six generations of mTep1-deficient mice. Here we show that the levels of the telomerase RNA and its association with the telomerase RNP are also unaffected in mTep1(-/-) mice. Although vaults purified from the livers of mTep1(-/-) mice appear structurally intact by both negative stain and cryoelectron microscopy, three-dimensional reconstruction of the mTep1(-/-) vault revealed less density in the cap than previously observed for the intact rat vault. Furthermore, the absence of TEP1 completely disrupted the stable association of the vault RNA with the purified vault particle and also resulted in a decrease in the levels and stability of the vault RNA. Therefore, we have uncovered a novel role for TEP1 in vivo as an integral vault protein important for the stabilization and recruitment of the vault RNA to the vault particle.

Animals↗
PubMed2000

RNA location and modeling of a WD40 repeat domain within the vault.

The vault complex is a ubiquitous 13-MDa ribonucleoprotein assembly, composed of three proteins (TEP1, 240 kDa; VPARP, 193 kDa; and MVP, 100 kDa) that are highly conserved in eukaryotes and an untranslated RNA (vRNA). The vault has been shown to affect multidrug resistance in cancer cells, and one particular component, MVP, is thought to play a role in the transport of drug from the nucleus. To locate the position of the vRNA, vaults were treated with RNases, and cryo-electron microscopy (cryo-EM) was performed on the resulting complexes. Using single-particle reconstruction techniques, 3,476 particle images were combined to generate a 22-A-resolution structure. Difference mapping between the RNase-treated vault and the previously calculated intact vault reconstructions reveals the vRNA to be at the ends of the vault caps. In this position, the vRNA may interact with both the interior and exterior environments of the vault. The finding of a 16-fold density ring at the top of the cap has allowed modeling of the WD40 repeat domain of the vault TEP1 protein within the cryo-EM vault density. Both stoichiometric considerations and the finding of higher resolution for the computationally selected and refined "barrel only" images indicate a possible symmetry mismatch between the barrel and the caps. The molecular architecture of the complex is emerging, with 96 copies of MVP composing the eightfold symmetric barrel, and the vRNA together with one copy of TEP1 and four predicted copies of VPARP comprising each cap.

Animals↗
PubMed1999

Review: resolution issues in single-particle reconstruction.

Specific factors that affect the resolution of single-particle reconstructions are discussed. We present reconstructions of six particles (DNA-dependent protein kinase catalytic subunit, alphaB-crystallin, the ribonucleoprotein vault, hepatitis A virus, adenovirus type 2, and the adenovirus type 12/alpha(v)beta5 integrin complex), which have a variety of symmetries (asymmetric to 60-fold) and a wide range of molecular masses (470 kDa to 150 MDa). In the case of icosahedral viruses, we have found that applying a "soft" mask to remove regions of disordered density improves the resolution given by the Fourier shell correlation 0.5 criterion. This masking procedure is also useful during refinement to improve the quality of the reference model and thus aid in precise alignment of the particle images. For asymmetric particles, we note that image classification, although often a necessary step to generate a first reconstruction, can limit the achievable resolution. The diameter of the particle and the available computational power can also affect the resolution, as can structural variability within the particle.

Adenoviridae↗
PubMed1999

Structure of the vault, a ubiquitous celular component.

BACKGROUND: The vault is a ubiquitous and highly conserved ribonucleoprotein particle of approximately 13 MDa. This particle has been shown to be upregulated in certain multidrug-resistant cancer cell lines and to share a protein component with the telomerase complex. Determination of the structure of the vault was undertaken to provide a first step towards understanding the role of this cellular component in normal metabolism and perhaps to shed some light on its role in mediating drug resistance. RESULTS: Over 1300 particle images were combined to calculate an approximately 31 A resolution structure of the vault. Rotational power spectra did not yield a clear symmetry peak, either because of the thin, smooth walls or inherent flexibility of the vault. Although cyclic eightfold (C8) symmetry was imposed, the resulting reconstruction may be partially cylindrically averaged about the eightfold axis. Our results reveal the vault to be a hollow, barrel-like structure with two protruding caps and an invaginated waist. CONCLUSIONS: Although the normal cellular function of the vault is as yet undetermined, the structure of the vault is consistent with either a role in subcellular transport, as previously suggested, or in sequestering macromolecular assemblies.

Animals↗
PubMed1998

Cryoelectron microscopic examination of human immunodeficiency virus type 1 virions with mutations in the cyclophilin A binding loop.

The human immunodeficiency virus type 1 capsid protein contains a conserved P217X4PX2PX5P231 motif. Mutation at Pro-222 decreases virion incorporation of cyclophilin A, while mutation at Pro-231 abolishes infectivity. Although viral RNA incorporation and protease cleavage of the Gag precursor were not affected by these mutations, cryoelectron microscopy revealed a loss of virion maturation in P231A particles.

Binding Sites↗
PubMed1996

Pedestrian-motor vehicle trauma: an analysis of injury profiles by age.

BACKGROUND: Pedestrian-motor vehicle trauma (PMVT) is a common mechanism of injury in urban populations. STUDY DESIGN: We performed a retrospective review of 273 PMVT victims (16 percent of all patients with blunt injuries) seen at a Level I trauma center over a three-year period. Patients were analyzed by age and grouped as children (age younger than 16 years), adults (age 16 to 59 years), or elderly (age older than 59 years). RESULTS: Children constituted 27 percent of the patients, adults 54 percent, and elderly 19 percent. This mixture had significantly more children and elderly than the population at large or the entire blunt trauma population at our hospital. The majority of patients (66 percent) were male, with females outnumbering males only in the elderly group. Elderly patients were more frequently admitted to the intensive care unit (ICU) and had significantly longer ICU and hospital stays. Injury Severity Scores were successively higher in each age group and significantly higher in the elderly. Extremity trauma was most common in all three groups, followed by head injuries. The elderly patients were more prone to chest and pelvic injuries and the children most often had femur fractures. Operations were performed in 22 percent of the patients; orthopedic procedures were most frequent. The mortality rate was 6 percent, with 69 percent of the deaths occurring during the initial resuscitation efforts. The mortality rate was significantly higher in the elderly patients (13 percent). The majority of accidents occurred during nighttime hours, especially in the adult group. Half of the accidents occurred on the weekend, with the greatest number on Saturday. One-third of the accidents occurred during the months of October to December. CONCLUSIONS: Pedestrian-motor vehicle trauma is a common injury, with distinct epidemiological features that may be useful in accident prevention strategies.

Accidents, Traffic↗
PubMed1996

Matrix-induced liver cell aggregates (MILCA) for bioartificial liver use.

Ex vivo reproduction of liver microstructure using isolated hepatocytes is critical for bioartificial liver use. We have developed a method of producing matrix-induced liver cell aggregates (MILCA) using a small number of collagen-coated beads as a nidus for formation of hepatocyte aggregates. Porcine hepatocytes were obtained by EDTA/collagenase digestion. Cell viability was assessed by trypan blue exclusion and LDH release. Cytochrome P-450 activity was determined at 4 and 24 hours by measuring the formation of 7-hydroxycoumarine (7-HC) from 7-ethoxycoumarine (7-EC). At 4 hours, the viability of MILCA was 92 +/- 2%, LDH release was 100 +/- 22 U/L and 7-HC formation was 140 +/- 34 nM/g cells. At 24 hours, MILCA viability remained greater than 90%, but 7-HC formation was lower than that of parallel control monolayer hepatocyte cultures (194 +/- 43 vs 481 +/- 78 nM/g cells; p < 0.002). On transmission electron microscopy, MILCA ultrastructure resembled that of a normal liver (maintenance of cell polarity, gap junctions, bile canaliculi, intact organellae, glycogen granules). MILCA were subsequently inoculated into hollow-fiber bioreactors which were perfused for 6 hours with plasma recovered from patients with fulminant hepatic failure (n = 6; 5 x 10(9) cells/cartridge, recirculation of 350 ml of plasma at 400 ml/min). In these studies, lidocaine (20 micrograms/ml) was cleared in less than 3 hours and 7-HC production at 6 hours was 71 +/- 8 nM/g cells. Other MILCA effects noted in this system included lowering of plasma lactate, bilirubin and ammonia and increase in the level of several non-essential amino acids.

Alginates↗
PubMed1992

Incidental preclinical hyperparathyroidism identified during thyroid operations.

The entity of preclinical hyperparathyroidism has never been clearly investigated. The authors believe that the incidence of pathologic abnormalities of the parathyroid glands before the development of any symptoms or hypercalcemia (serum calcium > 12.0 mg/dl) is more frequent than has been reported. Over a 14-year period, parathyroid glands were examined during thyroid operations in over 800 patients. Serum calcium and phosphorous levels were measured in all patients preoperatively. Thirty-six patients had additional parathyroid operations for a preclinical form of hyperparathyroidism, defined by abnormal appearing parathyroid glands at the time of thyroid surgery. None of the 36 patients had symptoms of hyperparathyroidism preoperatively. Nine patients had borderline hypercalcemia (serum calcium 10.6 to 12.0 mg/dl), and the remainder were considered normocalcemic. The average age was 53 (range 21 to 75) with a male to female ratio of 1:3. Nine of the 36 patients had thyroid cancer. There were eight patients with parathyroid adenoma and 28 patients with parathyroid hyperplasia. Of 13 patients who had a history of neck irradiation, five had parathyroid adenoma and eight had parathyroid hyperplasia. Only two patients with parathyroid hyperplasia remain on calcium medication. Since preoperative normocalcemia does not preclude the presence of parathyroid pathology, the authors urge careful identification and examination of the parathyroid glands during thyroid operations. It adds little time to the procedure. Excision of parathyroid disease along with the thyroid gland can be performed safely and prevents the need for further operation with its associated morbidity.

Adult↗
PubMedDate not supplied

Automated liver cell processing facilitates large scale isolation and purification of porcine hepatocytes.

An automated method for large scale isolation and purification of porcine hepatocytes is described. Liver cells were harvested by a two-step portal vein perfusion with ethylenediaminetetraacetate and collagenase. Hepatocyte purification was carried out using either a standard manual processing method (Procedure A) or an automated processing method using a filtration chamber and a programmable cell washer (Procedure B). Both methods produced high cell yields (Procedure A: 1.30 +/- 0.55 x 10(10) viable hepatocytes/liver; Procedure B: 1.38 +/- 0.32 x 10(10) viable hepatocytes/liver) and viability (Procedure A: 89 +/- 6.5%; Procedure B: 92 +/- 3.9%). Hepatocyte purity was significantly greater after Procedure B than after Procedure A (93.1 +/- 3.1% versus 83.1 +/- 3%, p < 0.01). Isolated hepatocytes by either method were morphologically intact, as demonstrated by transmission electron microscopy showing integrity of plasma membranes and intracellular organelles. Cultured hepatocytes isolated by either method were functionally intact, although those isolated by Procedure A showed significantly lower activity of microsomal 7-ethoxycoumarin-O-deethylase activity (p < 0.05) and mitochondrial succinate dehydrogenase activity (p < 0.01). In conclusion, use of the automated hepatocyte processing method resulted in efficient large scale preparation of porcine hepatocytes, with higher purity and greater retention of differentiated liver metabolic functions, and was found to be less time consuming and less labor intensive.

7-Alkoxycoumarin O-Dealkylase↗