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Biomedical subjects

Y C Kwok

Publications and source records attributed to Y C Kwok.

At least 19 recordsLinked to original sources
PubMed2001

Shah convolution differentiation Fourier transform for rear analysis in microchip capillary electrophoresis.

This paper first reports the application of Shah convolution differentiation Fourier transform for rear analysis. Rear analysis eliminates the need to create a well-defined and reproducible sample plug, thus making the operation simpler. The number of solution reservoirs, for microchip capillary electrophoresis (CE), could be reduced from the usual four to three. Sample bias in CE could be avoided too. The separation channel was first filled with the fluorescent sample solution, and subsequently flushed out with the buffer. The rear of each analyte zone gives rise to its flight of sigmoid-shaped steps in the time-domain. The time-domain detector signal was first differentiated and then Fourier transform was performed. The Fourier transform results were represented in the form of a magnitude plot. It is proposed that this would be as equally applicable to other separation techniques (e.g., chromatography) and detection methods (e.g., absorption).

Electrophoresis, Capillary↗
PubMed2001

Velocity measurement of particles flowing in a microfluidic chip using Shah convolution Fourier transform detection.

A noninvasive radiative technique, based on Shah convolution Fourier transform detection, for velocity measurement of particles in fluid flows in a microfluidic chip, is presented. It boasts a simpler instrumental setup and optical alignment than existing measurement methods and a wide dynamic range of velocities measurable. A glass-PDMS microchip with a layer of patterned Cr to provide multiple detection windows which are 40 microns wide and 70 microns apart is employed. The velocities of fluorescent microspheres, which were electrokinetically driven in the channel of the microfluidic chip, were determined. The effects of increasing the number of detection windows and sampling period were investigated. This technique could have wide applications, ranging from the determination of the velocity of particles in pressure-driven flow to the measurement of electrophoretic mobilities of single biological cells.

Algorithms↗
PubMed2001

Shah convolution Fourier transform detection: multiple-sample injection technique.

For the direct measurement of electrophoretic mobility, multiple-point (Shah function) detected, time-domain detector signals were converted into frequency-domain plots by means of Fourier transformation. Multiple sample plugs (up to a maximum of three) were introduced into the separation channel and the resultant time-domain signals were then Fourier-transformed. The multiple-sample injection technique has been successfully demonstrated for a one-component system and a separation. Though the number of fluorescing zones flowing through the illuminated length of the channel is greater than the number of analytes in the solution, Shah convolution Fourier transform detection (SCOFT) is able to identify the number of fluorescent species in the solution based on their migration velocities. The height of the fundamental peak increases as the number of injected sample plugs is increased. More importantly, the signal-to-noise ratio (S/N) is found to be proportional to the number of injected sample plugs. With these findings, the multiple-sample injection technique certainly has got many potential applications in trace analysis. The technique would be equally applicable to other separation techniques (e.g., high-performance liquid chromatography) and detection methods (e.g., absorption, refractive index).

Electrophoresis↗
PubMed1998

Application of dynamic liquid-phase microextraction to the analysis of chlorobenzenes in water by using a conventional microsyringe.

A dynamic liquid-phase microextraction technique combined with gas chromatography/mass spectrometry (GC/MS) is described for the extraction of 10 chlorobenzenes from water samples into 1 microL of organic solvent by using a conventional microsyringe. The effects of extraction solvent, plunger movement pattern, sampling volume, number of samplings, and salt concentration on the extraction performance were investigated. Good repeatabilities of extraction were obtained, with the RSD values below 5.3% except for hexachlorobenzene (9.3%). By using a sampling volume of 6 microL and 15 samplings, detection limits were found to be between 0.02 and 0.05 microgram/L under GC/MS-selective ion monitoring mode.

Chlorobenzenes↗
PubMed1995

Antifreeze proteins in winter rye are similar to pathogenesis-related proteins.

The ability to control extracellular ice formation during freezing is critical to the survival of freezing-tolerant plants. Antifreeze proteins, which are proteins that have the ability to retard ice crystal growth, were recently identified as the most abundant apoplastic proteins in cold-acclimated winter rye (Secale cereale L.) leaves. In the experiments reported here, amino-terminal sequence comparisons, immuno-cross-reactions, and enzyme activity assays all indicated that these antifreeze proteins are similar to members of three classes of pathogenesis-related proteins, namely, endochitinases, endo-beta-1,3-glucanases, and thaumatin-like proteins. Apoplastic endochitinases and endo-beta-1,3-glucanases that were induced by pathogens in freezing-sensitive tobacco did not exhibit antifreeze activity. Our findings suggest that subtle structural differences may have evolved in the pathogenesis-related proteins that accumulate at cold temperatures in winter rye to confer upon these proteins the ability to bind to ice.

Adaptation, Biological↗
PubMed1987

Tyrosine phosphorylation of two cytosolic proteins of 50 kDa and 35 kDa in rat liver by insulin-receptor kinase in vitro.

Insulin-receptor tyrosine kinase can phosphorylate a variety of artificial substrates in vitro. Its physiological substrate(s), however, remains unknown. In the present study, we show that immobilized insulin receptors phosphorylate tyrosine residues of two cytosolic proteins of 50 kDa and 35 kDa in rat liver. Phosphorylation of these two proteins required Mn2+- or Mg2+-ATP as the phosphate donor. Phosphorylation was time- and temperature-dependent. Furthermore, the rate of phosphorylation of the two proteins was related to the autophosphorylated state of the insulin receptor. The pI of the phosphorylated 50 kDa and 35 kDa proteins was 5.4 and 5.6 respectively. These proteins were present in low abundance. They were not related to each other, nor to the insulin receptor, as demonstrated by in-gel proteolytic digestion and by immunoprecipitation using antibodies produced against them. They were specific substrates for the insulin receptor kinase, since they were not phosphorylated by epidermal-growth-factor-receptor kinase. These observations suggest that the 50 kDa and 35 kDa cytosolic proteins may be endogenous substrates for the insulin-receptor kinase.

Adenosine Triphosphate↗
PubMed1987

Angiotensin receptors in resting smooth muscle are the low affinity sites observed in binding studies.

Angiotensin receptors in rat uterine smooth muscle have been investigated by [125I]angiotensin II binding studies in membrane preparations. Scatchard analysis of binding data has demonstrated the presence of low and high affinity angiotensin binding sites with KLD = 3.0 X 10(-8) and KHD = 5.0 X 10(-10)M respectively. These values are identical to our previously reported values for the EC50 and dissociation constant, respectively, obtained from bioassays on intact uterine tissues. The antagonist [Sar1, Ile8]angiotensin II also demonstrates a binding affinity in uterine membranes (pKD = 8.7) which is not significantly different from its apparent binding affinity (pA2 = 8.6) in responding tissues. Taken in conjunction with our previously published bioassay data the present binding studies suggest that the resting state of the angiotensin receptor in smooth muscle is a low affinity state, and that interaction with ANG II induces a portion of the receptors into a high affinity "excited" state. The antagonist [Sar1, Ile8]angiotensin II apparently binds with higher affinity than angiotensin II to the low affinity (resting) state of the receptor.

Angiotensin II↗
PubMed1986

Kinetic properties of the insulin receptor tyrosine protein kinase: activation through an insulin-stimulated tyrosine-specific, intramolecular autophosphorylation.

The insulin receptor is an insulin-activated, tyrosine-specific protein kinase. Previous studies have shown that autophosphorylation of tyrosine residues on the Mr 95,000 is associated with an activation of the protein kinase activity toward exogenous protein substrates. We have employed the highly purified insulin receptor, immobilized on insulin-Sepharose or eluted in an active form, to define the metal/ATP requirements for kinase activation, the relationship of receptor autophosphorylation to activation, and the kinetic properties of the autophosphorylated, activated receptor kinase. Prior incubation of the immobilized receptor with 2 mM ATP, 10 mM Mg (or 10 mM Mn), followed by removal of these reactants, served to abolish the upward curvilinearity in the rate of histone 2b (tyrosine) phosphorylation measured subsequently. This treatment also markedly increased the rate of histone 2b phosphorylation as compared to that observed with the unmodified, immobilized receptor, as estimated under conditions that per se minimized further activation. The extents of maximal activation of receptor histone 2b (tyrosine) kinase obtained on preincubation with MgATP or MnATP are identical; however, the affinity of the receptor for MnATP is approximately 10-fold higher than that for MgATP. The higher affinity of the receptor for MnATP is observed for both autophosphorylation/autoactivation and histone 2b tyrosine kinase activity (Km MnATP approximately 0.01 mM; Km MgATP approximately 0.1 mM). Autophosphorylation/autoactivation per se does not significantly alter the apparent affinity for MeATP (or protein substrate, as previously reported) but increases Vmax. Activation of receptor histone 2b (tyrosine) kinase is due to tyrosine-specific autophosphorylation of the Mr 95,000 (beta) subunit; thus the extent of total 32P incorporation into the beta subunit correlates precisely with the extent of kinase activation, both over time and at a wide variety of Me2+ ATP concentrations. Sequential treatment of the autophosphorylated receptor with elastase and trypsin yields a single, basically charged 32P-peptide, Mr less than 2000. The functional properties of the unphosphorylated and fully phosphorylated receptor were compared after elution from insulin-Sepharose. The insulin binding characteristics of the two forms of the receptor were indistinguishable; the kinase properties differed greatly; whereas the histone 2b activity of the unphosphorylated receptor was low in the basal state, and activated 10-fold by insulin, the fully autophosphorylated receptor exhibits maximal histone 2b kinase in the basal state and is unaffected by insulin addition.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1984

Insulin-stimulated tyrosine protein kinase. Characterization and relation to the insulin receptor.

Utilizing histone phosphorylation as the basis for a quantitative assay, the insulin-stimulated protein kinase in human placenta has been characterized. The kinase copurifies through wheat germ agglutinin-Sepharose and DEAE-cellulose in constant ratio to the insulin binding function. Both activities are bound to the same extent on insulin-Sepharose, and the immobilized kinase, after extensive washing, exhibits activity versus histone, which closely approaches that of the insulin-stimulated, solubilized kinase. In addition, the bound kinase retains the ability to phosphorylate the Mr = 95,000 subunit of the bead-bound receptor. Elution of the beads with sodium dodecyl sulfate yields on electrophoresis two major peptides of Mr = 130,000 and 95,000. Thus, insulin binding and insulin-stimulated histone kinase copurify in a constant stoichiometric ratio in close physical relation and are likely functional expressions of the same molecule. After the DEAE step, the insulin-stimulated kinase phosphorylates histone subfraction 2b exclusively on tyrosine residues. Insulin increases the Vmax for H2b by 3-5-fold and increases the rate of the histone phosphorylation in direct correspondence to the steady state level of specifically bound insulin. ATP is the preferred phosphate donor. The reaction is supported by either Mn2+ or Mg2+. At [ATP] less than 0.5 mM, insulin-stimulated kinase is substantially higher with Mn2+ as the sole divalent cation, as compared to Mg2+. At [ATP] greater than or equal to 0.5 mM, the rates observed with Mn2+ have plateaued, whereas the rates in the presence of Mg2+ show a continued increase such that maximal activity is seen with Mg2+ and 2-3 mM ATP. Under these conditions, the estimated turnover number of the kinase ranges between 30 and 100 pmol of 32P transferred per min/pmol of insulin bound. Thus, the tyrosine kinase activity of the insulin receptor is quantitatively comparable to that estimated for several serine protein kinases and is unlikely to reflect the side reaction of another enzymatic function.

Amino Acids↗
PubMed1984

Photoaffinity labeling of the rat isolated portal vein: determination of affinity constants and "spare" receptors for angiotensins II and III.

Photoaffinity labeling of the isolated rat portal vein with [azidobenzoic acid, isoleucine]angiotensin II resulted in selective partial inactivation of angiotensin receptors without affecting norepinephrine receptors. Establishment of dose-response curves to angiotensin II and III before and after photoaffinity labeling has permitted the calculation of "spare" receptors and affinity constants for angiotensins II and III. Spare receptors appear to exist for angiotensin II (greater than 60%) but not for angiotensin III. Furthermore, the data indicate that angiotensin III has a higher binding affinity (K = 6 X 10(-8) M) than angiotensin II (K = 3 X 10(-7) M), but is considerably less potent than angiotensin II in eliciting the contractile response. If angiotensins II and III act at the same receptors in the portal vein, angiotensin III could inhibit the constricting action of angiotensin II and thereby play a role in the angiotensin-mediated control of portal venous capacity.

Affinity Labels↗
PubMed1982

Extended chain analogs of [arginine8]vasopressin as model prohormones: investigation of precursor-processing enzymes in extracts of the rat hypothalamus and neural lobe.

Analogs of [arginine8]vasopressin (AVP) in which the peptide chain was elongated from the N-terminus by the addition of Ala-Arg-Arg-, Ala-Ala-Phe-, Pro-Arg-Val-, Pro-Ala-Arg-Arg, and Pro-Ala-Ala-Phe-, and from the C-terminus by the addition of -Ala-Met-Ala-NH2 and -Gly-Arg-Arg-Ala-NH2 were synthesized by the solid phase method and purified by Sephadex G-15 chromatography. At the final step of the synthesis, the extent of formation of the intramolecular disulfide bond was found to be sequence dependent. These peptides were incubated with extracts of the rat hypothalamus (supraoptic region) and neural lobe and with isolated neurosecretory granules from the neural lobe, and the release of vasopressin was measured by the rat pressor assay. All peptides resisted conversion to the hormone in the presence of tissue extracts, except (Ala-Ala-Phe)-AVP which was converted to AVP in the presence of all three tissue extracts at pH 4.7 but not at pH 8.0. When these peptides were treated with trypsin, chymotrypsin, or leucine aminopeptidase at pH 8.0, only the action of chymotrypsin on [Ala-Ala-Phe]AVP resulted in AVP formation. Evidence obtained using lysosomal enzyme markers suggested that the converting enzyme activity in neurosecretory granule preparations was not of lysosomal origin.

Amino Acid Sequence↗
PubMed1980

Cleavage of protected amino acids and peptides from benzyl ester resins by transesterification with potassium cyanide in benzyl alcohol.

The nature of the protected amino acid or peptide attached to the Merrifield resin influences the rate of KCN-catalyzed transesterification in benzyl alcohol. Although crown ether complexes of KCN do not expedite the reaction, sterically hindered amino acids are readily transesterified in the presence of saturating concentrations of KCN at elevated temperatures.

Amino Acids↗