Biomedical subjects

N Chen

Publications and source records attributed to N Chen.

At least 127 records · Page 7Linked to original sources

PubMed1998

Preparative electrophoresis in "sieving media" of subcellular-sized particles.

The commercial gel electrophoresis apparatus with intermittent scanning of the migration path and preparative capacity (HPGE-1000, LabIntelligence) is applicable to polymer solutions as well as gels. Unresolved rat liver microsomes can be isolated from 11-15% polyvinylpyrrolidone (PVP) solution by means of a syringe. The automated band isolation technique applied under resolving conditions in dilute polymer solutions allowed for the sequential isolation of three microsome components with 85, 76 and 75% recovery, respectively, under strict control of the dimensions of the volumetric collection module of the HPGE-1000 apparatus. Separations of unlabeled microsomes and sea urchin egg components in dilute polymer solutions have been performed, using detection by "fluorescence reduction". The unlabeled major component of a sea urchin egg homogenate has been isolated from electrophoresis in 1.5% PVP (Mr = 10(6)) solution in 25-50% yield (0.24-4 microg/8 lanes of the HPGE-1000 apparatus). However, since separations of both microsomes and sea urchin egg granules in dilute polymer solutions are restricted to a narrow range of polymer concentrations, their retardation coefficients, KR = d(log mobility)/d(polymer concentration), are not ascertained.

Animals↗

PubMed1998

Preparative application of commercial automated gel electrophoresis apparatus to subcellular-sized particles: sequential isolations, fractions re-run, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, yield and purity.

The analytical and preparative potential of automated gel electrophoresis apparatus with intermittent fluorescence scanning of the migration path, the HPGE-1000 apparatus (LabIntelligence, Belmont, CA) was further developed in application to subcellular-sized particles. Resolution between two rat liver microsome components in agarose (MetaPhor) gel electrophoresis was found to increase with decreasing agarose concentration to 0.04%. It was less, even in an agarose solution at that low concentration, than that in laterally aggregated 4% polyacrylamide gel. The three components of the microsomal preparation were sequentially isolated from 0.6 and 0.8% agarose gel electropherograms. One fraction when re-electrophoresed was found to exhibit the original mobility and did not give rise to the other components. Yields of each component were near-quantitative after one or two electroelution steps. Based on protein content, no impurities could be detected in two of the microsome fractions; the third fraction contained 2% of nonmicrosome impurity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of all three microsome fractions were indistinguishable from one another and from that of the unfractionated microsome preparation.

Animals↗

PubMed1998

Mass spectrometric analysis of integral membrane proteins: application to complete mapping of bacteriorhodopsins and rhodopsin.

Integral membrane proteins have not been readily amenable to the general methods developed for mass spectrometric (or internal Edman degradation) analysis of soluble proteins. We present here a sample preparation method and high performance liquid chromatography (HPLC) separation system which permits online HPLC-electrospray ionization mass spectrometry (ESI-MS) and -tandem mass spectrometry (MS/MS) analysis of cyanogen bromide cleavage fragments of integral membrane proteins. This method has been applied to wild type (WT) bacteriorhodopsin (bR), cysteine containing mutants of bR, and the prototypical G-protein coupled receptor, rhodopsin (Rh). In the described method, the protein is reduced and the cysteine residues pyridylethylated prior to separating the protein from the membrane. Following delipidation, the pyridylethylated protein is cleaved with cyanogen bromide. The cleavage fragments are separated by reversed phase HPLC using an isopropanol/acetonitrile/aqueous TFA solvent system and the effluent peptides analyzed online with a Finnigan LCQ Ion Trap Mass Spectrometer. With the exception of single amino acid fragments and the glycosylated fragment of Rh, which is observable by matrix assisted laser desorption ionization (MALDI)-MS, this system permits analysis of the entire protein in a single HPLC run. This methodology will enable pursuit of chemical modification and crosslinking studies designed to probe the three dimensional structures and functional conformational changes in these proteins. The approach should also be generally applicable to analysis of other integral membrane proteins.

Amino Acid Sequence↗

PubMed1998

Unilateral cricothyroid contraction and glottic configuration.

It is frequently stated that unilateral cricothyroid muscle (CT) paralysis can be diagnosed by physical examination, noting rotation of the glottis, and shortening and vertical displacement of the ipsilateral vocal fold. These signs, however, are inconsistently observed, and there is considerable controversy regarding the direction of glottic rotation. To determine the effects of CT contraction on three-dimensional glottic configuration, we performed computerized tomography on cadaver larynges before and after simulated CT contraction. Radiopaque makers were used to compute distances. Unilateral CT contraction equally increased the length of both membranous vocal folds, and rotated the posterior glottis less than 1 mm. CT contraction neither adducted the vocal processes, nor significantly their altered vertical level. These results suggest that unilateral CT paralysis cannot be diagnosed on the basis of any clinically apparent change in glottal configuration.

Cricoid Cartilage↗

PubMed1998

Integrin-mediated adhesion and signalling in ovarian cancer cells.

The integrins are a family of cell surface receptors which mediate cellular adhesion and signalling events. Our goal was to evaluate integrin function and signalling pathways in ovarian cancer cells. Ovarian cancer cell lines, NIH:OVCAR-3 and NIH:OVCAR-5, exhibited distinct extracellular matrix (ECM) binding preferences which were mediated primarily through beta1 integrin interactions. Western blot analysis was used to identify changes in cellular phosphotyrosine, focal adhesion kinase (FAK) and mitogen activated protein (MAP) kinase. Tyrosine phosphorylation of integrin-associated phosphoproteins was not enhanced in either cell type in response to adhesion onto ECM components or receptor crosslinking. FAK expression was greater in NIH:OVCAR-5 cells while MAP kinase activity was higher in NIH:OVCAR-3 cells. The data suggest that these two ovarian cancer cell lines exhibit specific ECM binding preferences and distinct differences in phosphotyrosine, focal adhesion and MAP kinase expression profiles.

Antigens, CD↗

PubMed1998

Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle.

Mutation of RPE65 can cause severe blindness from birth or early childhood, and RPE65 protein is associated with retinal pigment epithelium (RPE) vitamin A metabolism. Here, we show that Rpe65-deficient mice exhibit changes in retinal physiology and biochemistry. Outer segment discs of rod photoreceptors in Rpe65-/- mice are disorganized compared with those of Rpe65+/+ and Rpe65+/- mice. Rod function, as measured by electroretinography, is abolished in Rpe65-/- mice, although cone function remains. Rpe65-/- mice lack rhodopsin, but not opsin apoprotein. Furthermore, all-trans-retinyl esters over-accumulate in the RPE of Rpe65-/- mice, whereas 11-cis-retinyl esters are absent. Disruption of the RPE-based metabolism of all-trans-retinyl esters to 11-cis-retinal thus appears to underlie the Rpe65-/- phenotype, although cone pigment regeneration may be dependent on a separate pathway.

Animals↗

PubMed1998

Differentiation-specific increase in ALA-induced protoporphyrin IX accumulation in primary mouse keratinocytes.

A treatment regimen that takes advantage of the induction of intracellular porphyrins such as protoporphyrin IX (PPIX) by exposure to exogenous 5-amino-laevulinic acid (ALA) followed by localized exposure to visible light represents a promising new approach to photodynamic therapy (PDT). Acting upon the suggestion that the effectiveness of ALA-dependent PDT may depend upon the state of cellular differentiation, we investigated the effect of terminal differentiation upon ALA-induced synthesis of and the subsequent phototoxicity attributable to PPIX in primary mouse keratinocytes. Induction of keratinocyte differentiation augmented intracellular PPIX accumulation in cells treated with ALA. These elevated PPIX levels resulted in an enhanced lethal photodynamic sensitization of differentiated cells. The differentiation-dependent increase in cellular PPIX levels resulted from several factors including: (a) increased ALA uptake, (b) enhanced PPIX production and (c) decreased PPIX export into the culture media. Simultaneously, steady-state levels of coproporphyrinogen oxidase mRNA increased but aminolaevulinic acid dehydratase mRNA levels remained unchanged. From experiments using 12-o-tetradecanoylphorbol-13-acetate, transforming growth factor beta 1 and calcimycin we demonstrated that the increase in PPIX concentration in terminally differentiating keratinocytes is calcium- and differentiation specific. Stimulation of the haem synthetic capacity is seen in primary keratinocytes, but not in PAM 212 cells that fail to undergo differentiation. Interestingly, increased PPIX formation and elevated coproporphyrinogen oxidase mRNA levels are not limited to differentiating keratinocytes; these were also elevated in the C2C12 myoblast and the PC12 adrenal cell lines upon induction of differentiation. Overall, the therapeutic implications of these results are that the effectiveness of ALA-dependent PDT depends on the differentiation status of the cell and that this may enable selective targeting of several tissue types.

Aminolevulinic Acid↗

PubMed1998

Voltammetric studies on mechanisms of dopamine efflux in the presence of substrates and cocaine from cells expressing human norepinephrine transporter.

The effects of substrates m-tyramine and beta-phenethylamine, as well as cocaine, on the DA efflux from a cell line stably expressing the human norepinephrine transporter (hNET) were investigated by using rotating disk electrode voltammetry. Both the substrates and cocaine induced apparent DA efflux in a concentration-dependent manner. Their EC50 values for inducing DA efflux were similar to their IC50 values for inhibiting DA uptake. The substrate-induced DA efflux was inhibited by various NET blockers, enhanced by raising the internal [Na+] with Na+,K+-ATPase inhibition, but was insensitive to membrane potential-altering agents valinomycin, veratridine, and high [K+]. The initial rate of m-tyramine-induced DA efflux was related to preloaded [DA] in a manner defined by a Michaelis-Menten expression. In contrast, DA efflux in the presence of cocaine displayed a much slower efflux rate, lower efficacy, was not stimulated by elevated internal [Na+], and was nonsaturable with preloaded [DA]. Single exponential kinetic analysis of the entire time course of the DA efflux showed that the apparent first-order rate constant for m-tyramine-induced DA efflux declined with increased preloaded [DA], whereas that for the DA efflux in the presence of cocaine was unchanged with varying preloaded [DA]. These results suggest that the substrates stimulate the NET-dependent DA efflux by increasing the accessibility of the NET to internal DA, whereas cocaine "uncovers" NET-independent DA efflux by reducing the accessibility of diffused/leaked external DA to the NET.

Adrenergic Uptake Inhibitors↗

PubMed1998

Vanadium induces AP-1- and NFkappB-dependent transcription activity.

Vanadate has been reported to be involved in the causation of cancer. In this study, we found that both AP-1 and NFkappaB activities were increased after treatment with sodium vanadate in JB6 cells. Maximum induction of AP-1 and NFkappaB appeared at 48 to 72 h. Phosphorylations of Erks and p38 kinases were markedly increased at 100 microM of vanadate, while phosphorylation of JNKs was not affected. Vanadate also enhances the phosphorylation of IkappaBalpha. These results suggest that the activation of AP-1 and NFkappaB by vanadate may be mediated through enhancement of phosphorylation of Erk/p38 kinases and IkappaBalpha, respectively.

Animals↗

PubMed1998

Objective evaluation of oral and maxillofacial sensory nerves.

OBJECTIVE: The purpose of this article is to recommend a set of methods for the clinical objective evaluation of neurosensory nerves and to establish criteria for the clinical objective evaluation of oral and maxillofacial regions in a Chinese population. METHODS: Sensations in the oral and maxillofacial regions of 35 healthy subjects were objectively and quantitatively evaluated by means of two-point discrimination, static light touch, brush directional stroke, and thermal discrimination. RESULTS: Criteria for the clinical neurosensory evaluation were established for the sensations of the oral and maxillofacial regions of healthy Chinese. CONCLUSIONS: Two-point discrimination, static light touch, brush directional discrimination, and thermal discrimination can provide objective, quantitative information for the evaluation of sensory nerve impairment. These convenient methods represent the important tools in the diagnosis and treatment of sensory nerve impairment in oral and maxillofacial regions.

Adolescent↗

PubMed1998

A clinicopathological study of Alport syndrome and detection of type IV collagen chains in Alport patients.

OBJECTIVE: To summarize the clinical and pathological findings of Alport syndrome (AS), detect the distribution of type IV collagen within basement membrane of patients with AS and evaluate the diagnostic value of indirect immunofluorescence (iIF) study of type IV collagen in AS. METHODS: Fourteen patients belonging to 12 families were collected from January 1990 to June 1996. The clinical examinations include biochemical examination, audiometry and ocular examination. IIF technique was used to detect the location of chains of type IV collagen in 6 renal and 5 skin specimens from 8 Alport patients. RESULTS: Among fourteen patients, 11 were male and 3 female (mean age 29.4 years). Microscopic hematuria was found in 13 patients, and recurrent gross hematuria in 7. All had proteinuria. Three patients presented nephrotic syndrome. Slowly progressive renal failure occurred in 10 of 11 males (11-39 years) and 1 female (40 years). Sensorineural deafness was observed in 9 patients particularly high frequency sound. Anterior lenticonus were presented in 2. Five families transmitted as X-linked dominant (XD) trait and 3 autosomal dominant, 3 autosomal recessive inheritance. In 7 renal biopsies, the findings by light microscopy mostly revealed focal and segmental sclerosis glomerulonephritis (4/7). The results of IF were negative in 4. Ultrastructural studies showed variable thickening, thinning of glomerular basement membrane (GBM) in 7 specimens with lamellation and basket wearing of GBM in 1. Using the iIF technique, the alpha 3, 4, 5 (IV) chains were observed to be absent within both GBM and EBM of 4 male XD-AS patients. Six patients were treated with hemodialysis, 2/6 with transplantation. CONCLUSION: Alport syndrome (AS) is a heterogeneous hereditary disease characterized by progressive hematuric nephritis with or without sensorineural hearing loss and ocular defects. Ultrastructural alterations of GBM are helpful to the diagnosis of AS. IIF study suggests that type IV collagen in basement membrane of AS was abnormal and iIF study of type IV collagen chains distribution is useful for confirming the diagnosis of AS.

Adolescent↗

PubMed1998

[Experimental study of dahuang zhechong pills in the treatment of immunological liver fibrosis in rats].

OBJECTIVE: To study the effect of DHZC on liver fibrosis. METHOD: Immuno-injured fibrosis in rats was induced by bovine serum albumin and DHZC was given during and after the model production respectively. Hepatic collagen contents, pathology of liver and serum hyaluronic acid were measured. Colchicine was used as control. RESULT: It shows that the liver fibrosis rates of the DHZC treated group during and after the model production were 72.7% and 71.2% respectively, while those of the untreated and colchicine group were 92.3%-100%. Compared with the untreated group, there was a remarkable decrease of hepatic collagen contents in the DHZC-treated rats. CONCLUSION: DHZC have thus been proved helpful in reversing liver fibrosis to some extent.

Animals↗

PubMed1998

[Quantitative studies of taste and fungiform papillae on the anterior human tongue].

OBJECTIVE: To study qantitatively in the taste and fungiform papillae on the anterior human tongue. METHODS: Sixty normal adults were evaluated the spatial taste and the fungiform papilla density at spatially-matched sides on the right and left sides of the anterior tongue. The taste stimulative solutions were delivered to a spatially-matched flow chamber attached to the surface of the right or left anterior tongue. RESULTS: (1) There is no statistical difference in number of the fungiform papillae, taste threshold and magnitude matching between right/left, male/female and races; (2) There is a significant difference in fungiform papilla density and taste functions in different individuals; (3) The fungiform papilla density is the key to taste function on 2/3 anterior of tongue, and is positively correlated to it; (4) The use of a spatially-matched flow chamber is a valid test system for overall, objective and accurate evaluating taste on the 2/3 anterior of tongue, and can be used to study quantitatively the fungiform papilla density. CONCLUSION: It is of clinical significance for the diagnosis of taste diseases and the dynamic studies of the taste and the fungiform papillae after lingual nerve injury and repair.

Adolescent↗

PubMed1997

Capillary electrophoresis of DNA fragments in 9 to 20% uncrosslinked polyacrylamide gels: unique separating capacity hypothetically related to maintenance of random-coil DNA conformation independently of gel concentration.

DNA fragments (0.1 to 2 kb) were separated by capillary electrophoresis (CE) in 9 to 20% uncrosslinked polyacrylamide gels with a resolving power ranging from 3 to o.1 million theoretical plates/meter across that DNA size range. The unique feature of electrophoresis in 18 to 20% uncrosslinked polyacrylamide is that it provides a method capable of resolving charge isomeric species of DNA fragments (0.4 to 2 kb), confirming a previous report by Heiger et al. [Heiger DN, Cohen AS, Karger BL. J Chromatogr 516 (1990) 33-48]. A similarly unique resolving capacity of uncrosslinked polyacrylamide gels for DNA previously reported is that for heteroduplex DNA [Pulyaeva H, Zakharov SF, Garner MM, Chrambach A. Electrophoresis 15 (1994) 1095-1100] matched by crosslinked gels only in the presence of denaturants [Peeters AV, Kotze MJ. PCR Methods Appl 4 (1994) 188-190; Ganguly A, Rock MJ, Prockop DJ. Proc Natl Acad Sci USA 90 (1993) 10 325-10 329]. A clue as to the cause of that unique resolving capacity of crosslinked polyacrylamide is provided by the finding in the present study of a single, gel concentration independent KR [retardation coefficient, d(log mobility)/d(gel concentration)] for the DNA fragments, which contrasts with the decrease of KR with gel concentration observed for crosslinked polyacrylamide across a wide concentration range [Orban L, Chrambach A. Electrophoresis 12 (1991) 241-246; Tietz D, Chrambach A. Electrophoresis 14 (1993) 185-190]. Since the decrease of KR with gel concentration correlates with a decrease in equivalent molecular radius [Tietz D, Chrambach A. Electrophoresis 14 (1993) 185-190], it has been interpreted as being due to the transition from a random-coiled to a stretched DNA conformation upon passage through gels of increasing concentration. Since in uncrosslinked gels the decrease of KR does not occur, it is correspondingly assumed that the random-coil conformation of DNA is maintained in those gels in the investigated concentration range up to 20%. The maintenance of random-coil conformation [Tietz D, Chrambach A. Electrophoresis 14 (1993) 185-190]. The effect of denaturants in allowing for resolution of heteroduplex DNA in crosslinked gels [Peeters AV, Kotze MJ. PCR Methods Appl 4 (1994) 188-190; Ganguly A, Rock MJ, Prockop DJ, Proc Natl Acad Sci USA 90 (1993) 10 325-10 329] supports that hypothesis of the enhanced resolving power of electrophoresis in gels that maintain random-coiled DNA within the gel concentration range used.

Acrylic Resins↗

PubMed1997

Glutamate-194 to cysteine mutation inhibits fast light-induced proton release in bacteriorhodopsin.

Substitution of glutamic acid-194, a residue on the extracellular surface of bacteriorhodopsin, with a cysteine inhibits the fast light-induced proton release that normally is coupled with the deprotonation of the Schiff base during the L to M transition. Proton release in this mutant occurs at the very end of the photocycle and coincides with deprotonation of the primary proton acceptor, Asp-85, during the O to bR transition. the E194C mutation also results in a slowing down of the photocycle by about 1 order of magnitude as compared to the wild type and produces a strong effect on the pH dependence of dark adaptation that is interpreted as a drastic reduction or elimination of the coupling between the primary proton acceptor Asp-85 and the proton release group. These data indicate that Glu-194 is a critical component of the proton release complex in bacteriorhodopsin.

Bacteriorhodopsins↗

PubMed1997

Proton uptake and release are rate-limiting steps in the photocycle of the bacteriorhodopsin mutant E204Q.

In the absence of the putative proton release group, E204, the second half of the photocycle of the E204Q mutant of bacteriorhodopsin is slowed down more than 10-fold compared to the wild type. The effects of pH and D2O on the M decay and O formation rates in E204Q suggest that proton uptake occurs concurrently with the N <--> O transition, possibly coupled with the thermal reisomerization of the retinal. Hence, one of the rate-limiting steps in the slow E204Q photocycle is proton uptake from the outside medium, coincident with the decay of the slow component of M (the N <--> O transition). The second rate-limiting step is the long lifetime of decay of the O state, due to a high activation barrier for the deprotonation of D85 in the O --> bR step of the E204Q photocycle. Addition of the weakly acidic anions azide, cyanate, or formate accelerates the decay of the O intermediate, and restores the total photocycling time to that observed in the wild-type pigment, by accelerating the deprotonation of D85. We also find that azide similarly accelerates the decay of O in the wild type under conditions in which E204 does not deprotonate during the photocycle (pH < 6). It has previously been shown that azide and other weak acids can influence proton transfers in the cytoplasmic half of the protein [Tittor, J., Soell, C., Oesterhelt, D., Butt, H.-J., & Bamberg, E. (1989) EMBO J. 8, 3477-3482]; we suggest that these weak acids can affect proton transfers in the extracellular half of the protein as well.

Azides↗

PubMed1997

Preparative electrophoresis in a concentrated polymer solution: automated procedure for microsome isolation.

A recently introduced automated gel electrophoresis apparatus with an intermittent scanning of the migration path, the HPGE 1000 apparatus (LabIntelligence, Belmont, CA) has a unique preparative feature: A recovery cup can be automatically positioned on the band of interest, which is then electroeluted with fluorescence monitoring of recovery. Electroelution time is extended stepwise until quantitative recovery is attained. This preparative procedure has been extended from electrophoresis in gels to that of subcellularsized particles in concentrated polymer solutions Chang et al., Electrophoresis 17, 776-780, 1996). However, that application required manual rather than computer-directed positioning of the recovery chamber over the band. The present report details a modification of instrumentation and procedure by which automated operation was restored. The modified procedure extends the benefits of automation and known quantitative recovery to isolation of particles too large to enter into gels.

Animals↗

PubMed1997

Isolation of subcellular-sized particles separated by electrophoresis in dilute polymer solution, using commercial electrophoresis apparatus with intermittent scanning of fluorescence.

Resolution of subcellular-sized particles in electrophoresis employing semi-dilute polymer solutions as "sieving media" improves as the polymer concentration is decreased. Therefore, the previously reported conditions of preparative electrophoresis of microsomes, using concentrated (12%) polyvinylpyrrolidone (PVP) solutions, while solving the problem of non-entrance of large particles into "sieving media", do not provide adequate resolving capacity, as exemplified by failure of the microsome preparation used, to resolve in the manner of gels or dilute solutions. The present report provides the conditions under which the HPGE-1000 apparatus can be preparatively applied when the electrophoretic separation is effectively conducted in a dilute polymer solution. The isolation of three microsome components under those conditions constitutes the first application of "particle sieving", i.e., a separation due preponderantly to size and shape differences, at a preparative scale.

Animals↗