In JoVE (1)

Other Publications (3)

Articles by William J. Spencer in JoVE

Other articles by William J. Spencer on PubMed

Alpha-lactalbumin in Human Milk Alters the Proteolytic Degradation of Soluble CD14 by Forming a Complex

Pediatric Research. Dec, 2010  |  Pubmed ID: 20717070

Mother's milk represents a foundational step in the proper development of newborn immunity. This is achieved, in part, through the action of numerous regulatory proteins such as soluble cluster of differentiation 14 (sCD14) found in significant quantities in human milk (~25-50 μg/mL). In adults, CD14 stimulates cytokine production in response to lipopolysaccharide (LPS), the major lipid component found in the outer membrane of Gram-negative bacteria. However, the fate and function of sCD14 in the neonatal gastrointestinal (GI) tract are unknown and may function differently from adults. Therefore, we administered human sCD14 to experimental animals and observed that it persisted in the upper GI tract after feeding. In our search for potential proteolytic protectants, immunoprecipitation of sCD14 from human milk revealed a 15-kD novel protein that copurified with sCD14. Mass spectrometry analysis of the protein identified alpha-lactalbumin. CD14 was also identified by immunoblot after immunoprecipitation of alpha-lactalbumin from milk. In vitro digestion assays revealed that purified alpha-lactalbumin decreases the proteolytic degradation of human milk derived sCD14 in vitro, suggesting a mechanism by which this key LPS receptor may remain functional in the neonate gut.

(14)C Radiolabeling of Proteins to Monitor Biodistribution of Ingested Proteins

Analytical Biochemistry. Mar, 2011  |  Pubmed ID: 21094630

The economical preparation of microgram quantities of (14)C-labeled proteins by in vacuo methylation with methyl iodide is described. The (14)C radiolabeling was achieved by the covalent attachment of [(14)C]methyl groups onto amino and imidazole groups by reaction in vacuo with [(14)C]methyl iodide. The method was tested by investigating the biodistribution of (14)C in rats that were fed (14)C-labeled human soluble cluster of differentiation 14 (CD14) protein, a receptor for bacterial lipopolysaccharide. Two other control proteins, bovine serum albumin (BSA) and casein, were also labeled with (14)C and used for comparative analysis to determine the following: (i) the efficacy and cost efficiency of the in vacuo radiolabeling procedure and (ii) the extent of incorporation of the (14)C label into the organs of orogastrically fed 10-day-old Sprague-Dawley rats. [(14)C]BSA, [(14)C]casein, and [(14)C]CD14 were individually prepared with specific radioactivities of 34,400, 18,800, and 163,000 disintegrations per minute (dpm)/microg, respectively. It was found that the accumulation of (14)C label in the organs of [(14)C]CD14-fed rats, most notably the persistence of (14)C in the stomach 480 min postgavage, was temporally and spatially distinct from [(14)C]BSA and [(14)C]casein-fed rats.

Maternal Separation and Gastrointestinal Transit Time in Neonate Rats

Laboratory Animals. Oct, 2011  |  Pubmed ID: 21828078

Gastrointestinal transit times (GItts) were compared in separate litters of 10- and 15-day-old Sprague Dawley rats using barium sulphate. By tracking the leading front of the bolus on radiographs, the gastrocaecal transit times in pups were estimated. To measure the total GItt, the duration from orogastric gavage until an observable defecation of barium sulphate was recorded. The gastrocaecal times for 10-day-old pups maintained with their dam (n = 5) ranged from 4-5 h and those removed from the dam ranged from 2.5-5 h. For 15-day-old pups with their dam (n = 6) and without dam (n = 5), gastrocaecal times ranged from 4-6 h and 3.5-5 h, respectively. Ten-day-old pups that remained with the dam had a GItt of 13.8 ± 0.9 h and those kept in the absence of the dam had a time of 9.3 ± 0.7 h. This decrease (P < 0.05) in GItt in the absence of the dam was age-dependent in 10-day-old pups, and was not observed (P > 0.05) in 15-day-old pups. The results provide a basis, for the design of future studies involving neonate rat metabolism, to include maternal presence.

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