Latex particles with thermo-flocculation and magnetic properties for immobilization of alpha-chymotrypsin.

Core-shell-type latex particles composed of styrene, N-isopropylacrylamide (NIPAAm), and N-acryloxysuccinimide (NAS) were synthesized by surfactant-free emulsion polymerization. The latex particles show thermo-flocculation behavior due to the presence of temperature-sensitive monomer NIPAAm and could be used for immobilization of alpha-chymotrypsin through covalent bonding with the reactive ester groups of NAS. Enzyme recycle could be accomplished in this immobilized enzyme system by sedimentation of the thermo-flocculated latex particles in 20 min at 30 degrees C by raising the salt (NaCl) concentration to 0.5 M. To further enhance the sedimentation rate, ultrafine magnetite particles were prepared and included during polymerization to produce magnetic temperature-sensitive latex particles (MTLP), which could be recovered 6 times faster after thermo-flocculation by applying a low magnetic field. However, a higher salt concentration was necessary to flocculate the MTLP under the same condition as a result of its increased surface hydrophilicity, which originates from different polymerization conditions and the incorporation of magnetite. The immobilized enzyme shows high activity even against macromolecular substrates (hemoglobin and casein) owing to limited diffusion resistance, with full activity retention for nonmagnetic latex but one-half reduction in activity if the magnetic property was introduced. Optimal enzyme immobilization pH and enzyme loading were determined, and properties of the immobilized enzyme were characterized. The immobilized enzyme was used in 10 repeated batch hydrolyses of casein with successive flocculation/dispersion cycles and showed less than 15% activity decrease at the end. Overall, introducing the magnetic property to the latex could effectively enhance the solid-liquid separation rate after thermo-flocculation and maintain enzyme activity after repeated use but adversely influence the activity of the immobilized enzyme.

Perinatal ontogeny of brain growth in the domestic pig.

The perinatal development of the brain is highlighted by a growth spurt whose timing varies among species. The growth of the porcine cerebrum was investigated from the third trimester of gestation (70 days postconception) through the first 3.5 weeks of postnatal life (140 days postconception). The shape of the growth curves for cerebrum weight, total protein mass, total cell number (estimated by DNA content), and myelination (estimated by cholesterol accretion) were described. The growth velocity of cerebrum weight had two peaks, one at 90 days and the other at 130 days postconception, whereas that of total protein was greatest from 90 to 130 days postconception, and that of total DNA was greatest between 90 and 110 days and again at 130 days postconception. The growth velocity for total cholesterol continued to increase during the entire period, suggesting that myelination continued after the growth spurts for cells (protein and DNA). The growth velocity patterns observed in these contemporary pigs suggest that this species may be an appropriate model for human brain development, not only in the perinatal pattern of increase in mass of the cerebrum, as established previously, but also with regard to the patterns of cellular development and myelination.

Pigs fed cholesterol neonatally have increased cerebrum cholesterol as young adults.

Sixty-eight female neonatal pigs selected for seven (Experiment 1) or eight (Experiment 2) generations for high (HG) or low (LG) plasma cholesterol were used to test the hypothesis that neonatal dietary cholesterol fed during the first 4 or 8 wk of postnatal life increases the cholesterol content of the cerebrum in young adulthood following free access to a high-fat (15%), high-cholesterol (0.5%) diet from 8 to 20 or 24 wk of age. Pigs were removed from their dams at 1 d of age and given free access to a sow-milk replacer diet containing 9.5% coconut fat and 0 or 0.5 % cholesterol. All pigs (except four HG and four LG pigs in Experiment 2, which were deprived of cholesterol throughout the study) were fed the high-fat, high-cholesterol diet from 8 wk to termination at 20 or 24 wk of age. Cerebrum weight and cholesterol concentration were higher in pigs fed cholesterol neonatally than in those deprived of cholesterol neonatally in both experiments, but weight and cholesterol concentration were unaffected by genetic line. Cholesterol concentrations in longissimus and semitendinosus muscles and in subcutaneous fat were unaffected by diet or genetic line. We conclude that dietary cholesterol deprivation during the first 4 to 8 wk of life in piglets is associated with lower cholesterol concentration and total content in the young adult cerebrum than in pigs supplemented with cholesterol in early life. These data support previous observations and suggest the possibility of a metabolic need for neonatal dietary cholesterol in normal brain development.

Alleles of the cholesterol 7 alpha-hydroxylase (CYP7) gene in pigs selected for high or low plasma total cholesterol.

Crossbred pigs were selected for high (HTC) or low (LTC) plasma total cholesterol (TC). Pigs from the seventh (n = 51) and eighth (n = 92) generations were used to determine restriction fragment length polymorphisms (RFLP). Using TaqI restriction enzyme digestion, the frequencies of two alleles (2.8- or 5.0-kb fragments) of the cholesterol 7 alpha-hydroxylase (CYP7) gene were determined in the two populations as a potential indicator of TC concentration at 8 weeks of age. Only the 2.8-kb fragment allele was present in the 26 HTC pigs tested in Generation 7. In the LTC pigs both the 2.8- and 5.0-kb alleles were present in 12 pigs, and only the 5.0-kb allele was present in 13 pigs. The allele frequencies of the 2.8 and 5.0 fragments, respectively, were .26 and .74 in LTC pigs and 1.00 and 0 in HTC pigs. There was an association (P < .001) between the 5.0- and 2.8-kb CYP7 alleles, respectively, and low and high TC concentrations. In Generation 8, all HTC pigs were homozygous for the 2.8-kb allele. The 5.0 kb allele was present in all LTC pigs tested and was homozygous in 57% of LTC pigs. Mean plasma TC was 105.0 mg/dl in 30 pigs homozygous for the 2.8-kb allele in Generation 8; means for LTC pigs were 53.5 and 60.4 mg/dl in 35 pigs homozygous for the 5.0-kb allele and in 27 heterozygous pigs, respectively. High TC was associated with the presence of the 2.8-kb allele, and low TC was associated with the presence of the 5.0-kb allele in both Generations 7 and 8. We conclude that TaqI RFLP analysis of the CYP7 gene is a reliable indicator for TC in these swine.

Divergent concentrations of plasma metabolites in swine selected for seven generations for high or low plasma total cholesterol.

It was reported previously that selection for high (HG) or low (LG) plasma total cholesterol (TC) at 8 wk of age in a composite four-breed swine population resulted after four generations in divergent mean concentrations in the selected lines. The data revealed a significant positive correlation between body weight (BW) and TC concentration at 8 wk of age and differential responses in litter size, backfat depth, and carcass length at 6 mo of age. We report here the relationship between plasma TC concentration and other plasma traits related to growth and metabolism in the seventh generation of selection in these two lines of pigs. We measured plasma concentrations of TC, HDL cholesterol (HDL-C), triglycerides (TG), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), urea nitrogen (urea N), and three transaminases (alanine aminotransferase, ALT; aspartate aminotransferase, AST; gamma glutamyltransferase, GGT) in seventh-generation male and female pigs at 8 wk of age. Birth weight (1.48 vs 1.38 kg), 8 wk BW (14.85 vs 12.00 kg), TC (116.8 vs 63.6 mg/dL), HDL-C (43.9 vs 25.5 mg/dL), TG (50.5 vs 33.0 mg/dL), and ALP (78.3 vs 44.9 units/L) were higher (P < .01) in HG than in LG pigs, whereas ALB (3.2 vs 3.4 g/dL), ALT (43.0 vs 45.9 units/L), and AST (53.0 vs 62.2 units/L) were lower in HG than in LG pigs (P < .05). At 8 wk, overall plasma TC concentration was correlated with BW (r = .34, P < .01) and with ALP (r = .23, P < .05) but was not related to ALT, AST, or GGT. Plasma TP urea N, and GGT were unaffected by genetic line on sex. We conclude that the difference between HG and LG pigs in TC concentration in generation 4 at 8 wk of age has persisted but not broadened in pigs of generation 7, that changes in plasma ALP, ALT, and AST may have occurred in response to selection for high or low plasma TC, and that ALP is correlated with plasma TC concentration.

Response to dietary fat and cholesterol in young adult boars genetically selected for high or low plasma cholesterol.

Twenty-eight young adult boars (age 7 to 8 mo) genetically selected for four generations for high (HG) or low (LG) plasma cholesterol were studied to assess dietary and genetic effects and their interactions on cholesterol metabolism. Boars within a genetic group were paired according to their plasma cholesterol concentration at 8 wk of age and were fed for 15 wk (2.7 kg/d) one of two diets (16.5% fat and 1,156 mg of cholesterol/kg diet, HD; or 3.1% fat and 0 cholesterol diet, LD) in a 2 x 2 factorial arrangement. Plasma total cholesterol (P < .01) and HDL-cholesterol (P < .01) concentrations were higher in boars fed HS (P < .01) and in HG boars (P < .01). There was a trend toward a diet x genotype interaction for plasma HDL-cholesterol (P < .06). Plasma insulin concentration tended to be lower in boars fed HD (P < .09) or HG boars (P < .10). There was a diet x genotype interaction for plasma glucagon (P < .04) concentration and a trend toward an interaction for insulin (P < .07). After 15 wk, all boars were killed by electrocution and exsanguination and measurements were taken. Backfat thickness was greater (P < .01) and carcass length (P < .01) and weights of the four lean cuts (P < .02) were lower in LG than in HG boars. No microscopic atherosclerotic plaques were observed in aorta or coronary arteries. Cholesterol concentration in subcutaneous fat was lower (P < .04) in LG boars, suggesting that cholesterol content of depot fat can be reduced by genetic selection in swine.

A series of point mutations reveal interactions between the calcium-binding sites of calmodulin.

Calmodulin is a member of the "EF-hand" family of Ca(2+)-binding proteins. It consists of two homologous globular domains, each containing two helix-loop-helix Ca(2+)-binding sites. To examine the contribution of individual Ca(2+)-binding sites to the Ca(2+)-binding properties of CaM, a series of four site-directed mutants has been studied. In each, the glutamic acid at position 12 in one of the four Ca(2+)-binding loops has been changed to a glutamine. One-dimensional 1H-NMR has been used to monitor Ca(2+)-induced changes in the mutant proteins, and the spectral changes observed for each mutant have been compared to those for wild-type CaM. In this way, the effect of each mutation on both the mutated site and the other Ca(2+)-binding sites has been examined. The mutation of glutamate to glutamine at position 12 in any of the EF-hand Ca(2+)-binding loops greatly decreases the Ca(2+)-binding affinity at that site, yet differs in the overall effects on Ca2+ binding depending on which of the four sites is mutated. When the mutation is in site I, there is only a small decrease in the apparent Ca(2+)-binding affinity of site II, and vice versa. Mutation in either site III or IV results in a large decrease in the apparent Ca(2+)-binding affinities of the partner C-terminal site. In both the N- and C-terminal domains, evidence for altered conformational effects in the partners of mutated sites is presented. In the C-terminus, the conformational consequences of mutating site III or site IV are strikingly different.