Biomedical subjects

W C Law

Publications and source records attributed to W C Law.

9 recordsLinked to original sources

PubMed2005

Real-time optical biosensor based on differential phase measurement of surface plasmon resonance.

We report a real-time differential phase measurement technique which can be implemented in optical surface plasmon resonance biosensors. The important feature of our design is that sensitivity has been greatly improved by measuring the differential phase change between the s and p-polarizations. Real-time measurement capability is achieved by using a phase extracting routine which continuously monitors the waveforms captured by two photo-detectors. Measurement capability of our setup is demonstrated through real-time monitoring of bovine serum albumin (BSA)/anti-BSA binding reaction. The estimated sensitivity of our current setup is 7.4 ng/ml.

Biosensing Techniques↗

PubMed2004

Highly sensitive differential phase-sensitive surface plasmon resonance biosensor based on the Mach-Zehnder configuration.

A high-sensitivity surface plasmon resonance (SPR) biosensor based on the Mach-Zehnder interferometer design is presented. The novel feature of the new design is the use of a Wollaston prism through which the phase quantities of the p and s polarizations are interrogated simultaneously. Since SPR affects only the p polarization, the signal due to the s polarization can be used as the reference. Consequently, the differential phase between the two polarizations allows us to eliminate all common-path phase noise while keeping the phase change caused by the SPR effect. Experimental results obtained from glycerin-water mixtures indicate that the sensitivity limit of our scheme is 5.5 x 10(-8) refractive-index units per 0.01 degrees phase change. To our knowledge, this is a significant improvement over previously obtained results when gold was used as the sensor surface. Such an improvement in the sensitivity limit should allow SPR biosensors to become a possible replacement for conventional biosensing techniques based on fluorescence. Monitoring of the bovine serum albumin (BSA) binding reaction with BSA antibodies is also demonstrated.

Antigen-Antibody Complex↗

PubMed2001

Analysis of corticosteroids in equine urine by liquid chromatography-mass spectrometry.

A liquid chromatography-mass spectrometry (LC-MS) method for the analysis of corticosteroids in equine urine was developed. Corticosteroid conjugates were hydrolysed with beta-glucuronidase; free and enzyme-released corticosteroids were then extracted from the samples with ethyl acetate followed by a base wash. The isolated corticosteroids were detected by LC-MS and confirmed by LC-MS-MS in the positive atmospheric pressure chemical ionisation mode. Twenty-three corticosteroids (comprising hydrocortisone, deoxycorticosterone and 21 synthetic corticosteroids), each at 5 ng/ml in urine, could easily be analysed in 10 min.

Adrenal Cortex Hormones↗

PubMed1990

Substrate specificities and mechanism in the enzymatic processing of vitamin A into 11-cis-retinol.

The biosynthesis of 11-cis-retinol in the retinal pigment epithelium requires two consecutive enzymatic reactions. The first involves the esterification of all-trans-retinol by lecithin retinol acyltransferase (LRAT). The second reaction involves the direct conversion of an all-trans-retinyl ester into 11-cis-retinol by an isomerase-like enzyme. This latter reaction couples the free energy of hydrolysis of an ester to the thermodynamically uphill trans to cis conversion, thus providing the energy to drive the latter process. In this paper both enzymes are studied with respect to their substrate specificities to provide information on mechanism. The isomerase is shown to be highly specific with respect to the ionylidene ring system and substitution at C15, whereas sterically bulkier substituents at C9 and C11 are permitted. C5 and C13 demethyl retinoids are isomerized, removing from consideration isomerization mechanisms involving C-H abstraction at the C5 or C13 methyl groups of the retinoid. On the other hand, C9 demethyl retinoids are not isomerized. A C-H abstraction mechanism is unlikely at the C9 methyl group as well, because no kinetic deuterium isotope effect is found with all-trans-19,19,19-trideuterioretinoids and isomerization of unlabeled retinoids occurs without the incorporation of deuterium when the isomerization is performed in D2O. LRAT proved to be broadly specific for retinols but was relatively inert with other hydrophobic alcohols including cholesterol. The enzyme is also highly specific for phosphatidylcholine analogues versus other potential membranous acyl donors such as phosphatidylethanolamine and phosphatidylserine.

Acyltransferases↗

PubMed1989

The molecular basis of retinoic acid induced night blindness.

Drugs which affect the processing of vitamin A in the retina or pigment epithelium can cause ocular toxicity. It is shown here that the retinoic acids, which are used in the treatment of skin disorders and which cause night blindness, inhibit the ocular retinol dehydrogenases in an in vitro system. This is shown to lead to a decrease in the formation of the visual chromophore 11-cis-retinal, thus explaining why night blindness might occur.

Alcohol Oxidoreductases↗

PubMed1989

Membranes as the energy source in the endergonic transformation of vitamin A to 11-cis-retinol.

The eye needs to biosynthesize 11-cis-retinoids because the chromophore of rhodopsin is 11-cis-retinal. The critical metabolic step is the endergonic isomerization of free all-trans-retinol (vitamin A) into 11-cis-retinol. This isomerization process can take place in isolated membranes from the retinal pigment epithelium in the absence of added energy sources. Specific binding proteins probably do not serve as an energy source, and since all of the reactions in the visual cycle are shown here to be reversible, trapping reactions also do not participate in the isomerization reaction. One previously unexplored possibility is that the chemical energy in the bonds of the membrane itself may drive the isomerization reaction. A group transfer reaction is proposed that forms a retinyl ester from a lipid acyl donor and vitamin A. This transfer can drive the isomerization reaction because the all-trans-retinyl ester is isomerized directly to 11-cis-retinol. Thus, the free energy of hydrolysis of the ester is coupled to the thermodynamically uphill trans to cis isomerization. The prediction of an obligate C-O bond cleavage in the vitamin A moiety during isomerization is borne out. Although the natural substrate for isomerization is not known, all-trans-retinyl palmitate is processed in vitro to 11-cis-retinol by pigment epithelial membranes.

Amphibians↗

PubMed1988

Stereochemical inversion at C-15 accompanies the enzymatic isomerization of all-trans- to 11-cis-retinoids.

all-trans-Retinol (vitamin A) is processed by membranes from the pigment epithelium of the amphibian or bovine eye to form 11-cis-retinoids. When the isomerization reaction is performed with either [15(S)-3H,14C]-all-trans-retinol or [15(R)-3H,14C]-all-trans-retinol as substrate, the resultant 11-cis-retinals, formed by the in vitro enzymatic oxidation of the retinols, retain their 3H in the former case and lose it in the latter. The ocular all-trans- (pro-R specific) and 11-cis-retinol (pro-S specific) dehydrogenases operate with different stereochemistries with respect to the prochiral methylene hydroxyl centers of their substrates. Inversion of stereochemistry at the prochiral retinol centers was shown to accompany the isomerization process in both the amphibian and bovine systems. The 11-cis-retinol formed from [15(S)-3H,14C]-all-trans-retinol was chemically isomerized with I2 to produce [15(R)-3H,14C]-all-trans-retinol. The 11-cis-retinol formed from [15(R)-3H,14C]-all-trans-retinol was chemically isomerized with I2 to produce [15(S)-3H,14C]-all-trans-retinol. The stereochemistry at the prochiral center of retinol is not affected by the I2-catalyzed double-bond isomerization process and, hence, inversion of stereochemistry at C-15 must accompany isomerization. The same inverted stereochemistry was found with the associated retinyl palmitates. Possible mechanistic reasons for the observed inversion of stereochemistry during isomerization are discussed.

Alcohol Dehydrogenase↗

PubMed1987

Biochemical characterization of the retinoid isomerase system of the eye.

We have previously shown that membranes from the retinal pigment epithelium can transform added all-trans-retinol into a mixture of 11-cis-retinoids, demonstrating the "missing reaction" in the visual cycle for the first time (Bernstein, P. S., Law, W. C., and Rando, R. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1849-1853). In this article, this isomerase activity is further characterized. Double-label experiments with [15-3H]- and [15-14C]all-trans-retinol as the substrate show that the tritium label is retained in the 11-cis-retinol and 11-cis-retinyl palmitate products. This requires that isomerization occur at the alcohol level of oxidation. All-trans-retinyl esters, such as the palmitate, acetate, butyrate, and hexanoate esters, are not directly transformed into their 11-cis counterparts by the membranes. The data are consistent with the presence of an all-trans-retinol isomerase enzyme system or enzyme complex, which produces 11-cis-retinol. Other isomeric retinols were tested for substrate activity. Neither 9-cis-retinol(al) nor 13-cis-retinol were processed by the isomerase. Since the membranes containing the isomerase possess other retinol metabolizing activities, such as retinyl ester synthetase and dehydrogenase activities, further purification was attempted. Appreciable quantities of all detergents tested led to the disappearance of isomerase activity, and high salt or EDTA did not dissociate isomerase activity from the membranes. However, extensive sonication of the membranes did produce a 100,000 x g supernatant fraction of light membranes depleted of other all-trans-retinol processing activities. The isomerase activity in these membranes was saturable with all-trans-retinol, as required for a biologically significant process, and showed a Vmax of 5 pmol/h/mg of protein, a KM of 0.8 microM, and a pH optimum of 8. The isomerase was destroyed by proteinase K, by phospholipase C, by heating, or by ethanol at concentrations greater than 1%. The addition of high energy compounds, such as MgATP, MgGTP, or palmitoyl-CoA, did not appear to stimulate isomerase activity in the 100,000 x g supernatant.

Animals↗

PubMed1987

Isomerization of all-trans-retinoids to 11-cis-retinoids in vitro.

The key biochemical process of the vertebrate visual cycle required for rhodopsin regeneration, 11-cis-retinoid production from all-trans-retinoids, is shown to occur in vitro. A 600 X g supernatant from a frog retina/pigment epithelium homogenate transforms added all-trans-[3H]retinol, in a time-dependent fashion, to a mixture of 11-cis-retinol, 11-cis-retinal, and 11-cis-retinyl palmitate. 13-cis-Retinoids are formed in only minor amounts by nonspecific processes. Studies using washed particulate fractions of the 600 X g supernatant indicate that all-trans-[3H]retinol is isomerized to 11-cis-retinoids much more effectively than is all-trans-[3H]retinal or all-trans-[3H]retinyl palmitate. The 11-cis-retinoid biosynthetic activity is heat-labile, sedimentable by high-speed centrifugation, and largely found in the pigment epithelium rather than in the neural retina.

Animals↗