Mouldable, thermoplastic, glue-on frog-supportive shoes change hoof kinetics in normal and obese Shetland ponies.

BACKGROUND: Obesity and hyperinsulinaemia are frequently encountered in the equine population and risk factors for the development of laminitis. There are many options for hoof support that claim a beneficial effect, but often the scientific evidence is scarce. OBJECTIVES: To quantify the effect of frog-supportive shoes on hoof kinetics in normal and obese ponies. STUDY DESIGN: Controlled in vivo trial. METHODS: Ten Shetland mares (n = 10) with a normal (n = 5) or obese (n = 5) body condition were led over a dynamically calibrated pressure plate before (T0), immediately after (T1) and 72 h (T2) after application of the shoes. The following locomotor variables were measured: stance duration (StDur), vertical impulse (VI), peak vertical force (PVF), time to PVF and time from PVF to lift off. The hoof print was divided into a toe and heel region and the StDur toe-heel index was calculated. The toe-heel hoof balance curves of the vertical force were plotted throughout the stance phase. RESULTS: The VI and PVF increased significantly 72 h after application of the shoes, when compared with T0 and T1. The StDur toe-heel index and toe-heel balance curves were significantly different between the normal and obese ponies. These variables became more comparable between the groups after application of the frog-supportive shoes. MAIN LIMITATIONS: It would have been interesting to measure the effect of the shoe in patients with acute laminitis. However, this would have had major welfare implications. CONCLUSIONS: The obese ponies moved more carefully than the normal group, demonstrated by a decreased loading of the toe area. The data illustrate that the ponies became more comfortable 72 h after application of the shoes, with a pronounced effect in the obese group. Thus, these results suggest that frog-supportive shoes could be beneficial, especially for obese ponies.

Demonstrating Functional Equivalence of Pilot and Production Scale Freeze-Drying of BCG.

Process analytical technology (PAT)-tools were used to monitor freeze-drying of Bacille Calmette-Guérin (BCG) at pilot and production scale. Among the evaluated PAT-tools, there is the novel use of the vacuum valve open/close frequency for determining the endpoint of primary drying at production scale. The duration of primary drying, the BCG survival rate, and the residual moisture content (RMC) were evaluated using two different freeze-drying protocols and were found to be independent of the freeze-dryer scale evidencing functional equivalence. The absence of an effect of the freeze-dryer scale on the process underlines the feasibility of the pilot scale freeze-dryer for further BCG freeze-drying process optimization which may be carried out using a medium without BCG.

Trypsin diminishes the rat potency of polio serotype 3.

This study addresses observations made in view of testing in practice the guideline in the European Pharmacopoeia (EP) on omitting the rat potency test for release of polio containing vaccines. In general, use of the guideline is valid and the D-antigen ELISA can indeed be used as an in vitro alternative for the in vivo test. However, the set-up of the ELISA is crucial and should include detection of antigenic site 1 in polio serotype 3 as destruction of that site by trypsin results in a reduced rat potency. Antigenic site 1 in polio serotype 2 may also be modified by trypsin, but the cleavage of viral protein 1 (VP1) did not affect the rat potency. Therefore, any antigenic site, except site 1, can be used for detection of polio serotype 2. It is advised to include testing of the effect of trypsin treatment in the EP-guideline. This allows polio vaccine manufacturers to check whether their in-house ELISA needs improvement.

Development of a fast ELISA for quantifying polio D-antigen in in-process samples.

A fast ELISA was developed and qualified for analysis of polio D-antigen. The original 20 h-protocol was optimized by minimizing the total incubation time to 1 h, and by replacing the signal reagent 3,3',5,5'-tetramethylbenzidine by a chemiluminogenic signal reagent with a theoretical low intrinsic background and high dynamic range.

On a revised mechanism of side product formation in the lignin peroxidase catalyzed oxidation of veratryl alcohol.

The O2-dependent formation of side products during the oxidation of veratryl alcohol (VA) by lignin peroxidase has previously been proposed to start with the attack of H2O on the VA radical cation (VA*+). This initial reaction is unlikely since it would also lead to side product formation in the absence of O2, which is not the case. In the current mechanism VA* reacts first with O2, whereafter H2O attacks. Furthermore, this paper describes an alternative explanation for the inhibitory effect of Mn2+ on VA side product formation. It is proposed that Mn2+ reduces reactive intermediates back to VA.

Amino acid-catalyzed conversion of citral: cis-trans isomerization and its conversion into 6-methyl-5-hepten-2-one and acetaldehyde.

Under alkaline conditions, amino acids or proteins catalyze the deacetylation of citral, a major aroma component, resulting in methylheptenone and acetaldehyde formation. 3-Hydroxycitronellal is an intermediate in this reaction. Amino acids also catalyze the cis-trans isomerization of the pure isomers of citral, geranial, and neral. Most likely the amino acids are involved in stabilizing intermediates of the isomerization and deacetylation reaction of citral. On the basis of the findings presented, some consequences for the application of citral, or its isomers, in food are discussed.

Veratryl alcohol-mediated oxidation of isoeugenyl acetate by lignin peroxidase.

The mechanism of the veratryl alcohol (VA)-mediated oxidation of isoeugenyl acetate (IEA) by lignin peroxidase, and the subsequent spontaneous Calpha-Cbeta cleavage of IEA to vanillyl acetate were studied. IEA oxidation only occurred in the presence of VA. It probably did not bind to lignin peroxidase as evidenced by an unaffected Km for VA in the presence of IEA, and by the fact that a 10-fold molar excess of the unreactive IEA counterpart, eugenyl acetate, did not affect the IEA oxidation rate. IEA was very efficient in recycling VA. Up to 34 mol of IEA were oxidized per mol VA. Formation of the predominant VA oxidation product, veratraldehyde, was postponed until IEA was almost completely oxidized. Together these findings suggest that IEA was oxidized by VA.+ rather than directly by lignin peroxidase. Thus, VA functioned as a redox mediator during IEA oxidation which is remarkable considering the high calculated ionization potential of 8.81 eV. Regardless of the presence of O2, approximately 2 mol of IEA were consumed per mol H2O2, which indicated that IEA was enzymatically oxidized by one electron to the putative radical cation (IEA.+). After formation of IEA.+, a series of O2-dependent chemical reactions were responsible for Calpha-Cbeta cleavage to the major oxidation product vanillyl acetate, as evidenced by the observation that an N2 atmosphere did not inhibit IEA oxidation, but almost completely inhibited vanillyl acetate formation. GC-MS analyses revealed that under an air atmosphere 1-(4'-acetoxy-3'-methoxyphenyl)-2-propanone, 1-(4'-acetoxy-3'-methoxyphenyl)-1-hydroxy-2-propanone, and 1-(4'-acetoxy-3'-methoxyphenyl)-2-hydroxy-1-propanone were also formed. Formation of the latter two was diminished under an N2 atmosphere.

Calculated ionisation potentials determine the oxidation of vanillin precursors by lignin peroxidase.

In view of the biocatalytic production of vanillin, this research focused on the lignin peroxidase (LiP) catalysed oxidation of naturally occurring phenolic derivatives: O-methyl ethers, O-acetyl esters, and O-glucosyl ethers. The ionisation potential (IP) of a series of model compounds was calculated and compared to their experimental conversion by LiP, defining a relative IP threshold of approximately 9.0 eV. Based on this threshold value only the O-acetyl esters and glucosides of isoeugenol and coniferyl alcohol would be potential LiP substrates. Both O-acetyl esters were tested and were shown to be converted to O-acetyl vanillin in molar yields of 51.8 and 2.3%, respectively.

Purification and characterization of two lignin peroxidase isozymes produced by Bjerkandera sp. strain BOS55.

The white-rot fungus Bjerkandera sp. strain BOS55 excretes at least seven lignin peroxidase (LiP) isozymes. Two of these, LiP-2 and LiP-5 (molecular weight 40-42 kDa), were purified to homogeneity. Both isozymes had the same N-terminal amino acid sequence which showed strong homology with LiP isozymes produced by other white-rot fungi. The kinetics of both isozymes were similar. LiP-5 oxidized veratryl alcohol optimally only in the presence of H2O2 near pH 3.0 (16.7 U/mg) and LiP-2 did this below pH 2.5 (33.8 U/mg). Also at normal physiological pHs for fungal growth (pH 5.0-6.5) both isozymes were still active. Further characterization of LiP-2 and LiP-5 revealed that the Km for H2O2 strongly decreased with increasing pH. As a result of this the catalytic efficiency (TN/Km) calculated on the basis of the Km for H2O2 in the oxidation of veratryl alcohol was constant over wide pH range.

Interference of peptone and tyrosine with the lignin peroxidase assay.

The N-unregulated white rot fungus Bjerkandera sp. strain BOS55 was cultured in 1 liter of peptone-yeast extract medium to produce lignin peroxidase (LiP). During the LiP assay, the oxidation of veratryl alcohol to veratraldehyde was inhibited due to tyrosine present in the peptone and the yeast extract.

Clinic administration--Malayan experience.

PIP: Criteria for starting a new family planning clinic in Malaya is described. In villages where there are 7-15 new mothers each month, there will probably be 5-7 acceptors, justifying a monthly visit from a National Family Planning Board nurse. The health subcenter is the best place to hold the clinic and it should be held on the same days as the postnatal clinic. The nurse explains the program and trains the staff to distribute oral contraceptives and keep records. Factors to be considered in the establishment of the clinic include finances, supply systems, staff development, and an evaluation system.^ieng