Biomedical subjects

N Chen

Publications and source records attributed to N Chen.

At least 109 records · Page 6Linked to original sources

PubMed1999

Transient and persistent tetrodotoxin-sensitive sodium currents in squid olfactory receptor neurons.

Squid olfactory receptor neurons are primary bipolar sensory neurons capable of transducing water-born odorant signals into electrical impulses that are transmitted to the brain. In this study, we have identified and characterized the macroscopic properties of voltage-gated Na+ channels in olfactory receptor neurons from the squid Lolliguncula brevis. Using whole-cell voltage-clamp techniques, we found that the voltage-gated Na+ channels were tetrodotoxin sensitive and had current densities ranging from 5 to 169 pA pF-1. Analyses of the voltage dependence and kinetics revealed interesting differences from voltage-gated Na+ channels in olfactory receptor neurons from other species; the voltage of half-inactivation was shifted to the right and the voltage of half-activation was shifted to the left such that a "window-current" occurred, where 10-18% of the Na+ channels activated and did not inactivate at potentials near action potential threshold. Our findings suggest that in squid olfactory neurons, a subset of voltage-gated Na+ channels may play a role in generating a pacemaker-type current for setting the tonic levels of electrical activity required for transmission of hyperpolarizing odor responses to the brain.

Animals↗

PubMed1999

Two groups control light-induced Schiff base deprotonation and the proton affinity of Asp85 in the Arg82 his mutant of bacteriorhodopsin.

Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 micros to 75 micros with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.

Absorption↗

PubMed1999

Codon usage in Plasmodium vivax nuclear genes.

Codon usage in Plasmodium vivax nuclear genes was analysed and compared with that in Plasmodium falciparum nuclear genes. Preferred codons were determined for P. vivax. Unlike P. falciparum, P. vivax genes are about 15% less A+T rich in the coding regions, with no obvious A+T bias at the third position of the codons. The amino-acid composition of P. vivax gene products is also different from that of P. falciparum. These results provide valuable information to facilitate gene cloning as well as expression and transfection studies for P. vivax.

Amino Acids↗

PubMed1999

Structure of the TRAIL-DR5 complex reveals mechanisms conferring specificity in apoptotic initiation.

TRAIL, an apoptosis inducing ligand, has at least four cell surface receptors including the death receptor DR5. Here we report the crystal structure at 2.2 A resolution of a complex between TRAIL and the extracellular region of DR5. TRAIL forms a central homotrimer around which three DR5 molecules bind. Radical differences in the surface charge of the ligand, together with variation in the alignment of the two receptor domains confer specificity between members of these ligand and receptor families. The existence of a switch mechanism allowing variation in receptor domain alignment may mean that it is possible to engineer receptors with multiple specificities by exploiting contact positions unique to individual receptor-ligand pairs.

Amino Acid Sequence↗

PubMed1999

BPD-MA-mediated photosensitization in vitro and in vivo: cellular adhesion and beta1 integrin expression in ovarian cancer cells.

Benzoporphyrin derivative monoacid (BPD-MA) photosensitization was examined for its effects on cellular adhesion of a human ovarian cancer cell line, OVCAR 3, to extracellular matrix (ECM) components. Mild BPD-MA photosensitization (approximately 85% cell survival) of OVCAR 3 transiently decreased adhesion to collagen IV, fibronectin, laminin and vitronectin to a greater extent than could be attributed to cell death. The loss in adhesiveness was accompanied by a loss of beta1 integrin-containing focal adhesion plaques (FAPs), although beta1 subunits were still recognized by monoclonal antibody directed against human beta1 subunits. In vivo BPD-MA photosensitization decreased OVCAR 3 adhesiveness as well. Photosensitized adhesion was reduced in the presence of sodium azide and enhanced in deuterium oxide, suggesting mediation by singlet oxygen. Co-localization studies of BPD-MA and Rhodamine 123 showed that the photosensitizer was largely mitochondrial, but also exhibited extramitochondrial, intracellullar, diffuse cytosolic fluorescence. Taken together, these data show that intracellular damage mediated by BPD-PDT remote from the FAP site can affect cellular-ECM interactions and result in loss of FAP formation. This may have an impact on long-term effects of photodynamic therapy. The topic merits further investigation.

Animals↗

PubMed1999

Life without huntingtin: normal differentiation into functional neurons.

Huntington disease (HD) is a neurodegenerative disorder associated with polyglutamine expansion in a recently identified protein, huntingtin. Huntingtin is widely expressed and plays a crucial role in development, because gene-targeted HD-/- mouse embryos die early in embryogenesis. To analyze the function of normal huntingtin, we have generated HD-/- embryonic stem (ES) cells and used an in vitro model of ES cell differentiation to analyze their ability to develop into neuronal cells. Expression analysis of wild-type ES cells revealed that huntingtin is expressed at all stages during ES cell differentiation with high expression in neurons. Expression levels increased with the maturation of differentiating neurons, demonstrating that expression of huntingtin is developmentally regulated in cell culture and resembles the pattern of expression observed in differentiating neurons in the mouse brain. It is interesting that HD-/- ES cells could differentiate into mature postmitotic neurons that expressed functional voltage- and neurotransmitter-gated ion channels. Moreover, both excitatory and inhibitory spontaneous postsynaptic currents were observed, indicating the establishment of functional synapses in the absence of huntingtin. These results demonstrate that huntingtin is not required for the generation of functional neurons with features characteristic of postmitotic neurons in the developing mouse brain.

Animals↗

PubMed1999

Subtype-specific enhancement of NMDA receptor currents by mutant huntingtin.

Evidence suggests that NMDA receptor-mediated neurotoxicity plays a role in the selective neurodegeneration underlying Huntington's disease (HD). The gene mutation that causes HD encodes an expanded polyglutamine tract of >35 in huntingtin, a protein of unknown function. Both huntingtin and NMDA receptors interact with cytoskeletal proteins, and, for NMDA receptors, such interactions regulate surface expression and channel activity. To determine whether mutant huntingtin alters NMDA receptor expression or function, we coexpressed mutant or normal huntingtin, containing 138 or 15 glutamine repeats, respectively, with NMDA receptors in a cell line and then assessed receptor channel function by patch-clamp recording and surface expression by western blot analysis. It is interesting that receptors composed of NR1 and NR2B subunits exhibited significantly larger currents when coexpressed with mutant compared with normal huntingtin. Moreover, this effect was selective for NR1/NR2B, as NR1/NR2A showed similar currents when coexpressed with mutant versus normal huntingtin. However, ion channel properties and total surface expression of the NR1 subunit were unchanged in cells cotransfected with NR1/NR2B and mutant huntingtin. Our results suggest that mutant huntingtin may increase numbers of functional NR1/NR2B-type receptors at the cell surface. Because NR1/NR2B is the predominant NMDA receptor subtype expressed in medium spiny neostriatal neurons, our findings may help explain the selective vulnerability of these neurons in HD.

Cell Line↗

PubMed1999

Cationic modulation of human dopamine transporter: dopamine uptake and inhibition of uptake.

Effects of cations on dopamine (DA) uptake into cells expressing the human dopamine transporter and on inhibition of DA uptake by various substrates and inhibitors were investigated by using rotating disk electrode voltammetry. The Na(+) dependence of DA uptake varied with Na(+) substitutes, hyperbolic with Li(+), almost linear at 1 microM DA but hyperbolic at 8 microM DA with choline, and sigmoidal with K(+). With Na(+) substituted by Li(+), K([DA]) decreased and V(app) remained constant with increasing [Na(+)], whereas K([Na+]) decreased and V(app) increased with increasing [DA], suggesting an ordered sequence with Na(+) binding before DA. Similar trends for the Na(+)-DA interactions were observed in the presence of cocaine. Cocaine inhibited DA uptake solely by increasing K([DA]), with its K(i) not significantly different at 55 and 155 mM [Na(+)], whereas it inhibited Na(+) stimulation by reducing V(app) more than K([Na+]) at 1 microM DA, and V(app) only and less potently at 8 microM DA. Thus, cocaine may compete with DA, not with Na(+), for the transporter, and might not follow a strictly ordered reaction with Na(+). With Na(+) substituted by K(+), K([DA]) or K([Na+]) became insensitive to Na(+) or DA. K(+) impaired the DA uptake mainly by reducing V(app,) but affected cocaine inhibition by elevating K(i). Despite their different patterns for inhibiting DA uptake, nontransportable inhibitors cocaine, methylphenidate, mazindol, and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenyl-2-propyl)piperazi ne (GBR12909) showed similarly modest Na(+) dependence in their K(i) values. In contrast, substrates DA, m-tyramine, and amphetamine displayed a similarly stronger Na(+) requirement for their apparent affinities.

Carrier Proteins↗

PubMed1999

A human prolactin antagonist, hPRL-G129R, inhibits breast cancer cell proliferation through induction of apoptosis.

Human breast cancer is the predominant malignancy and the leading cause of cancer death in women from Western societies. The cause of breast cancer is still unknown. Recently, the association between human prolactin (hPRL) activity and breast cancer has been reemphasized. Biologically active hPRL has been found to be produced locally by breast cancer cells that contain high levels of PRL receptor. A high incidence of mammary tumor growth has also been found in transgenic mice overexpressing lactogenic hormones. More importantly, it has been demonstrated that the receptors for sex steroids and PRL are coexpressed and cross-regulated. In this study, we report that we have designed and produced a hPRL antagonist, hPRL-G129R. By using cell proliferation assays, we have demonstrated that: (a) hPRL and E2 exhibited an additive stimulatory effect on human breast cancer cell (T-47D) proliferation; (b) hPRL-G129R possessed an inhibitory effect on T-47D cell proliferation; and (c) when antiestrogen (4-OH-tamoxifen) and anti-PRL (hPRL-G129R) agents were added together, an additive inhibitory effect was observed. We further investigated the mechanism of the inhibitory effects of hPRL-G129R in four hPRLR positive breast cancer cell lines. We report that hPRL-G129R is able to induce apoptosis in all four cell lines in a dose-dependent manner as determined by the Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. The apoptosis is induced within 2 h of treatment at a dose as low as 50 ng/ml. We hope that the hPRL antagonist could be used to improve the outcome of human breast cancer therapy in the near future.

Amino Acid Sequence↗

PubMed1999

Neurosensory changes following orthognathic surgery.

Much variability exists among studies of neurosensory disturbance following facial surgery. This diversity of findings may be a function of the different surgical procedures and measurement methods used. The present study compared 3 methods of assessing neurosensory loss following surgical orthodontics. Two objective tests and 1 subjective test were administered to 24 patients preoperatively and 4 weeks postoperatively. These included measures of 2-point discrimination, pressure-pain thresholds, and perceived sensation changes in specific facial regions. Postoperatively, all patients needed greater separation on the 2-point discrimination test in the lower facial regions, but not in the upper regions. Bilateral sagittal split osteotomy patients, especially males, required greater separation on these lower sites. Pressure-pain thresholds were not significantly impaired in most patients. Those who underwent combined maxillary and mandibular procedures experienced lower thresholds on the lower lip, while bilateral sagittal split osteotomy patients reported lower thresholds on the upper lip. The 2-point discrimination test was consistent with patients' self-ratings of neurosensory problems using facial maps, but the pressure-pain test was not. The majority of patients reported changed sensation in the lower facial regions postoperatively, regardless of surgery type. Examiners were less likely to rate these same facial regions as different in sensory acuity. Male patients were more likely to report sensory loss or pain postoperatively. These findings suggest that self-reports of neurosensory change following orthognathic surgery are consistent with tests of 2-point discrimination and somewhat higher than examiner ratings, but the objective test of pressure-pain thresholds in this study was least sensitive to neurosensory changes.

Discrimination, Psychological↗

PubMed1999

Expression, purification, and bone-inducing activity of recombinant human bone morphogenetic protein-3 mature peptide.

It was inferred that the mature peptide of human bone morphogenetic protein-3 (hBMP-3m) consists of the carboxyl terminal 127 amino acid residues of hBMP-3. A plasmid, pDH-B3m, was constructed by inserting the cDNA sequences encoding hBMP-3m into pDH, a PL-containing expression vector. pDH-B3m was transformed into Escherichia coli DH5 alpha. The highest expression level of recombinant hBMP-3m (rhBMP-3m) could be reached after 6 hours of induction at 42 degrees C, accounting for 28% of the total bacterial proteins. The rhBMP-3m was found in the inclusion bodies. After being washed and partially purified, the inclusion bodies were solubilized in urea and purified efficiently through ion-exchange chromatography. The purity of the rhBMP-3m was at least 95%. The rhBMP-3m was refolded by dilution method and then 1 mg was implanted into mouse thigh muscle to assay its activity. A classic pattern of cartilaginous osteogenesis was observed. The results showed that the purified and refolded rhBMP-3m had ectopic bone-inducing activity.

Animals↗

PubMed1999

[Study on the mechanism of glutamate mediated neurotoxicity by cortical neuron culture technique in vitro].

This study was intended to further explain the mechanism of glutamate (Glu) mediated neurotoxicity. The concentrations of TCa and Ca2+ i were measured in 32 cortical neuron cultures, which were divided into four groups: normal control group (n = 8); 0.5 mmol/L Glu group (Glu group n = 8); 0.5 mmol/L Glu + 100 mmol/L Nimodipine group (antagonist I, n = 8); 0.5 mmol/L Glu + 12 mumol/L MK-801 group (antagonist II, n = 8). The results showed that TCa and Ca2+ i concentrations in Glu group were significantly higher than those in normal control group (P < 0.05); TCa and Ca2+i in both antagonist I and II groups were evidently lower than those in Glu group (P < 0.05); No difference was found between antagonist I and II groups. The results suggest that Glu neurotoxicity is due to the intracellular calcium overload, which may be from the pathway of voltage-dependent calcium channels (VDCCs) and NMDA receptor-operated channels (NROCs).

Animals↗

PubMed1999

[Pattern recognition applied to the Pu I even-parity energy level].

The partial least square (PLS)-back propagation network (BPN) method is applied to assigning the electronic configurations of Pu I even-parity levels and the assignments agree well with the experimental results. It is shown that PLS-BPN can be used as a tool to assign complicated spectral levels.

English Abstract↗

PubMed1998

Human immunodeficiency virus type 1 Vif-derived peptides inhibit the viral protease and arrest virus production.

Human immunodeficiency virus type 1 (HIV-1) Vif protein is required for productive HIV-1 infection of peripheral blood lymphocytes and macrophages in cell culture and for pathogenesis in the SCID-hu mouse model of HIV-1 infection. Vif inhibits the viral protease (PR)-dependent autoprocessing of truncated HIV-1 Gag-Pol precursors expressed in bacterial cells and efficiently inhibits the PR-mediated hydrolysis of peptides in cell-free systems. The obstructive activity of Vif has been assigned to the 92 amino acids residing at its N'-terminus (N-Vif). To determine the minimal Vif sequence required to inhibit PR, we synthesized overlapping peptides derived from N-Vif. These peptides were then assessed, using two in vitro and two in vivo systems: (i) inhibition of purified PR, (ii) binding of PR, (iii) inhibition of the autoprocessing of the Gag-Pol polyprotein expressed by a vaccinia virus vector, and (iv) inhibition of mature virus production in human cells. The peptides derived from two regions of N-Vif encompassing residues Tyr-30-Val-65 and Asp-78-Val-98, inhibited PR activity in both the in vitro and the in vivo assays. Thus, these peptides can be used as lead compounds to design new PR inhibitors.

Amino Acid Sequence↗

PubMed1998

Cocaine acts as an apparent competitive inhibitor at the outward-facing conformation of the human norepinephrine transporter: kinetic analysis of inward and outward transport.

The inhibition by cocaine of inward and outward transport of dopamine (DA) at the cloned human norepinephrine transporter (hNET) and the relationship of the inhibitory patterns of cocaine to the conformational requirements of the transporter were investigated. This was done using rotating disk electrode voltammetry in transfected cells. The uphill uptake of external DA, the lack of inhibition by internal substrates on DA uptake, and the accelerated exchange of internal DA by external m-tyramine support a carrier model in which the hNET alternates between outward-facing and inward-facing conformations. Cocaine exhibited competitive inhibition of DA uptake, which was insensitive to intracellular substrates. In contrast, the inhibition by cocaine of the m-tyramine-induced DA efflux appeared noncompetitive relative to intracellular DA, but competitive relative to extracellular m-tyramine. Simultaneous measurement of m-tyramine uptake and accompanying DA efflux at various concentrations of intracellular DA showed that cocaine did not alter the ratio of DA efflux to m-tyramine uptake. Moreover, cocaine displayed similar potency for inhibiting DA uptake and efflux. Additionally, the inhibition profile of cocaine was unrelated to the addition time of cocaine, simultaneously with or earlier than a substrate. All of the findings are consonant with a competitive interaction between cocaine and substrates at the outward-facing conformation of the hNET. This action directly prevents the inward transport of external substrates, thereby inhibiting the outward transport of internal substrates by reducing the availability of the inward-facing conformation. Consequently, the experimental inhibition pattern of cocaine depends on the conformation of the hNET to which the transported substrate is exposed.

Animals↗

PubMed1998

A dynein heavy chain homologue gene in Hexamita inflata.

A gene encoding an unusually small dynein heavy chain homologue, hDYHH, was cloned from the genome of a free-living diplomonad, Hexamita inflata (Hi). The open reading frame (ORF) of hDYHH is 867bp and encodes a polypeptide of 289 amino acids (aa), hDYHH. hDYHH is homologous to the region around the third P-loop ATP-binding site of several dynein heavy chain polypeptides that are around 4000aa. Northern blot analysis showed that hDYHH is expressed in vivo and that the mRNA length (approximately 1.8kb) is consistent with the gene length (1.67kb). Southern blot analysis indicated that there are hDYHH homologues within the Hi genome, possibly including a longer dynein heavy chain gene. An hDYHH homologue was also identified in Hexamita pusilla (Hp). hDYHH is the first full-length protein-encoding gene cloned from Hexamita.

Amino Acid Sequence↗

PubMed1998

Enhanced detection sensitivity of "fluorescence reduction" by shifting the analyte absorbance spectrum and use of a fluorescent paper with higher signal/noise ratio.

Nonfluorescing protein bands can be detected by the fluorescence optics of the commercial gel electrophoresis apparatus with automated scanning of the migration path (HPGE-1000, LabIntelligence, Belmont CA), taking advantage of the decrease of emission from a fluorescent paper placed below the gel by the absorbance of proteins ("fluorescence reduction"). That decrease of fluorescence gives rise to an inverted protein peak. Nonfluorescent colorless proteins appear to reduce the intensity of light emitted from the fluorescent paper due to absorbance of incident and emitted light. When the absorbance spectrum only slightly overlaps with the excitation and emission spectra of the fluorescent paper, that reduction is weak, and detection sensitivity in that application is consequently only 1/30 of that of fluorescent proteins. By contrast, when the protein is colored so that its absorbance spectrum overlaps widely with the excitation and emission spectra of the fluorescent paper, the sensitivity of "fluorescence reduction" equals 1/4 to 1/5 of that obtained for fluorescent proteins. Bands detected by "fluorescence reduction" provide a quantitative measure of protein load and mobility. The area of the inverted bands is proportional to protein loads up to 16 microg/lane of the gel tray. A theory of "fluorescence reduction" is presented which accounts for the existence of a linear relationship between band area and load.

Animals↗

PubMed1998

Separation and microgram-scale isolation of sea urchin egg granules by electrophoresis in polyvinylpyrrolidone solution, using horizontal gel electrophoresis apparatus with fluorescence detector.

A homogenate of sea urchin (Lytechinus pictus) eggs rich in exocytotic membrane vesicles (granules) was subjected to analytical and preparative electrophoresis in the commercial automated horizontal gel electrophoresis apparatus (HPGE-1000, LabIntelligence, Belmont, CA) capable of intermittent scanning of the migration path, using buffered solutions of polyvinylpyrrolidone (PVP). The nonfluorescent granules were detected by "fluorescence reduction", i.e., a decrease of fluorescence intensity due to the absorbance and/or light scattering properties of the particle. Granules migrated at linear migration rates in buffers ranging from 0 to 2.5% PVP. Two bands were observed and optimally separated in 1.5% PVP solution. As shown by sodium dodecyl sulfate (SDS)-polypeptide patterns, the material recovered from the bands was qualitatively indistinguishable from the two major fractions A and C of granules previously separated by free-flow electrophoresis in the absence of polymer. Ferguson plot analysis failed to provide the sizes of the granules in view of the narrow PVP concentration range available for mobility measurement and the unavailability of chemically homogeneous size standards.

Animals↗