Non-Invasive Cattle Body Temperature Measurement Using Infrared Thermography and Auxiliary Sensors.

To achieve a sensitive and accurate method in body temperature measurement of cattle, this study explores the uses of infrared thermography (IRT), an anemometer, and a humiture meter as a multiple sensors architecture. The influence of environmental factors on IRT, such as wind speed, ambient temperature, and humidity, was considered. The proposed signal processes removed the IRT frames affected by air flow, and also eliminated the IRT frames affected by random body movement of cattle using the frame difference method. In addition, the proposed calibration method reduced the impact of ambient temperature and humidity on IRT results, thereby increasing the accuracy of IRT temperature. The difference of mean value and standard deviation value between recorded rectal reference temperature and IRT temperature were 0.04 °C and 0.10 °C, respectively, and the proposed system substantially improved the measurement consistency of the IRT temperature and reference on cattle body temperature. Moreover, with a relatively small IRT image sensor, the combination of multiple sensors architecture and proper data processing still achieved good temperature accuracy. The result of the root-mean-square error (RMSE) was 0.74 °C, which is quite close to the accurate result of the IRT measurement.

The role of factor XIa (FXIa) catalytic domain exosite residues in substrate catalysis and inhibition by the Kunitz protease inhibitor domain of protease nexin 2.

To select residues in coagulation factor XIa (FXIa) potentially important for substrate and inhibitor interactions, we examined the crystal structure of the complex between the catalytic domain of FXIa and the Kunitz protease inhibitor (KPI) domain of a physiologically relevant FXIa inhibitor, protease nexin 2 (PN2). Six FXIa catalytic domain residues (Glu(98), Tyr(143), Ile(151), Arg(3704), Lys(192), and Tyr(5901)) were subjected to mutational analysis to investigate the molecular interactions between FXIa and the small synthetic substrate (S-2366), the macromolecular substrate (factor IX (FIX)) and inhibitor PN2KPI. Analysis of all six Ala mutants demonstrated normal K(m) values for S-2366 hydrolysis, indicating normal substrate binding compared with plasma FXIa; however, all except E98A and K192A had impaired values of k(cat) for S-2366 hydrolysis. All six Ala mutants displayed deficient k(cat) values for FIX hydrolysis, and all were inhibited by PN2KPI with normal values of K(i) except for K192A, and Y5901A, which displayed increased values of K(i). The integrity of the S1 binding site residue, Asp(189), utilizing p-aminobenzamidine, was intact for all FXIa mutants. Thus, whereas all six residues are essential for catalysis of the macromolecular substrate (FIX), only four (Tyr(143), Ile(151), Arg(3704), and Tyr(5901)) are important for S-2366 hydrolysis; Glu(98) and Lys(192) are essential for FIX but not S-2366 hydrolysis; and Lys(192) and Tyr(5901) are required for both inhibitor and macromolecular substrate interactions.

A Glu113Ala mutation within a factor VIII Ca2+-binding site enhances cofactor interactions in factor Xase.

We recently identified an acidic-rich segment in the A1 domain of factor VIII (residues 110-126) that functions in the coordination of Ca(2+), an ion necessary for cofactor activity [Wakabayashi et al. (2004) J. Biol. Chem. 279, 12677-12684]. Mutagenesis studies showed that replacement of residue Glu113 with Ala (E113A) yielded a factor VIII point mutant possessing increased specific activity as determined by a one-stage clotting assay. Mutagenesis at this site suggested that substitution with relatively small, nonpolar residues was well tolerated, whereas replacement with a number of polar or charged residues appeared detrimental to activity. Ala substitution resulted in the greatest enhancement, yielding an approximately 2-fold increased specific activity. Time course experiments following reaction with thrombin revealed similar rates of activation and inactivation of E113A as observed for the wild type. Results from factor Xa generation assays showed minimal differences in kinetic parameters and factor IXa affinity for E113A and wild-type factor VIIIa when run in the presence of synthetic phospholipid vesicles, whereas factor VIIIa E113A displayed an approximately 4-fold greater affinity for factor IXa compared with factor VIIIa wild type in reactions run on the platelet membrane surface. This latter effect may be attributed, in part, to a 2-fold increased affinity of factor VIIIa E113A for the platelet membrane. Considering that low levels of factors VIIIa and IXa are generated during clotting in plasma, the increased cofactor specific activity observed for E113A factor VIII may result from its enhanced affinity for factor IXa on the physiological membrane.

A first-order-like state transition for recombinant protein folding.

Normally, proteins will aggregate and precipitate by direct folding processes. In this study, we report that quasi-static processes can restore both the structure and bio-function of two kinds of fish recombinant growth hormones (Plecoglossus altivelis and Epinephelus awoara). The conformational changes and the particle-size-distribution (PSD) of each refolding intermediate can be monitored by circular dichroism spectroscopy (CD) and dynamic light scattering (DLS), respectively. Conformation analysis of the CD spectra of the refolding intermediates indicated that the secondary structures were restored in the initial refolding intermediate. However, the tertiary interactions of the proteins were restored during the last two refolding stages, as elucidated by thermal stability tests. This is consistent with a sequential model. DLS analysis suggested that the average hydrodynamic radii of the refolding intermediates shrank to their native-like sizes after the first refolding stage. This is consistent with a collapse model. After comparison with the data on the direct folding process, it is concluded that the denaturant-containing protein folding reaction is a first-order-like state transition process.

Protein folding by a quasi-static-like process: a first-order state transition.

In this paper we report that quasi-static-like processes, in which stable intermediates were introduced carefully and deliberately, may be used to reversibly unfold and refold purified native porcine growth hormone. Through circular dichroism (CD) and dynamic light scattering (DLS), we were able to study the secondary structure conformational changes, tertiary structure thermal stabilities, and the particle size distributions of both the intermediates and the final folded product. The CD data showed that the secondary structure was restored in the initial folding stage, whereas the tertiary structure within the protein was restored one step before the last folding stage, as elucidated by thermal stability experiments. DLS analysis suggested that the average hydrodynamic radii of the folding intermediates shrunk to nativelike size immediately after the first folding stage. Our data suggested that the denaturant-containing protein folding reaction is a first-order-like state transition process. This quasi-static-like process may be useful in the prevention of aggregate formation in protein purification and thus can be used in protein engineering to improve the overall yield from harvesting proteins.