Human Cellular Retinol Binding Protein II Forms a Domain-Swapped Trimer Representing a Novel Fold and a New Template for Protein Engineering

Domain-swapping is a mechanism for evolving new protein structure from extant scaffolds, and has been an efficient protein-engineering strategy for tailoring functional diversity. However, domain swapping can only be exploited if it can be controlled, especially in cases where various folds can coexist. Herein, we describe the structure of a domain-swapped trimer of the iLBP family member hCRBPII, and suggest a mechanism for domain-swapped trimerization. It is further shown that domain-swapped trimerization can be favored by strategic installation of a disulfide bond, thus demonstrating a strategy for fold control. We further show the domain-swapped trimer to be a useful protein design template by installing a high-affinity metal binding site through the introduction of a single mutation, taking advantage of its threefold symmetry. Together, these studies show how nature can promote oligomerization, stabilize a specific oligomer, and generate new function with minimal changes to the protein sequence.     

Chemistry of agaroids,carrageenans and furcellarans

The differences in the fine structure of these algal polysaccharides which are all polydisperse linear galactans are described. The effect of these differences on the conformation of the macromolecules and consequently on the physical properties, in particular the gelling properties, of the individual polymers is discussed. The evidence that gel formation occurs through the association of chain segments into a three dimensional framework which is held together by hydrogen bonds is reviewed.     

Survival and Metabolic Activity of Listeria monocytogenes on Ready‐to‐Eat Roast Beef Stored at 4 °C

Three brands of commercial roast beef were purchased and artificially inoculated with a 5‐strain Listeria monocytogenes cocktail at 2 inoculation levels (approximately 3 and 6 Log CFU/g). Although all 3 brands contained sodium diacetate and sodium lactate, inoculated Listeria cocktail survived for 16 d in all 3 brands; significant increases in L. monocytogenes numbers were seen on inoculated Brand B roast beef on days 12 and 16. Numbers of L. monocytogenes increased to 4.14 Log CFU/g for the 3 Log CFU/g inoculation level and increased to 7.99 Log CFU/g for the 6 Log CFU/g inoculation level by day 16, with the pH values being 5.4 and 5.8 respectively. To measure the cell viability in potential biofilms formed, an Alamar blue assay was conducted. Brand B meat homogenate had the highest metabolic activities (P < 0.05). By comparing its metabolic activities to Brands A and C and the inoculated autoclaved meat homogenates, results indicated that the microflora present in Brand B may be the reason for high metabolic activities. Based on the denaturing gradient gel electrophoresis and the Shannon–Wiener diversity index analysis, the “Brand” factor significantly impacted the diversity index (P = 0.012) and Brand B had the highest microflora diversity (Shannon index 1.636 ± 0.011). Based on this study, results showed that antimicrobials cannot completely inhibit the growth of L. monocytogenes in ready‐to‐eat roast beef. Native microflora (both diversity and abundance), together with product formula, pH, antimicrobial concentrations, and storage conditions may all impact the survival and growth of L. monocytogenes.     

Estrogen content of dairy and swine wastes

Naturally occurring estrogens in animal wastes may cause negative environmental impacts, yet their abundance in animal waste treatment and storage structures is poorly documented. To better quantify estrogen concentrations in animal wastes, multiple waste samples were collected from treatment and storage structures at dairy and swine facilities and analyzed for concentrations of 17beta-estradiol (E2), estrone (E1), and 17alpha-estradiol by gas chromatography-mass spectroscopy and by enzyme linked immunosorbent assay (E2 only). Mass ratios of each estrogen to the macronutrients nitrogen, phosphorus, and potassium were also determined. Because manure application rates are typically macronutrient-based, estrogen to macronutrient ratios are proportional to areal mass application rates of estrogen to fields. Swine farrowing waste (from farrowing sows and piglets) had the highest ratios of E2 to macronutrients. Mean ratios in swine farrowing waste were roughly twice those in swine finishing waste (from growing male and nonpregnant female animals) and more than four times higher than those in dairy waste (from lactating cows in various stages of their reproductive cycles); these differences were statistically significant (alpha = 0.05). Estrone followed a similar trend. In contrast, ratios of 17alpha-estradiol to macronutrients were highest in dairy operations. These results can be used to better predict estrogen loading rates on fields receiving swine and dairy wastes.     

Occurrence, synthesis, and mammalian cell cytotoxicity and genotoxicity of haloacetamides: an emerging class of nitrogenous drinking water disinfection byproducts

The haloacetamides, a class of emerging nitrogenous drinking water disinfection byproduct (DBPs), were analyzed for their chronic cytotoxicity and for the induction of genomic DNA damage in Chinese hamster ovary cells. The rank order for cytotoxicity of 13 haloacetamides was DIAcAm > IAcAm > BAcAm > TBAcAm > BIAcAm > DBCAcAm > CIAcAm > BDCAcAm > DBAcAm > BCAcAm > CAcAm > DCAcAm > TCAcAm. The rank order of their genotoxicity was TBAcAm > DIAcAm approximately equal to IAcAm > BAcAm > DBCAcAm > BIAcAm > BDCAcAm > CIAcAm > BCAcAm > DBAcAm > CAcAm > TCAcAm. DCAcAm was not genotoxic. Cytotoxicity and genotoxicity were primarily determined by the leaving tendency of the halogens and followed the order I > Br > > Cl. With the exception of brominated trihaloacetamides, most of the toxicity rank order was consistent with structure-activity relationship expectations. For di- and trihaloacetamides, the presence of at least one good leaving halogen group (I or Br but not Cl) appears to be critical for significant toxic activity. Log P was not a factor for monohaloacetamides but may play a role in the genotoxicity of trihaloacetamides and possible activation of dihaloacetamides by intracellular GSH and -SH compounds.