Search PubMed⌕ Search

Biomedical subjects

S He

Publications and source records attributed to S He.

At least 73 records · Page 4Linked to original sources
PubMed2001

[A study of different intracellular signal transduction pathways on phagocytosis of fibronectin by retinal pigment epithelial cells].

OBJECTIVE: To determine the phagocytosis of fibronectin (FN) by retinal pigment epithelial (RPE) cells and its related intracellular signal transduction pathways. METHODS: Fluorescent latex beads were coated by FN and then incubated with RPE cells at 37C for 3 hours. Phagocytosis was quantified by a flow cytometric assay. Experiments were also performed in the presence of inhibitors of various intracellular signaling pathways [tyrosine kinase (TK), phosphatidylinositol 3-kinase (PI 3-K), protein kinase C (PKC)]. RESULTS: FN-coated beads produced significantly increase in the phagocytic index (P < 0.05) when compared to the uncoated control. The PKC inhibitors, phorbol 12-myristate 13-acetate (PMA, 100 nmol/L) or calphostin C (400 nmol/L), non-specifically increased the phagocytosis of both FN-coated (P < 0.01) and uncoated beads (P < 0.01). Inhibitors of TK (genistein, 100 microgram/ml) and PI 3-K (wortmannin, 5 micromol/L), significantly inhibited FN phagocytosis (P < 0.001) but did not affect the uncoated control. While a combination of different inhibitors (genistein plus wortmannin, genistein plus PMA, wortmannin plus PMA) was used, the results showed that genistein or wortmannin can counteract the effect of PMA, and that genistein plus wortmannin have an additive inhibitory effect. CONCLUSIONS: Our results suggest that the FN phagocytosis by RPE cells appear to be regulated, at least in part, by some signal transduction pathways. The knowledge of the signaling pathways that mediate FN phagocytosis by RPE cells may provide novel therapeutic targets for molecular pharmacology of proliferative RPE disorders such as proliferative vitreoretinopathy.

Androstadienes↗
PubMed2001

[Effect of high-vaccum-manual-chop technique in phacoemulsification].

OBJECTIVE: To study the effect of high-vaccum-manual-chop technique to reduce complications related to phacoemulsification. METHODS: The correlation between the relative energy complex parameter (RECP) and the postoperative corneal edema rate (CER) in manual-chop and phaco-divide groups was statistically studied. RESULTS: The intraoperative mean relative ultrasound power and the mean time of ultrasound phacoemulsification were increased with the increase of the lens nucleus hardness. A significant difference existed between the mean time of the ultrasound phacoemulsification of the two groups (t = 3.1467, P < 0.01). The comparison between the mean relative ultrasound power of the two groups has no significant difference (t = 0.1874, P > 0.05). Positive correlation was observed between RECP and CER (r = 0.97, P < 0.01). CONCLUSION: High-vaccum-manual-chop technique may reduce the postoperative complications related to phacoemulsification, and RECP can be taken as a reliable index to evaluate the safety of phacoemulsification.

Adult↗
PubMed2001

[The preparation and study on hepatic targeting tendency of galactosyl-anti-CD3-McAb in mice].

OBJECTIVE: This study sought to reduce the recurrence rate of primary liver cacer (PLC) after hepatectomy by increasing the concentration of infiltrating lymphocytes(TILs) at the target organ. METHODS: It has been reported that galactosyl-anti-CD3-McAb has an obvious hepatic targeting tendency in vitro. On the basis of that work, the present authors conducted a study in mice. Galactosyl-anti-CD3-McAb was prepared and its carbohydrate density was measured with the phenol-sulphyric acid method, and then anti-CD3-McAb (labeled with 125I) and galactosyl-anti-CD3-McAb (labeled with 131I) were infused respectively via the peripheral vein and the radioactivity in each organ was measured. RESULTS: The carbohydrate density of galactosyl-anti-CD3-McAb in this experiment was 58.12, which effectively guaranteed the specific binding between galactosyl-anti-CD3-McAb and hepatic binding protein(HBP). It was also noticed that anti-CD3-McAb tended to aggregate in the lungs while being infused via the peripheral vein, and that galactosyl-anti-CD3-McAb had an obvious hepatic targeting tendency in vivo and it stayed in liver for quite a long period. CONCLUSIONS: Galactosyl-anti-CD3-McAb has an obvious hepatic targeting tendency in vivo while being infused through peripheral vein; this may be due to the specific binding between galactose and HBP.

Animals↗
PubMed2000

Functionally inert HIV-specific cytotoxic T lymphocytes do not play a major role in chronically infected adults and children.

The highly sensitive quantitation of virus-specific CD8(+) T cells using major histocompatibility complex-peptide tetramer assays has revealed higher levels of cytotoxic T lymphocytes (CTLs) in acute and chronic virus infections than were recognized previously. However, studies in lymphocytic choriomeningitis virus infection have shown that tetramer assays may include measurement of a substantial number of tetramer-binding cells that are functionally inert. Such phenotypically silent CTLs, which lack cytolytic function and do not produce interferon (IFN)-gamma, have been hypothesized to explain the persistence of virus in the face of a quantitatively large immune response, particularly when CD4 help is impaired. In this study, we examined the role of functionally inert CTLs in chronic HIV infection. Subjects studied included children and adults (n = 42) whose viral loads ranged from <50 to >100,000 RNA copies/ml plasma. Tetramer assays were compared with three functional assays: enzyme-linked immunospot (Elispot), intracellular cytokine staining, and precursor frequency (limiting dilution assay [LDA]) cytotoxicity assays. Strong positive associations were observed between cell numbers derived by the Elispot and the tetramer assay (r = 0.90). An even stronger association between tetramer-derived numbers and intracellular cytokine staining for IFN-gamma was present (r = 0.97). The majority (median 76%) of tetramer-binding cells were consistently detectable via intracellular IFN-gamma cytokine staining. Furthermore, modifications to the LDA, using a low input cell number into each well, enabled LDAs to reach equivalence with the other methods of CTL enumeration. These data together show that functionally inert CTLs do not play a significant role in chronic pediatric or adult HIV infection.

Adult↗
PubMed2000

Functional differences between cardiac and renal isoforms of the rat Na+-Ca2+ exchanger NCX1 expressed in Xenopus oocytes.

The transcript of the Na+-Ca2+ exchanger gene NCX1 undergoes alternative splicing to produce tissue-specific isoforms. The cloned NCX1 isoforms were expressed in Xenopus oocytes and studied using a two-electrode voltage clamp method to measure Na+-Ca2+ exchanger activity. The cardiac isoform (NCX1.1) expressed in oocytes was less sensitive to depolarizing voltages and to activation by [Ca2+]i than the renal isoform (NCX1.3). The cardiac isoform of NCX1 is more sensitive to activation by protein kinase A (PKA) than the renal isoform which may be explained by preferential phosphorylation. The cardiac isoform of NCX1 is phosphorylated to a greater extent than the renal isoform. The action of PKA phosphorylation which increases the activity of the cardiac isoform of the Na+-Ca2+ exchanger in oocytes was confirmed in adult rat ventricular cardiomyocytes by measuring Na+-dependent Ca2+ flux. We conclude that alternative splicing of NCX1 confers distinct functional characteristics to tissue-specific isoforms of the Na+-Ca2+ exchanger.

Animals↗
PubMed2000

The synthesis, testing and use of 5-fluoro-alpha-D-galactosyl fluoride to trap an intermediate on green coffee bean alpha-galactosidase and identify the catalytic nucleophile.

5-Fluoro-alpha-D-galactopyranosyl fluoride was synthesized and its interaction with the active site of an alpha-galactosidase from green coffee bean (Coffea arabica), a retaining glycosidase, characterized kinetically and structurally. The compound behaves as an apparently tight binding (Ki = 600 nM) competitive inhibitor, achieving this high affinity through reaction as a slow substrate that accumulates a high steady-state concentration of the glycosyl-enzyme intermediate, as evidenced by ESiMS. Proteolysis of the trapped enzyme coupled with HPLC/MS analysis allowed the localization of a labeled peptide that was subsequently sequenced. Comparison of this sequence information to that of other members of the same glycosidase family revealed the active site nucleophile to be Asp145 within the sequence LKYDNCNNN. The importance of this residue to catalysis has been confirmed by mutagenesis studies.

Amino Acid Sequence↗
PubMed2000

Identification of Asp197 as the catalytic nucleophile in the family 38 alpha-mannosidase from bovine kidney lysosomes.

Bovine kidney lysosomal alpha-mannosidase is a family 38 alpha-mannosidase involved in the degradation of glycoproteins. The mechanism-based reagent, 5-fluoro-beta-L-gulosyl fluoride, was used to trap a glycosyl-enzyme intermediate, thereby labelling the catalytic nucleophile of this enzyme. After proteolytic digestion and high performance liquid chromatography/tandem mass spectrometry (MS) analysis, a labelled peptide was localised, and the sequence: HIDPFGHSRE determined by fragmentation tandem MS analysis. Taking into consideration sequence alignments of this region with those of other alpha-mannosidases of the same family, this result strongly suggests that the catalytic nucleophile in this enzyme is Asp197.

Amino Acid Sequence↗
PubMed2000

Identification of Glu-519 as the catalytic nucleophile in beta-mannosidase 2A from Cellulomonas fimi.

Incubation of the beta-mannosidase Man2A from Cellulomonas fimi with 2-deoxy-2-fluoro-beta-D-mannosyl fluoride (2FMan beta F) resulted in time-dependent inactivation of the enzyme (inactivation rate constant k(i)=0.57 min(-1), dissociation constant for the inactivator K(i)=0.41 mM) through the accumulation of a covalent 2-deoxy-2-fluoro-alpha-D-mannosyl-beta-mannosidase 2A (2FMan-Man2A) enzyme intermediate, as observed by electrospray ionization mass spectrometry. The stoichiometry of inactivation was 1:1. Removal of excess inactivator and regeneration of active enzyme by transglycosylation of the covalently attached inhibitor to gentiobiose [Glc beta(1-6)Glc] demonstrated that the covalent intermediate was catalytically competent. Comparison by MS of the peptic digests of 2FMan-Man2A with peptic digests of native Man2A revealed a peptide of m/z 1520 that was unique to 2FMan-Man2A, and one of m/z 1036.5 that was unique to a Man2A peptide. Their sequences, determined by collision-induced fragmentation, were CSEFGFQGPPTW and FGFQGPPTW, corresponding to residues 517-528 and 520-528 of Man2A respectively. The difference in mass of 483.5 between the two peptides equals the sum of the masses of the tripeptide CSE plus that of 2-fluoromannose. It was concluded that in 2FMan-Man2A, the 2-fluoromannose esterified to Glu-519 blocks hydrolysis of the Glu-519-Phe-520 peptide bond, and that Glu-519 is the catalytic nucleophile in this enzyme. This residue is conserved in all members of family 2 of the glycosyl hydrolases. This represents the first ever labelling and identification of an active-site nucleophile in a beta-mannosidase.

Actinomycetales↗
PubMed2000

Dendritic computation of direction selectivity by retinal ganglion cells.

Direction-selective ganglion cells (DSGCs) in the retina respond strongly when stimulated by image motion in a preferred direction but are only weakly excited by image motion in the opposite null direction. Such coding represents an early manifestation of complex information processing in the visual system, but the cellular locus and the synaptic mechanisms have yet to be elucidated. We recorded the synaptic activity of DSGCs using strategies to observe the asymmetric inhibitory inputs that underlie the generation of direction selectivity. The critical nonlinear interactions between the excitatory and inhibitory inputs took place postsynaptically within the dendrites of the DSGCs.

Action Potentials↗
PubMed2000

Visible binocular beats from invisible monocular stimuli during binocular rivalry.

When two qualitatively different stimuli are presented at the same time, one to each eye, the stimuli can either integrate or compete with each other. When they compete, one of the two stimuli is alternately suppressed, a phenomenon called binocular rivalry [1,2]. When they integrate, observers see some form of the combined stimuli. Many different properties (for example, shape or color) of the two stimuli can induce binocular rivalry. Not all differences result in rivalry, however. Visual 'beats', for example, are the result of integration of high-frequency flicker between the two eyes [3,4], and are thus a binocular fusion phenomenon. It remains in dispute whether binocular fusion and rivalry can co-exist with one another [5-7]. Here, we report that rivalry and beats, two apparently opposing phenomena, can be perceived at the same time within the same spatial location. We hypothesized that the interocular difference in visual attributes that are predominantly processed in the Parvocellular pathway will lead to rivalry, and differences in visual attributes that are predominantly processed in the Magnocellular pathway tend to integrate. Further predictions based on this hypothesis were tested and confirmed.

Humans↗
PubMed2000

Identification of Asp-130 as the catalytic nucleophile in the main alpha-galactosidase from Phanerochaete chrysosporium, a family 27 glycosyl hydrolase.

Characterization of the complete gene sequence encoding the alpha-galactosidase from Phanerochaete chrysosporium confirms that this enzyme is a member of glycosyl hydrolase family 27 [Henrissat, B., and Bairoch, A. (1996) Biochem. J. 316, 695-696]. This family, together with the family 36 alpha-galactosidases, forms glycosyl hydrolase clan GH-D, a superfamily of alpha-galactosidases, alpha-N-acetylgalactosaminidases, and isomaltodextranases which are likely to share a common catalytic mechanism and structural topology. Identification of the active site catalytic nucleophile was achieved by labeling with the mechanism-based inactivator 2',4', 6'-trinitrophenyl 2-deoxy-2,2-difluoro-alpha-D-lyxo-hexopyranoside; this inactivator was synthesized by anomeric deprotection of the known 1,3,4,6-tetra-O-acetyl-2-deoxy-2, 2-difluoro-D-lyxo-hexopyranoside [McCarter, J. D., Adam, M. J., Braun, C., Namchuk, M., Tull, D., and Withers, S. G. (1993) Carbohydr. Res. 249, 77-90], picrylation with picryl fluoride and 2, 6-di-tert-butylpyridine, and O-deacetylation with methanolic HCl. Enzyme inactivation is a result of the formation of a stable 2-deoxy-2,2-difluoro-beta-D-lyxo-hexopyranosyl-enzyme intermediate. Following peptic digestion, comparative liquid chromatographic/mass spectrometric analysis of inactivated and control enzyme samples served to identify the covalently modified peptide. After purification of the labeled peptide, benzylamine was shown to successfully replace the 2-deoxy-2,2-difluoro-D-lyxo-hexopyranosyl peptidyl ester by aminolysis. The labeled amino acid was identified as Asp-130 of the mature protein by further tandem mass spectrometric analysis of the native and derivatized peptides in combination with Edman degradation analysis. Asp-130 is found within the sequence YLKYDNC, which is highly conserved in all known family 27 glycosyl hydrolases.

Amino Acid Sequence↗
PubMed2000

Spatial-temporal response characteristics of the ON-OFF direction selective ganglion cells in the rabbit retina.

We studied the response properties of the ON-OFF direction selective (DS) ganglion cells in the rabbit retina. The activities of the ganglion cells were recorded extracellularly with tungsten-in-glass electrodes. Drifting sine-wave gratings with various spatial and temporal frequencies were used to stimulate the ON-OFF DS cells. Individual ON-OFF DS cell yielded strongest response to a particular spatial frequency irrespective of temporal frequency. The optimal spatial wavelength increased with increasing eccentricity and its half-width approximately matched the size of the receptive field. The optimal spatial frequency implies that the directional inhibition is effective over a range of about a receptive field diameter. The characteristics of the ON-OFF DS cells suggest that they might function as local motion detectors.

Animals↗
PubMed2000

Condensation of DNA by multivalent cations: experimental studies of condensation kinetics.

DNA in viruses and cells exists in highly condensed, tightly packaged states. We have undertaken an in vitro study of the kinetics of DNA condensation by the trivalent cation hexaammine cobalt (III) with the aim of formulating a quantitative, mechanistic model of the condensation process. Experimental approaches included total intensity and dynamic light scattering, electron microscopy, and differential sedimentation. We determined the average degree of condensation, the distribution of condensate sizes, and the fraction of uncondensed DNA as a function of reaction time for a range of [DNA] and [Co(NH(3))(3+)(6)]. We find the following: (1) DNA condensation occurs only above a critical [Co(NH(3))(3+)(6)] for a given DNA and salt concentration. At the onset of condensation, [Co(NH(3))(3+)(6)]/[DNA-phosphate] is close to the average value of 0.54, which reflects the 89-90% charge neutralization criterion for condensation. (2) The equilibrium weight average hydrodynamic radius of the condensates first decreases, then increases with increasing [Co(NH(3))(3+)(6)] as they undergo a transition from intramolecular (monomolecular) to intermolecular (multimolecular) condensation. However, is insensitive to [DNA]. (3) The uncondensed DNA fraction decays approximately exponentially with time. The equilibrium uncondensed DNA fraction and relaxation time decrease with increasing [Co(NH(3))(3+)(6)] but are insensitive to [DNA]. (4) The condensation rate in its early stages is insensitive to [DNA] but proportional to [Co(NH(3))(3+)(6)](xs) = [Co(NH(3))(3+)(6)] - [Co(NH(3))(3+)(6)](crit). (5) Data for low [DNA] and low [Co(NH(3))(3+)(6)] at early stages of condensation are most reliable for kinetic modeling since under these conditions there is minimal clumping and network formation among separate condensates. A mechanism with initial monomolecular nucleation and subsequent bimolecular association and unimolecular dissociation steps with rate constants that depend on the number of DNA molecules in the condensate, accounts reasonably well for these observations.

Cobalt↗
PubMed2000

Endogenous dopaminergic regulation of horizontal cell coupling in the mammalian retina.

Horizontal cells in an isolated wholemount preparation of the mouse retina were injected with Lucifer yellow and neurobiotin to characterize both the pattern of gap junctional connectivity and its regulation by dopamine. The injected horizontal cells had a uniform morphology of a round cell body, a compact dendritic tree, and an axon, which could sometimes be traced to an expansive terminal system. The dendro-dendritic gap junctions between neighboring cells mediated both weak Lucifer yellow dye coupling and strong neurobiotin tracer coupling. The extent of the tracer coupling was decreased by either exogenous dopamine (100 microM) or cyclic adenosine monophosphate (cAMP) analogs and was significantly increased by the D1 antagonist SCH 23390 (10 microM). These results provide the first evidence in the mammalian retina that the gap junctions between horizontal cells are endogenously regulated by dopamine, which acts through D1 receptors to increase the intracellular cAMP. It has been proposed that the gap junctional coupling between horizontal cells is mediated by connexin 32 (Cx32), but the pattern and dopaminergic regulation of horizontal cell coupling were unaffected in Cx32-knockout mice, ruling out the possible involvement of Cx32. Every tracer-coupled horizontal cell showed calbindin immunoreactivity, and vice versa, providing strong evidence that the horizontal cells in the mouse retina comprise a single cell type. Like the axonless horizontal cells in other mammalian retinas, the axon-bearing horizontal cells in the mouse retina are coupled by gap junctions that are permeable to Lucifer yellow and dopamine sensitive, suggesting that the mouse horizontal cells have hybrid properties to compensate for the absence of axonless horizontal cells.

Animals↗
PubMed2000

Cloning, expression, characterization, and nucleophile identification of family 3, Aspergillus niger beta-glucosidase.

The beta-glucosidase from Aspergillus niger (CMI CC 324262) was purified, and an N-terminal sequence and two internal sequences were determined. BglI genomic gene and the cDNA were cloned from a genomic library and by reverse transcriptase-polymerase chain reaction, respectively. The cDNA was successfully expressed in Saccharomyces cerevisiae and Pichia pastoris. Sequence analysis revealed that the gene encodes a 92-kDa enzyme that is a member of glycosidase family 3. (1)H-NMR analysis of the reaction catalyzed by this enzyme confirmed that, in common with other family 3 glycosidases, this enzyme hydrolyzes with net retention of anomeric configuration. Accordingly, the enzyme was inactivated by 2-deoxy-2-fluoro beta-glucosyl fluoride, with kinetic parameters of k(i) = 4.5 min(-1), K(I) = 35.4 mM, through the trapping of a covalent glycosyl enzyme intermediate. The catalytic competence of this intermediate was demonstrated by the fact that incubation with linamarin resulted in reactivation, presumably via a transglycosylation mechanism. Peptic digestion of the 2-deoxy-2-fluoroglucosyl enzyme and subsequent analysis of high pressure liquid chromatography eluates by electrospray ionization triple quadrupole mass spectrometry in the neutral loss mode allowed the localization of a 2-deoxy-2-fluoroglucosyl-peptide. Sequence determination of this labeled peptide by tandem mass spectrometry in the daughter ion scan mode permitted the identification of Asp-261 as the catalytic nucleophile within the sequence VMSDW. Asp-261 is fully conserved within this family, consistent with its key role, and aligns with the aspartic acid residue previously identified in the Aspergillus wentii enzyme by labeling with conduritol B epoxide (Bause, E., and Legler, G. (1974) Hoppe-Seyler's Z. Physiol. Chem. 355, 438-442).

Amino Acid Sequence↗
PubMed2000

Mechanism of action and identification of Asp242 as the catalytic nucleophile of Vibrio furnisii N-acetyl-beta-D-glucosaminidase using 2-acetamido-2-deoxy-5-fluoro-alpha-L-idopyranosyl fluoride.

The novel mechanism-based reagent 2-acetamido-2-deoxy-5-fluoro-alpha-L-idopyranosykl fluoride has been synthesized, and the kinetic parameters K(M) = 0.23 mM and K(CAT)= 0.55 min(-1) for its hydrolysis by vibrio furnisi beta-N-acetylglucosaminidase (ExoII) HAVE been determined. Investigation of mixtures of enzyme with this slow substrate by electrospray mass spectrometry revealed a high steady-state population of the 2-acetamido-2-deoxy-5-fluoro-beta-L-idopyranosyl-enzyme, indicating that the hydrolytic mechanism of ExoII involves the formation and rate-determining hydrolysis of a glycosyl-enzyme intermediate. Analysis of a peptic digest of the glycosyl-enzyme by HPLC/ESMS/MS in the netural-loss mode permitted identification of a peptide bearing the 5-fluoro-sugar moiety. Tandem MS sequencing of the labeled peptide, in conjuction with multiple sequence alignmentsS of family 3 members, allowed the identification of ASP242 as the catalytic nucleophile within the sequence IVFSDDLSM.

Acetylglucosaminidase↗
PubMed2000

Visual motion of missing-fundamental patterns: motion energy versus feature correspondence.

Missing-fundamental gratings, generated by subtracting the fundamental Fourier components from square-wave gratings, appear to move backward when presented in quarter-cycle jumps, even though their edges and features all move forward. We used variants of these stimuli to test current models of motion perception. We found that missing-fundamental plaids, constructed from orthogonal missing-fundamental gratings, also appear to move backward. Forward motion was restored to missing-fundamental gratings and plaids by adding back small fractions of the original fundamental. In-phase and antiphase addition of the fundamental had similar effects on the perceived motion, despite having markedly different effects on the features, appearances and zero-crossings of the stimuli. The critical amplitude of fundamental needed to restore forward motion to plaids was the same as that needed to restore forward motion to their isolated component gratings, indicating that the plaids' emergent features, such as edge intersections and 'blobs', made little or no contribution to the perceived direction of motion in these stimuli. In two derivative experiments, missing-fundamental chromatic gratings and plaids, at approximate isoluminance, and missing-fundamental luminance barberpoles, also generated backward perceived motions, and these were also reversed by in-phase or antiphase addition of small amounts of fundamental.

Fourier Analysis↗