beta-N-acetylglucosaminidase and beta-glucuronidase activities in insulin-dependent diabetic subjects with retinopathy

The serum activities of two lysosomal enzymes, beta-N-acetylglucosaminidase (EC 3.2.1.30, NAG) and beta-glucuronidase (EC 3.2.1.31, GLU), were determined in 41 insulin-dependent diabetics, 27 age-matched non-diabetic first-degree relatives of the diabetics and 103 age-matched non-diabetic blood-donors. The diabetics were divided into three groups on the basis of ophthalmoscopy: (1) no retinal abnormalities; (2) non-proliferative retinopathy; and (3) proliferative retinopathy. The activities of both serum enzymes were higher in diabetics (NAG 21.39 +/- 5.99; GLU 2.19 +/- 1.01) than in their relatives (NAG 17.22 +/- 3.99; GLU 1.62 +/-0.61). The diabetics with non-proliferative retinopathy had higher serum enzyme levels (NAG 24.05 +/- 6.26; GLU 2.60 +/- 1.06) than diabetics without retinopathy (NAG 17.88 +/- 3.00; GLU 1.69 +/ 0.64), whereas no statistically significant difference was found in patients with the proliferative form of retinopathy (NAG 18.67 +/- 6.28; GLU 1.99 +/- 1.04). In diabetics a positive correlation was found between serum beta-N-acetylglucosaminidase activity and blood glucose (p < 0.01), but not between beta-glucuronidase and blood glucose. Furthermore, the activities of both enzymes in diabetics correlated with the plasma triglyceride level (p < 0.05 for both correlations). No correlation was found between the enzyme levels and signs of other diabetic late complications.     

Model-based mutagenesis to improve the enantioselective fractionation properties of an antibody

The binding affinity and specificity of recombinant antibodiescan be modified by site-directed mutagenesis. Here we have usedmolecular modelling of the variable domains of an enantiospecificantibody fragment to fine-tune its affinity so it is more suitablefor the fractionation of the drug enantiomers. We have shownearlier that the Fab fragment of this antibody specificallyrecognizes one enantiomer from the racemic mixture of a medicaldrug and that it can be used for the fractionation of theseenantiomers by affinity chromatography. However, the affinitywas unnecessarily high, requiring harsh elution conditions torelease the bound enantiomer. Thus, the continuous use of theantibody affinity columns was impossible. We made a homologymodel of the antibody and designed mutations to the antigen-bindingsite to decrease the affinity. Four out of five point mutationsshowed decreased affinity for the hapten. Two of the mutationswere also combined to construct a double mutant. The affinitycolumns made using one of the single mutants with lowered affinityand the double mutant were capable of multiple rounds of enantioseparation. Received April 23, 2003; revised September 25, 2003;; accepted October 21, 2003     

Emulsifier Composition of Solid Lipid Nanoparticles (SLN) Affects Mechanical and Barrier Properties of SLN‐Protein Composite Films

Protein films can be applied to improve food quality and to reduce packaging waste. To overcome their poor water barrier properties, lipids are often incorporated. The function of incorporated lipid depends on the interface between filler and matrix. This study aimed to tailor the properties of a protein–lipid film by designing the oil/water interface to see if the concept of inactive/active filler is valid. Therefore, we varied the emulsifier stabilizing solid lipid nanoparticles (SLN) to promote (via β‐lactoglobulin) or to minimize (via Tween 20) interactions between particle surface and protein. SLN were incorporated into protein films and film properties were determined. Addition of SLN led to significantly decreased water vapor permeability (WVP) of protein films. However, WVP was mainly affected by the emulsifiers and not by the lipid. Protein‐stabilized SLN (BS) replaced a lacking protein in the protein network and therefore did not influence the mechanical properties of the films at ambient temperature. BS‐composite films were temperature sensitive, as lipid and sucrose palmitate melted at temperatures above 40 °C. Tween 20‐stabilized SLN (TS) led to reduced tensile strengths, probably due to perturbative effects of TS and plasticizing effects of Tween 20. Dynamic mechanical analysis showed that TS and Tween 20 increased film mobility. Melting of lipid and emulsifiers, and temperature‐dependent behavior of Tween 20 led to a strong temperature dependence of the film stiffness. By designing the interface, particles can be used to tailor mechanical properties of protein films. Tuned edible films could be used to control mass transfers between foods.     

Different methods to calculate genomic predictions--comparisons of BLUP at the single nucleotide polymorphism level (SNP-BLUP), BLUP at the individual level (G-BLUP), and the one-step approach (H-BLUP)

Several strategies to use genomic data in predictions have been proposed. The aim of this study was to compare different genomic prediction methods. The response variables used in the genomic predictions were deregressed proofs, which were derived from 2 estimated breeding value (EBV) data sets. The full EBV data set from March 2010 included the EBV for production and mastitis traits for all Nordic red bulls. The reduced data set included the same animals as the full data set, but the EBV were predicted from a data set that excluded the last 5 yr of observations. Genomic predictions were obtained using different BLUP models: BLUP at the single nucleotide polymorphism level (SNP-BLUP), BLUP at the individual level (G-BLUP), and the one-step approach (H-BLUP). For the selection candidate bulls, the SNP-BLUP and G-BLUP models gave the same direct genomic breeding values (e.g., correlation of direct genomic breeding values between SNP-BLUP and G-BLUP for protein was 0.99), but slightly different from genomic EBV obtained from H-BLUP (correlations of SNP-BLUP or G-BLUP with H-BLUP were about 0.96). For all traits, SNP-BLUP and G-BLUP gave the same validation reliability, whereas H-BLUP led to slightly higher reliability. Therefore, the results support a slight advantage of using H-BLUP for genomic evaluation.     

Genetic and phenotypic relationships among milk yield and somatic cell count before and after clinical mastitis

This paper studies whether cows with originally lower somatic cell count (SCC) are more susceptible to clinical mastitis (CM) than cows with higher somatic cell count, and evaluates the correlations between CM, SCC, and milk yield. Data were extracted from the Finnish national milk-recording database and from the health recording system. First and second lactation records of 87,861 Ayrshire cows calving between January 1998 and December 2000 were included. Traits studied were incidence of CM, test-day SCC, and test-day milk yield before and following CM. Genetic parameters were estimated using multitrait REML with a sire model. Results did not indicate that cows with genetically low SCC would be more susceptible to CM. The genetic correlation between CM in the first and second lactation was reasonably high (0.73), suggesting that susceptibility to mastitis remains similar across lactations. The genetic correlation between CM and milk yield traits was positive (from 0.38 to 0.56), confirming the genetic antagonism between production and udder health traits. The genetic correlation between SCC and milk was positive in the first lactation, but negative, or near zero in the second lactation. This indicates that breeding for lower SCC might not affect milk production in later lactations. The results of this study support the use of SCC as an indicator of mastitis and a tool for selection for mastitis resistance.     

Sorption of Pb(II), Cr(III), Cu(II), As(III) to peat,and utilization of the sorption properties in industrial waste landfill hydraulic barrier layers

The low conductivity landfill barrier layers protect the groundwater and soil by limiting the water flow through the bottom layers of the landfill material. Many materials used in hydraulic barrier layers also have sorption properties which could be used to reduce environmental risks. The adsorption of lead, chromium, copper, and arsenic to peat was studied with a batch-type test and a column test for compacted peat, both without pH adjustment in acidic conditions. Peat adsorbed all the metals well, 40 000 mg/kg of lead, 13 000 mg/kg of chromium, and 8400 mg/kg of copper in the column test. Arsenic was only tested in a batch-type test, and in that peat adsorbed 60 mg/kg of arsenic. The column test showed heavy metals to be adsorbed on the surface layers of the compacted peat sample, on the first centimeter of the sample. The adsorption was much greater in the column test than in the batch-type test, partly due to the different pH conditions and the buffer capacity of the peat in the column test. The liquid/solid ratio of the column experiment represented a time period of approximately 40 years in a landfill, under Finnish climate conditions. The hydraulic conductivity of the peat decreased as it was compressed, but it already met the hydraulic conductivity limits set by European Union legislation for the hydraulic barrier layer (1 × 10⁻⁹ m/s at a pressure of 150 kPa for a 5-m layer), with a pressure of 50 kPa. The results show that peat would be an excellent material to construct compacted, low hydraulic conductivity layers with adsorption properties in, e.g. industrial waste landfills.     

Apolipoprotein E and A-IV polymorphisms in the Estonian population

A 41-year-old man was admitted to our hospital with chief complaints of painless tumor in the left scrotal contents and loss of body weight. As the tumor was suspected of being a neoplastic lesion, left radical inguinal orchiectomy was carried out. Histopathological examination showed metastatic adenocarcinoma of the bile duct. Metastatic carcinoma to the testicle, epididymis, and their tunics is unusual and testicular metastases from adenocarcinoma of the bile duct is extremely rare. A case of testicular metastases from carcinoma of the bile duct is reported and the literature reviewed.     

The active site of Trichoderma reesei cellobiohydrolase II: the role of tyrosine 169

Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanasecleaving primarily cellobiose units from the non-reducing endof cellulose chains. The ß-l,4 glycosidic bond iscleaved by acid catalysis with an aspartic acid, D221, as thelikely proton donor, and another aspartate, D175, probably ensuringits protonation and stabilizing charged reaction intermediates.The catalytic base has not yet been identified experimentally.The refined crystal structure of CBHII also shows a tyrosineresidue, Y169, located close enough to the scissile bond tobe involved in catalysis. The role of this residue has beenstudied by introducing a mutation Y169F, and analysing the kineticand binding behaviour of the mutated CBHII. The crystal structureof the mutated enzyme was determined to 2.0 Å resolutionshowing no changes when compared with the structure of nativeCBHII. However, the association constants of the mutant enzymefor cellobiose and cellotriose are increased threefold and for4-methylumbelliferyl cellobioside over 50-fold. The catalyticconstants towards cellotriose and cellotetraose are four timeslower for the mutant. These data suggest that Y169, on interactingwith a glucose ring entering the second subsite in a narrowtunnel, helps to distort the glucose ring into a more reactiveconformation. In addition, a change in the pH activity profilewas observed. This indicates that Y169 may have asecond rolein the catalysis, namely to affect the protonation state ofthe active site carboxylates, D175 and D221.     

Development of a high-throughput format for solid-phase extraction of enantiomers using an immunosorbent in 384-well plates

An antibody-based solid-phase extraction method for filtered 384-well plates was developed for a medical drug candidate having two enantiomeric forms in order to demonstrate the potential of the use of recombinant antibody fragments as specific and efficient immunosorbents. An immobilization method using a six-histidine tag of the antibody fragment and mild oxidation was applied in order to immobilize antibody fragments in an oriented and kinetically stable way that ensured high capacity of the antibody support. Phosphate buffer or plasma spiked with enantiomers were used as samples. Selective solid-phase extraction was followed by liquid chromatography-mass spectrometry analysis. Average recoveries for buffer and plasma samples ranged from 79 to 122% and 80 to 108%, respectively. Good linearity was observed in the concentration range of 30-3000 ng/mL of the enantiomer.     

The effect of short-term fasting, apolipoprotein E gene polymorphism, and sex on plasma lipids

Until today, malaria is still one of the most important diseases in Malaysia. This is because Malaysia is located within the equatorial zone with high temperatures and humidities, usually important for the transmission of malaria. The number of malaria cases were estimated to be around 300,000 before the launching of the Malaria Eradication Program (MEP). The program was successful in reducing the numbers progressively during the 1967-1982 period. During the period 1980-1991, the highest number of malaria cases recorded for the country was 65,283 in 1989 (16,902 in Peninsular Malaysia, 47,545 in Sabah and 836 in Sarawak) whilst the lowest was 22,218 (10,069 in Peninsular Malaysia, 11,290 in Sabah and 859 in Sarawak) in 1983. In Malaysia, there are 434 species of mosquitos, representing 20 genera. Of these, 75 species are Anopheles that comprise of 2 subgenus, i.e. Anopheles and Cellia. Of the 75 species, only 9 have been reported as vectors: An. maculatus, An balabacensis, An. dirus, An. letifer An. campestris, An. sundaicus, An. donaldi, An. leucophyrus and An. flavirostris. The behavior, seasonal abundance, biting activities and breeding sites of these species are discussed.