Biomedical subjects

N Chen

Publications and source records attributed to N Chen.

At least 145 records · Page 8Linked to original sources

PubMed1997

The resolution between two native proteins and between their sodium dodecyl sulfate-complexes in agarose and polyacrylamide gel electrophoresis.

Commercial gel electrophoresis apparatus with intermittent fluorescence scanning of the migration path (HPGE-1000 apparatus, LabIntelligence) makes it possible to measure band width and migration distance as a function of the duration of electrophoresis. As a result, resolution can be evaluated quantitatively and therefore different gel media can be compared objectively. The resolution of fluorescein carboxylate labeled conalbumin (molecular mass 86 kDa) and soybean trypsin inhibitor (22.7 kDa) in gel electrophoresis was found to increase as a function of the gel type in the order SeaKem GTG-, SeaKem Gold-agarose, 2% N,N'-methylenebisacrylamide cross-linked polyacrylamide, MetaPhor-XR-, and SeaPrep-agarose. The advantage in resolving capacity of SeaPrep agarose over the polyacrylamide gel was by a factor of up to five. The resolving capacity of the agaroses was in indirect relation to the degree of electroendosmosis. In all media, resolution increased with migration distance (time). The same proteins when reacted with sodium dodecyl sulfate (SDS) resolve (i) better at up to 6% SeaPrep agarose concentration than in polyacrylamide, as in the gel electrophoresis of the native proteins; (ii) less effectively, by contrast, at SeaPrep agarose concentrations > 6%, than in polyacrylamide gel; and (iii) significantly better in 4-6% SeaPrep agarose than in 4-6% SeaKem GTG agarose. Since Ferguson plot analysis in both agarose and polyacrylamide gels shows that the two SDS-proteins are larger than the native proteins with which they are complexed, the superiority of polyacrylamide gels above 7% appears to be correlated with the fact that its mean pore radius, estimated for both media using identical assumptions and identical rigid spherical standards - proteins, is approximately seven times larger than that of SeaPrep agarose in the concentration range of 3-8%, and that therefore the molecular "fit" in polyacrylamide is closer than that in SeaPrep agarose of the concentration range used. The dependence of resolution on the ratio of particle radius to mean pore radius ("fit") is also suggested by the fact that the two SDS-proteins resolve in a biphasic dependence on gel concentration in both agarose and polyacrylamide, with a maximum at 6% agarose and 10% polyacrylamide.

Conalbumin↗

PubMed1997

Mutation of a surface residue, lysine-129, reverses the order of proton release and uptake in bacteriorhodopsin; guanidine hydrochloride restores it.

K129 is a residue located in the extracellular loop connecting transmembrane helices D and E of bacteriorhodopsin. Replacement of K129 with a histidine alters the pKa's of two key residues in the proton transport pathway, D85, and the proton release group (probably E204); the resulting pigment has properties that differ markedly from the wild type. 1) In the unphotolyzed state of the K129H mutant, the pKa of D85 is 5.1 +/- 0.1 in 150 mM KCl (compared to approximately 2.6 in the wild-type bacteriorhodopsin), whereas the unphotolyzed-state pKa of E204 decreases to 8.1 +/- 0.1 (from approximately 9.5 in the wild-type pigment). 2) The pKa of E204 in the M state is 7.0 +/- 0.1 in K129H, compared to approximately 5.8 in the wild-type pigment. 3) As a result of the change in the pKa of E204 in M, the order of light-induced proton release and uptake exhibits a dependence on pH in K129H differing from that of the wild type: at neutral pH and moderate salt concentrations (150 mM KCl), light-induced proton uptake precedes proton release, whereas it follows proton release at higher pH. This pumping behavior is similar to that seen in a related bacterial rhodopsin, archaerhodopsin-1, which has a histidine in the position analogous to K129. 4) At alkaline pH, a substantial fraction of all-trans K129H pigment (approximately 30%) undergoes a conversion into a shorter wavelength species, P480, with pKa approximately 8.1, close to the pKa of E204. 5) Guanidine hydrochloride lowers the pKa's of D85 and E204 in the ground state and the pKa of E204 in the M intermediate, and restores the normal order of proton release before uptake at neutral pH. 6) In the K129H mutant the coupling between D85 and E204 is weaker than in wild-type bacteriorhodopsin. In the unphotolyzed pigment, the change in the pKa's of either residue when the other changes its protonation state is only 1.5 units compared to 4.9 units in wild-type bacteriorhodopsin. In the M state of photolyzed K129H pigment, the corresponding change is 1 unit, compared to 3.7 units in the wild-type pigment. We suggest that K129 may be involved in stabilizing the hydrogen bonding network that couples E204 and D85. Substitution of K129 with a histidine residue causes structural changes that alter this coupling and affect the pKa's of E204 and D85.

Bacteriorhodopsins↗

PubMed1997

A novel poxvirus gene and its human homolog are similar to an E. coli lysophospholipase.

A novel poxvirus gene has been characterized within the genome of ectromelia virus. It has significant similarity to a family of lysophospholipases suggesting that it may function in the degradation of lysophospholipids. Since these molecules are active in the stimulation of inflammation, we hypothesize that this gene may play a role in virus virulence. This gene is expressed early in the ectromelia virus replication cycle, before DNA replication. We have also characterized a human cDNA that encodes a protein which is 49.5% identical to the ectromelia virus protein. By its presence in multiple cDNA libraries, this human gene is known to be expressed in a variety of body tissues and is likely to function in the normal regulation of lysophospholipid levels. This family of proteins have conserved blocks of amino acids that are indicative of a serine-aspartic acid-histidine catalytic triad, similar to those used by true lipases and a number of esterases.

Amino Acid Sequence↗

PubMed1997

Chemosensory and somatosensory regeneration after lingual nerve repair in humans.

PURPOSE: The objective of these studies was to measure the impact of Class IV and V lingual nerve injuries on taste sensitivity and taste receptor density of the anterior tongue before and after microneurosurgical repair. MATERIALS AND METHODS: Citric acid detection threshold and suprathreshold magnitude response were measured on the anterior tongue in 12 adult volunteers with unilateral lingual nerve Class IV or V injuries. The right and left sides of the anterior tongue were tested at each session to assess the effect of nerve damage before and 1, 3, 6, and 12 months after repair. Whole-mouth threshold and suprathreshold scales of citric acid taste intensity were measured. The level of sensory impairment was scaled at each test session using a clinical neurosensory test algorithm. Finally, patients completed an 11-item instrument survey that queried the patient's perceived expectations of surgery on sensory, taste, and general health before surgery and the patient's perceived outcome of surgery at each postrepair session. The patient's perceived global satisfaction of surgery was also assessed. RESULTS: All 12 patients failed to detect and scale citric acid and had complete or severe sensory impairment on the injured side of the anterior tongue. One year after repair, 50% of the patients demonstrated a substantial increase in the number of fungiform papillae, pores, and ratio of pores/papilla at the same time as they demonstrated the ability to detect and scale citric acid. After repair, patients perceived the greatest improvements in the categories of eating, chewing, feeling, and taste, and the least in speech. CONCLUSION: Lingual nerve repair may result in significant changes in somatosensory and chemosensory function and taste bud anatomy on the anterior tongue over time.

Adult↗

PubMed1997

Lethal Giardia from a wild-caught sulphur-crested cockatoo (Cacatua galerita) established in vitro chronically infects mice.

An axenic culture of Giardia was established from a sample of infected intestine obtained following autopsy of a sulphur-crested cockatoo (Cacatua galerita). The cockatoo recently captured in the wild and with good muscle tone died along with several other cage mates, apparently of an overwhelming, acute infection of Giardia. Trophozoites which established in the traditional, axenic Giardia medium (TYI-S-33 with supplementary bile) were morphologically identical to G. duodenalis. When outbred Quackenbush Swiss neonatal mice were infected with trophozoites a chronic infection was established and parasites were still present at 38 days post-inoculation. Weight gain by infected mice was reduced by 20%, thus mimicking failure-to-thrive syndrome in children, and maximum parasite load was more than 3-fold higher in comparison with other G. duodenalis strains. Analysis of the electrophoretic karyotype, rDNA and hybridization studies together with Giemsa- and trichrome-stained samples, and scanning electron microscopy indicated that the bird-derived Giardia belonged to the duodenalis group. This is the first report of infection of mammals with Giardia isolated from a bird. These data may have potentially serious implications for contamination of watersheds and establishment of zoonotic infections.

Animals↗

PubMed1997

p27Kip1 induces an accumulation of the repressor complexes of E2F and inhibits expression of the E2F-regulated genes.

p27Kip1 is an inhibitor of the cyclin-dependent kinases and it plays an inhibitory role in the progression of cell cycle through G1 phase. To investigate the mechanism of cell cycle inhibition by p27Kip1, we constructed a cell line that inducibly expresses p27Kip1 upon addition of isopropyl-1-thio-beta-D-galactopyranoside in the culture medium. Isopropyl-1-thio-beta-D-galactopyranoside-induced expression of p27Kip1 in these cells causes a specific reduction in the expression of the E2F-regulated genes such as cyclin E, cyclin A, and dihydrofolate reductase. The reduction in the expression of these genes correlates with the p27Kip1-induced accumulation of the repressor complexes of the E2F family of factors (E2Fs). Our previous studies indicated that p21WAF1 could disrupt the interaction between cyclin/cyclin-dependent kinase 2 (cdk2) and the E2F repressor complexes E2F-p130 and E2F-p107. We show that p27Kip1, like p21WAF1, disrupts cyclin/cdk2-containing complexes of E2F-p130 leading to the accumulation of the E2F-p130 complexes, which is found in growth-arrested cells. In transient transfection assays, expression of p27Kip1 specifically inhibits transcription of a promoter containing E2F-binding sites. Mutants of p27Kip1 harboring changes in the cyclin- and cdk2-binding motifs are deficient in inhibiting transcription from the E2F sites containing reporter gene. Moreover, these mutants of p27Kip1 are also impaired in disrupting the interaction between cyclin/cdk2 and the repressor complexes of E2Fs. Taken together, these observations suggest that p27Kip1 reduces expression of the E2F-regulated genes by generating repressor complexes of E2Fs. Furthermore, the results also demonstrate that p27Kip1 inhibits expression of cyclin A and cyclin E, which are critical for progression through the G1-S phases.

Animals↗

PubMed1997

Telomeric organization of a variable and inducible toxin gene family in the ancient eukaryote Giardia duodenalis.

Giardia duodenalis is the best-characterized example of the most ancient eukaryotes, which are primitively amitochondrial and anaerobic. The surface of Giardia is coated with cysteine-rich proteins. One family of these proteins, CRP136, varies among isolates and upon environmental stress. A repeat region within the CRP136 family is interchangeable by a cassette-like mechanism, generating further diversity in repeat size, copy number, and sequence. Flanking the 5' region of the CRP136 family is a novel protein kinase gene and an ankyrin homolog, creating a conserved unit. A short spacer separates the ankyrin gene from the variable, tandem array of rDNA gene units at a common breakpoint within the large subunit gene, which is followed by the (TAGGG)n telomeric sequence. Transcriptional up-regulation of the CRP136 family is accompanied by a switch in mRNA length and promoter, of de novo expression, and suggests that CRP136 mRNA induction is under the control of a telomerically regulated position effect, which evolved very early in the eukaryotic lineage.

Animals↗

PubMed1997

Mutation of arginine 134 to lysine alters the pK(a)s of key groups involved in proton pumping by bacteriorhodopsin.

Arginine 134 is located near the extracellular surface of bacteriorhodopsin (bR) and may interact with one or more nearby glutamate residues. In the bR mutant R134K, light-induced Schiff-base deprotonation (formation of the M intermediate) exhibits several kinetic components and has a complex pH dependence. The kinetics and pH dependence of M formation were analyzed using the following general guidelines for interpreting M formation: (1) The fastest component of M formation reflects the redistribution of the Schiff-base proton to D85, the usual proton acceptor, in response to the change in the proton affinities of the Schiff base and D85 early in the photocycle; (2) Two additional components of M formation reflect transitions between spectroscopically similar substates of M. By applying these guidelines, supplemented by information about the pK(a)s of D85 and the proton release group from acid (purple-to-blue) and alkaline titrations of the absorption spectra of the unphotolyzed R134K pigment, we explain the pH dependence of M formation as being due to titration of the counterion, D85, and of the proton release group. We calculate, in R134K, that the pKa of D85 is 4.6 in the unphotolyzed state, while the pKa of the proton release group is 8.0 in the unphotolyzed state but drops to approximately 5.8 in the M intermediate. The same value for the pKa of the proton release group in the M intermediate is obtained when we use photocurrent measurements to monitor proton release. The altered values of these pK(a)s relative to the corresponding values in wild-type bR suggest that D85 and the proton release group are coupled more weakly in R134K than in the wild type.

Arginine↗

PubMed1997

Differential sensitivity of recombinant N-methyl-D-aspartate receptor subtypes to zinc inhibition.

Zinc has been shown to be present in synaptic vesicles of a subset of glutamatergic boutons and is believed to be core-leased with glutamate at these synapses. A variety of studies have suggested that zinc might play a role in modulation of excitatory transmission, as well as excitotoxicity, by inhibiting N-methyl-D-aspartate (NMDA)-type glutamate receptors. To further investigate the modulatory effects of zinc on NMDA receptors of different subunit compositions, we coexpressed the recombinant subunit NR1 with NR2A and/or NR2B in HEK 293 cells. In whole-cell patch-clamp recordings from these transfected cells, zinc inhibited peak glutamate-evoked current responses in a noncompetitive manner, but there were significant differences between the receptor subtypes in sensitivity to zinc inhibition. For NR1/NR2A, approximately 40% of the peak current was inhibited by zinc in a voltage-independent manner with an IC50 value of 5.0 +/- 1.6 nM and at a V(H) value of -60 mV; the remainder was blocked at a second, voltage-dependent site with an IC50 value of 79 +/- 18 microM. In contrast, NR1/NR2B currents showed nearly complete inhibition at a voltage-independent site with an IC50 value of 9.5 +/- 3.3 microM. Cells cotransfected with NR1, NR2A, and NR2B showed zinc sensitivity intermediate between that characteristic of NR1/NR2A and that of NR1/NR2B. Furthermore, zinc accelerated the macroscopic desensitization of both NR1/NR2A and NR1/NR2B in a dose-dependent manner, apparently independently of glycine-sensitive desensitization and Ca2(+)-dependent inactivation; maximal effects were to decrease desensitization time constants for NR1/NR2A by approximately 75% and for NR1/NR2B by approximately 90%. Differential modulation of NR1/NR2A and NR1/NR2B currents by zinc may play a role in regulating NMDA receptor-induced synaptic plasticity and neurotoxicity.

Animals↗

PubMed1997

Changes in length and spatial orientation of the vocal fold with arytenoid adduction in cadaver larynges.

Videoendoscopy suggests that arytenoid adduction (AA) surgery not only medializes the paralyzed vocal fold, but increases the length of its membranous portion so that it more closely resembles the normal side. This could represent either real length change or out-of-plane rotation. Computed tomography scanning was performed on adult male cadaver larynges before and after the AA procedure to measure changes in length and spatial orientation of the vocal fold. Three-dimensional coordinates of radiopaque markers on the anterior commissure, posterior glottic midline, and vocal processes were determined. The distance between the vocal processes was 3.9 mm before, and 0.8 mm after AA. The mean vocal fold length was 12.4 mm before, and 13.4 mm after AA (p = .14). The vocal process moved consistently caudally, an average of 3.5 mm (p = .02). The data suggest that clinically apparent vocal fold length changes with AA could be an illusion due to vertical displacement of the vocal process, and not actual lengthening.

Adult↗

PubMed1997

Characterization of voltage- and Ca(2+)-activated K+ channels in squid olfactory receptor neurons.

We performed whole-cell voltage-clamp experiments on isolated olfactory neurons from the squid Lolliguncula brevis. Total outward currents were composed of three identifiable K+ currents: a delayed rectifier K+ current that showed slow inactivation and was sensitive to 5 mmol l-1 tetraethylammonium; a rapidly inactivating, 4-aminopyridine (4-AP)-sensitive, A-type K+ current and a Ca(2+)-sensitive K+ current that was blocked by 200 nmol l-1 charybdotoxin and 10 mmol l-1 Cd2+ but was insensitive to apamin. The proportion of each current type varied from cell to cell, suggesting that responses to a given odorant would depend of the complement of channels present. The kinetics of the K+ currents were affected by temperature, with Q10 values ranging from 2 to 6. The identification and characterization of these K+ currents will greatly aid our understanding of action potential generation in these cells and will facilitate modelling of how odor responses are transduced and coded in squid olfactory receptor neurons.

Animals↗

PubMed1997

[Immunohistochemical study of type IV collagen chain distribution within basement membrane in patients with Alport syndrome].

To observe the distribution of type IV collagen chains within basement membrane in patients with Alport syndrome (AS), discuss the pathogenesis of AS and assess the diagnostic value of indirect immunofluorescent study of type IV collagen chains. By using indirect immunofluorescence technique, the distribution of different chains of type IV collagen in specimen of renal (6 specimens) and skin (5 specimens) basement membrane of 8 AS patients belonging to 7 kindreds were investigated. In 4 male patients with X-linked dominant (XD) AS, the specific monoclonal antibodies against NC1 domains of the alpha 3, alpha 4 and alpha 5 chains of type IV collagen failed to localize on glomerular basement membrane (GBM) and the alpha 5 chain of type IV collagen were also absent in the epidermal basement membrane (EBM). The results of immunofluorescent study were positive in 3 autosomal dominant and 1 autosomal recessive AS patients as same as that in control. AS presents a biochemical abnormality of type IV collagen. The pathogenesis of XD-AS may associate with the abnormal products of alpha 5 chain. The immunofluorescent study of type IV collagen chains distribution within GBM and EBM by using monoclonal antibodies is useful in confirming the diagnosis of AS, especially XD-AS.

Adolescent↗

PubMed1997

[Evaluating methods and effects of repairing injured lingual nerves on human].

We evaluated the regenerations of sense and taste on the anterior 2/3 of the tongue following repairing injured lingual nerve. Injured lingual nerves in 14 patients were repaired with anastomosis of the nerve epineurium. On the anterior 2/3 of the ipsilateral tongue, most of the fungiform papillae atrophied and disappeared, the sense and taste degenerated after severe injury to lingual nerve. Following repair of the injured lingual nerve with anastomosis of nerve epineurium, the papillae and their taste pores can regenerated, 50% of the patients recovered their tongue sense and 35.71% of the patients recovered their tongue taste 1 year after the repair. It is objective, accurate and reliable to evaluate the regenerations of sense and taste on the anterior 2/3 of the tongue after repair of injured lingual nerve by sensory test, taste evaluation, quantitative observation of fungiform papillae, and their taste pores.

Adult↗

PubMed1997

[Oral candidiasis in Sjögren's syndrome: prevalence, clinical features and treatment].

The clinical feature of 20 cases with oral candidiasis in Sjögren's syndrome (SS) were reported, and the treatment effect by topical nystatin smearing, 2% sodium bicarbonate solution rinsing, and transfer factor injecting for about 2 months were analysed. The results showed that the diagnostic criterion of oral candidiasis in SS included two aspects: the SS changes confirmed by labial gland biopsy and candidal hyphae and spores found in a smear. The results indicated that the comprehensive treatments were proved to be effective for oral candidiasis in SS.

Adolescent↗

PubMed1997

[Total synthesis of nordihydroguaiaretic acid].

beta-Keto ester(5) was obtained from vanilin through etherification, oxidation and condensation with acetoacetic ester, (5) on oxidative coupling reaction by NaOEt/I2 produced dimer (6) in high yield. Acid catalyzed cyclodehydration of (6) gave the furan derivative(7), and by a series of selective hydrogenation nordihydroguaiaretic acid, furoguaiacin dimethyl ether and dihydroguaiaretic acid dimethyl ether were synthesized.

Furans↗

PubMed1997

[Characteristics of virus growth factor (VGF) encoded by vaccinia virus Tian Tan strain].

Vaccinia virus growth factor (VGF) is encoded by an early gene located near the border between the terminal inverted repeats and the internal unique region of the viral genome. It has been suggested that secreted VGF may serve to stimulate the metabolism of neighboring uninfected cells, thereby increasing the capacity of these cells to support viral replication. In this study, we cloned and sequenced the VGF gene of vaccinia virus strain Tian Tap. The analytical results indicated that VGF of strain Tian Tan possessed the core sequences of Epidermal growth factor superfamily, and more closely related to that of strain WR than to those of other orthopoxvirus strains. Overall deduced amino acid divergence of 4.3% was observed between Tian Tan and WR VGF protein, while divergences ranged from 10% to 20% compared to other orthopoxviruses strains. Most of the amino acid substitutions were found within signal peptide and transmembrane regions, but were not present in secreted ligand domain. It was striking that the amino acid sequence deduced from the ORF TB22L which located within the right terminal region of genome was identical to VGF protein amino acids 67-140, suggesting that ORF TB22L may be derived from the seconed copy of VGF gene of strain Tian Tan.

Intercellular Signaling Peptides and Proteins↗

PubMed1996

Improved resolution in the gel electrophoresis of proteins by a periodically interrupted electric field.

The capability of the commercial gel electrophoresis apparatus with intermittent scanning of fluorescence (HPGE-1000, LabIntelligence) to provide time-dependent zone dispersion allows one to quantitate resolution. Using a model protein separation, that between phycoerythrin and fluorescein-labeled conalbumin, resolution was compared between separations conducted at a constant field strength of 80 V/cm and one conducted in 10-s pulses of the same field strength, interrupted periodically by 120 s in the absence of an electric field. Resolution was improved by a factor of two in the discontinuous application of the electric field compared to that obtained in its continuous application. Similarly, the intermittent application of 80 V/cm for 10 s, followed by 120-s pauses, gave rise to twice the resolution obtained from a continuous application of 7 V/cm.

Computer Graphics↗

PubMed1996

The role of interleukin-4 in the induction phase of allogeneic neonatal tolerance.

We previously reported that prolonged graft survival in neonatally tolerant mice was associated with enhanced Th2/Th1 cytokines. To determine whether Th2 CD4 cells function in tolerance, we examined whether we could prevent tolerance by blocking Th2 CD4 maturation, using anti-interleukin (IL)-4 monoclonal antibody treatment during neonatal antigen exposure. Anti-IL-4 treatment restored the ability BALB/c of mice to reject A/J skin grafts and blocked the induction of tolerance through multiple mechanisms. Anti-IL-4 treatment blocked the development of donor microchimerism and recovered the ability of mice to proliferate and to generate appropriate delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte (CTL) responses against A/J in a dose-dependent manner. Low-dose anti-IL-4 recovered DTH responses and interferon (IFN)-gamma production, but failed to completely prevent IL-4 production or to recover the CTL activity. No A/J-reactive IFN-gamma-producing CD8 cells were detected in these mice. In contrast, mice treated with higher doses of anti-IL-4 generated normal CTL responses against A/J, and contained A/J-reactive IFN-gamma-producing CD8 cells. The recovery of CTL responses and IFN-gamma-producing CD8 cells was associated with a more complete blocking of Th2 cytokine production. Therefore, the presence of IL-4 may play an important role in the induction of neonatal tolerance by shifting maturation of CD4 cells toward Th2 cells and away from Th1 cells, and also by preventing maturation of alloreactive CD8 CTL cells.

Animals↗