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Biomedical subjects

H M Farrell

Publications and source records attributed to H M Farrell.

At least 19 recordsLinked to original sources
PubMed2005

Contributions of terminal peptides to the associative behavior of alphas1-casein.

The N- and C-terminal segments of bovine alphas1-casein-B (f1-23 and f136-196) were characterized under conditions that promoted or inhibited self-association to determine the relative contributions of each fragment to the interaction of alphas1-casein with itself or with other caseins. In earlier studies of f1-23, nuclear magnetic resonance (NMR) data and circular dichroism (CD) spectra showed that its conformation was thermostable between 10 degrees and 25 degrees C. In contrast, NMR studies of f136-196 indicated temperature sensitivity between 10 and 60 degrees C, as did near-UV and far-UV CD data, suggesting a molten globule-like structure at higher temperatures. To compare the effects of temperature on conformational attributes of alphas1-casein and its terminal peptides, additional CD studies were conducted over a broader temperature range (10 to 70 degrees C). The far-UV CD spectra indicated little temperature sensitivity for alphas1-casein, and the N-terminal peptide remained thermostable. During molecular dynamics simulations, the N-terminal peptide conformation did not change significantly, but the conformation of the C-terminal peptide (f136-196) was dramatically altered. These changes are correlated with the thermal instability observed by both CD and NMR in f136-196. Analytical ultracentrifugation studies of the self-association reactions of genetic variants A, B, and C of alphas1-casein showed that at 37 degrees C the associative state is primarily dimeric; the amounts of higher order polymers significantly decreased when temperature was increased from 20 to 37 degrees C. In all 3 genetic variants, the C-terminal portion of the whole molecule showed thermal instability with respect to aggregation to higher polymers, confirming the predictions of CD data and molecular dynamics simulations. The temperature dependency of these conformational changes suggests a possible function for alphas1-casein in facilitating casein-casein interactions in casein micelle formation.

Caseins↗
PubMed2004

Nomenclature of the proteins of cows' milk--sixth revision.

This report of the American Dairy Science Association Committee on the Nomenclature, Classification, and Methodology of Milk Proteins reviews changes in the nomenclature of milk proteins necessitated by recent advances of our knowledge of milk proteins. Identification of major caseins and whey proteins continues to be based upon their primary structures. Nomenclature of the immunoglobulins consistent with new international standards has been developed, and all bovine immunoglobulins have been characterized at the molecular level. Other significant findings related to nomenclature and protein methodology are elucidation of several new genetic variants of the major milk proteins, establishment by sequencing techniques and sequence alignment of the bovine caseins and whey proteins as the reference point for the nomenclature of all homologous milk proteins, completion of crystallographic studies for major whey proteins, and advances in the study of lactoferrin, allowing it to be added to the list of fully characterized milk proteins.

Amino Acid Sequence↗
PubMed2003

Functionality of extrusion--texturized whey proteins.

Whey, a byproduct of the cheesemaking process, is concentrated by processors to make whey protein concentrates (WPC) and isolates (WPI). Only 50% of whey proteins are used in foods. In order to increase their usage, texturizing WPC, WPI, and whey albumin is proposed to create ingredients with new functionality. Extrusion processing texturizes globular proteins by shearing and stretching them into aligned or entangled fibrous bundles. In this study, WPC, WPI, and whey albumin were extruded in a twin screw extruder at approximately 38% moisture content (15.2 ml/min, feed rate 25 g/min) and, at different extrusion cook temperatures, at the same temperature for the last four zones before the die (35, 50, 75, and 100 degrees C, respectively). Protein solubility, gelation, foaming, and digestibility were determined in extrudates. Degree of extrusion-induced insolubility (denaturation) or texturization, determined by lack of solubility at pH 7 for WPI, increased from 30 to 60, 85, and 95% for the four temperature conditions 35, 50, 75, and 100 degrees C, respectively. Gel strength of extruded isolates increased initially 115% (35 degrees C) and 145% (50 degrees C), but gel strength was lost at 75 and 100 degrees C. Denaturation at these melt temperatures had minimal effect on foaming and digestibility. Varying extrusion cook temperature allowed a new controlled rate of denaturation, indicating that a texturized ingredient with a predetermined functionality based on degree of denaturation can be created.

Animals↗
PubMed2002

Secondary structural studies of bovine caseins: structure and temperature dependence of beta-casein phosphopeptide (1-25) as analyzed by circular dichroism, FTIR spectroscopy, and analytical ultracentrifugation.

The defining structural feature of all of the caseins is their common phosphorylation sequence. In milk, these phosphoserine residues combine with inorganic calcium and phosphate to form colloidal complexes. In addition, nutritional benefits have been ascribed to the phosphopeptides from casein. To obtain a molecular basis for the functional, chemical, and biochemical properties of these casein peptides, the secondary structure of the phosphopeptide of bovine beta-casein (1-25) was reexamined using Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies. Both methods predict secondary structures for the peptide which include polyproline II elements as well as beta-extended sheet and turn-like elements. These structural elements were highly stable from 5 degrees to 70 degrees C. Reexamination of previously published 1H NMR data using chemical shift indices suggests structures in accord with the CD and FTIR data. Dephosphorylation showed little or no secondary structural changes, as monitored by CD and FTIR, but the modified peptide demonstrated pronounced self-association. The polymers formed were not highly temperature sensitive, but were pressure sensitive as judged by analytical ultracentrifugation at selected rotor speeds. Molecular dynamics (MD) simulations demonstrated relatively large volume changes for the dephosphorylated peptide, in accord with the pressure dependent aggregation observed in the analytical ultracentrifuge data. In contrast the native peptide in MD remained relatively rigid. The physical properties of the peptide suggest how phosphorylation can alter its biochemical and physiological properties.

Amino Acid Sequence↗
PubMed2002

Molten globule structures in milk proteins: implications for potential new structure-function relationships.

Recent advances in the field of protein chemistry have significantly enhanced our understanding of the possible intermediates that may occur during protein folding and unfolding. In particular, studies on alpha-lactalbumin have led to the theory that the molten globule state may be a possible intermediate in the folding of many proteins. The molten globule state is characterized by a somewhat compact structure, a higher degree of hydration and side chain flexibility, a significant amount of native secondary structure but little tertiary folds, and the ability to react with chaperones. Purified alpha(s1)- and kappa-caseins share many of these same properties; these caseins may thus occur naturally in a molten globule-like state with defined, persistent structures. The caseins appear to have defined secondary structures and to proceed to quaternary structures without tertiary folds. This process may be explained, in part, by comparison with the architectural concepts of tensegrity. By taking advantage of this "new view" of protein folding, and applying these concepts to dairy proteins, it may be possible to generate new and useful forms of proteins for the food ingredient market.

Animals↗
PubMed2001

Solution structures of casein peptides: NMR, FTIR, CD, and molecular modeling studies of alphas1-casein, 1-23.

To determine its potential for interacting with other components of the casein micelle, the N-terminal section of bovine alphas1-casein-B, residues 1-23, was investigated with nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies, and molecular modeling. NMR data were not consistent with conventional alpha-helical or beta-sheet structures, but changes in N-H proton chemical shifts suggested thermostable structures. Both CD and FTIR predicted a range of secondary structures for the peptide (30-40% turns, 25-30% extended) that were highly stable from 5 degrees C to 25 degrees C. Other conformational elements, such as loops and polyproline II helix, were indicated by FTIR only. Molecular dynamics simulation of the peptide predicted 32% turns and 27% extended, in agreement with FTIR and CD predictions and consistent with NMR data. This information is interpreted in accord with recent spectroscopic evidence regarding the nature of unordered conformations, leading to a possible role of alphas1-casein (1-23) in facilitating casein-casein interactions.

Caseins↗
PubMed2001

Secondary structure of bovine alphaS2-casein: theoretical and experimental approaches.

Circular dichroism and Fourier transform infrared spectroscopy of bovine alphaS2-casein both report a 24 to 32% content of alpha-helix. A consensus of sequence based predictions for alpha-helix suggests a Lys77-Gln91 helix within the sequence (Ser61-Arg125). This motif is repeated at (Ser143-Leu207), and this region contains a longer Thr145-Leu177 predicted alpha-helix. A short, seven-member alpha-helix may also organize the N-terminal peptide that precedes the first phosphoserine [-Srp-]3 cluster. As was found for other caseins studied by these spectroscopic methods, a high degree of extended beta-sheet (approximately 30%) and turns (25 to 30%) are predicted for alphaS2-casein.

Animals↗
PubMed2000

Sugar-casein interaction in deuterated solutions of bovine and caprine casein as determined by oxygen-17 and carbon-13 nuclear magnetic resonance: a case of preferential interactions.

17O NMR spectroscopy and (13)C NMR spectroscopy have been used to study the mechanism of interaction of sugars with bovine and caprine caseins in D(2)O. The (17)O NMR relaxation results showed in all cases an increase in water of hydration, as a result of added sugar; this was predominantly associated with "trapped" water in the caseins. Analysis of the vir al coefficients, obtained from the (17)O relaxation data, suggested that preferential interactions occur in the sugar-protein solutions. This could be the result of either sugar binding or a solute-solute thermodynamic effect, preferential hydration. The addition of sugars to deuterated solutions of bovine casein and caprine casein high in alpha(s1)-casein had little or no effect on either line width or chemical shifts of the (13)C NMR spectra of these milk proteins. (13)C NMR studies of sucrose, at various concentrations (100-300 mM) in the presence of caprine casein high in alpha(s1)-casein, showed no changes in either chemical shifts or T(1) values. This indicates that the sugar molecules tumble isotropically and therefore neither bind to the protein nor affect viscosity in the protein-sugar studies. All of these data suggest that the preferential exclusion of the sugar from the domain of the caseins results in preferential hydration of the caseins.

Animals↗
PubMed2000

Influence of neutral salts on the hydrothermal stability of acid-soluble collagen.

The thermal stability of acid-soluble collagens was studied by circular dichroism (CD) spectroscopy. Adult bovine dermal collagen (BDC), rat-tail tendon collagen (RTC), and calf skin collagen (CSC) were compared. Despite some variability in amino acid composition and apparent molecular weight, the CD spectra for helical and unordered collagen structures were essentially the same for all the sources. The melting of these collagens occurs as a two-stage process characterized by a pretransition (Tp) followed by complete denaturation (Td). The characteristic temperatures vary with the source of the collagen; for mature collagens (BDC, RTC) Tp = 30 degrees C and Td = 36 degrees C, and for CSC Tp = 34 degrees C and Td = 40 degrees C. Neutral salts, NaCl or KCl, at low concentrations (0.02-0.2 M) appear to bind to the collagens and shift the thermal transitions of these collagens to lower temperatures.

Acids↗
PubMed1999

Conformational analysis of the hydrophobic peptide alphas1-casein(136-196).

Hydrophobic interactions are important in the self-association of milk proteins, including alphas1-casein. The extent to which casein interaction sites are influenced by local secondary structure is not widely known. Both primary amino acid sequence and local secondary structure are shown to affect the self-association of the hydrophobic peptide alphas1-casein(136-196). The peptide is aggregated at low concentrations (7 microM and above), as determined by 1H nuclear magnetic resonance (NMR) measurements at pH 6.0 in phosphate buffer. Increase in temperature is shown to induce side chain mobility (melting) as indicated by both 1H NMR and near-UV circular dichroism (CD) measurements. As determined by far-UV CD, there is also a loss in the global amount of extended structure with increasing temperature, while beta-turn structures and some aromatic dichroism are conserved at temperatures as high as 70 degrees C. Similar retention of structure occurs at pH 2 and in 6 M guanidine HCl. The observed stability of beta-turns and some side chains in alphas1-casein(136-196) supports previous assumptions that hydrophobic, proline-based turns are important interaction sites in the self-association of alphas1-casein, and possibly in the formation of the calcium transport complexes, the casein micelles. It may be speculated that these areas of the peptide represent a 'molten globule-like', heat stable, core structure for alphas1-casein.

Amino Acid Sequence↗
PubMed1999

Effect of self-association of alphas1-casein and its cleavage fractions alphas1-casein(136-196) and alphas1-casein(1-197),1 on aromatic circular dichroic spectra: comparison with predicted models.

The self-association of native alphas1-casein is driven by a sum of interactions which are both electrostatic and hydrophobic in nature. The dichroism of aromatic side chains was used to derive regio-specific evidence in relation to potential sites of alphas1-casein polymerization. Near-ultraviolet circular dichroism (CD) revealed that both tyrosine and tryptophan side chains play a role in alphas1-casein associations. Spectral evidence shows these side chains to be in an increasingly nonaqueous environment as both ionic strength and protein concentration lead to increases in the degree of self-association of the protein from dimer to higher oligomers. Near-UV CD investigation of the carboxypeptidase A treated peptide, alphas1-casein(1-197), indicated that the C-terminal residue (Trp199) may be superficial to these interactions, and that the region surrounding Trp164 is more directly involved in an aggregation site. Similar results for the cyanogen bromide cleavage peptide alphas1-casein(136-196) indicated the presence of strongly hydrophobic interactions. Association constants for the peptides of interest were determined by analytical ultracentrifugation, and also were approximated from changes in the near-UV CD curves with protein concentration. Sedimentation equilibrium experiments suggest the peptide to be dimeric at low ionic strength; like the parent protein, the peptide further polymerizes at elevated (0.224 M) ionic strength. The initial site of dimerization is suggested to be the tyrosine-rich area near Pro147, while the hydrophobic region around Pro168, containing Trp164, may be more significant in the formation of higher-order aggregates.

Amino Acid Sequence↗
PubMed1999

Characterization of the particles of purified kappa-casein: trypsin as a probe of surface-accessible residues.

kappa-Casein as purified from bovine milk exhibits a rather unique disulfide bonding pattern as revealed by SDS-PAGE. The disulfide-bonded caseins present range from dimer to octamer and above and preparations contain about 10% monomer. All of these heterogeneous polymers, however, self-associate into nearly spherical particles with an average diameter of 13 nm at pH 8.0, as revealed by negatively stained transmission electron micrographs and dynamic light scattering. The weight-average molecular weight of the aggregates at pH 8.0, as judged by analytical ultracentrifugation, is 648,000. Trypsin digestion at pH 8.0 was used to probe the surface groups of the kappa-casein A polymers. The reaction with trypsin was rapid and the peptides liberated were identified by separation with reverse-phase HPLC, amino acid analysis, and protein sequencing. The most rapidly released peptides (t1/2 < 30 sec) were from cleavage at Arg 97 and Lys residues 111 and 112. These results suggest a surface orientation for these residues, and the data are in accord with earlier proposed 3D predictive models for kappa-casein. It is speculated that Arg 97, together with adjacent His residues (98 and 100) and Lys residues 111 and 112, form two positively charged clusters on the surface of the otherwise negatively charged casein. These clusters bracket the neutral chymosin cleavage site (whose hydrolysis triggers a well-known digestive process) and so these clusters may facilitate docking of the substrate caseins with chymosin.

Amino Acid Sequence↗
PubMed1998

Environmental effects on disulfide bonding patterns of bovine kappa-casein.

Bovine kappa-casein, the stabilizing protein of the colloidal milk protein complex, has a unique disulfide bonding pattern. The protein exhibits varying molecular sizes on SDS-PAGE ranging from monomer to octamer and above in the absence of reducing agents. Heating the samples with SDS prior to electrophoresis caused an apparent decrease in polymeric distribution: up to 60% monomer after 30 min at 90 degrees C as estimated by densitometry of SDS-PAGE. In contrast, heating the samples without detergent at 90 or 37 degrees C caused a significant increase in high-molecular-weight polymers as judged by electrophoresis and analytical ultracentrifugation. In 6 M urea, the protein could be completely reduced, but upon dialysis, varying degrees of polymer reformation occurred depending on the dialysis conditions. Spontaneous reoxidation to polymeric forms is favored at low pH (<5.15) and low ionic strength. The results are discussed with respect to the influence of the method of preparation on the polymer size of kappa-caseins and on their resultant physical chemical properties.

Animals↗
PubMed1998

Changes in the secondary structure of bovine casein by Fourier transform infrared spectroscopy: effects of calcium and temperature.

Bovine casein submicelles and reformed micelles, produced by addition of Ca2+, were examined by Fourier transform infrared spectroscopy at 15 and 37 degrees C in aqueous salt solutions of K+ and Na+. Previous measurements of caseins, made in D2O and in the solid form, can now be made in a more realistic environment of H2O. When analyzed in detail, data obtained by Fourier transform infrared spectroscopy have the potential to show subtle changes in secondary structural elements that are associated with changes in protein environment. Electrostatic binding of Ca2+ to casein resulted in a redistribution of the components of the infrared spectra. Addition of Ca2+ in salt solutions of K+ and Na+ led to apparent decreases in large loop or helical structures at 37 degrees C with concomitant increases in the percentage of structures having greater bond energy, such as turns and extended helical structures. At 15 degrees C, Na+ and K+ have differential effects on the Ca(2+)-casein complexes. All of these observations are in accordance with the important role of serine phosphate side chains as sites for Ca2+ binding in caseins and the swelling of the casein structure upon incorporation into reformed micelles at 37 degrees C. This new open, hydrated structure is buttressed by a change in backbone as evidenced by a shift in absorbance to higher wave numbers (greater bond energies) as colloidal micelles are reformed.

Animals↗
PubMed1998

Fate of gamma-irradiated Listeria monocytogenes during refrigerated storage on raw or cooked turkey breast meat.

The radiation resistance and ability of Listeria monocytogenes ATCC 7644, 15313, 43256, and 49594 to multiply on irradiated, air-packed, refrigerated raw or cooked turkey breast meat nuggets (ca. 25 g) and ground turkey breast meat was investigated. Gamma-radiation D values for L. monocytogenes were significantly different on raw and cooked nuggets, 0.56 +/- 0.03 kGy and 0.69 +/- 0.03 kGy, respectively; but they were not significantly different (P < or = 0.05) on raw and cooked ground turkey meat. High populations (approximately 10(9) CFU/g) of L. monocytogenes declined during 14 days of storage at 4 degrees C in both irradiated and nonirradiated samples of raw but not of cooked ground turkey breast meat. A moderate inoculum (approximately 10(3) CFU/g) did not survive a radiation dose of 3 kGy. The population increased in cooked but not in raw samples of irradiated ground turkey meat stored at either 2 or 7 degrees C for 21 days. The D value changed significantly from 0.70 +/- 0.04 to 0.60 +/- 0.02 kGy when the product was cooked to an internal temperature of 80 degrees C before irradiation. Growth on either raw or cooked turkey meat did not alter the radiation resistance of L. monocytogenes. Analyses were performed for pH, aw, moisture, and reducing potential of raw and cooked turkey meat and for pH, amino acid profile, thiamine, and riboflavin contents of aqueous extracts of raw and cooked turkey meats without identifying the factor or factors involved in differences in the survival and multiplication of L. monocytogenes on raw and cooked meat.

Animals↗
PubMed1997

Bovine mammary explant versus primary cell cultures: effect of bovine somatotropin and insulin-like growth factor-I on DNA content and protein synthesis.

Cellular DNA, milk protein content, and protein secretion by bovine mammary explants were compared to cultures of confluent and growing primary bovine mammary secretory cells over 4 d. Explants were obtained at slaughter from eight Holstein cows (120 +/- 35 d lactation). Primary cells were grown to confluence, cryopreserved, thawed, and cultured through five passages. Explants and cells were cocultured with liver and adipose tissue in the presence of somatotropin, insulin-like growth factor-I, and somatotropin + insulin-like growth factor-I. Cellular DNA and milk proteins were assayed using fluorescent probes and flow cytometry. Media proteins were assayed by densitometer scanning of electrophoresis gel bands. DNA content of explant, confluent, and growing primary cells increased similarly through the 96 h incubation. DNA content in G0G1 phase was increased by: (a) insulin-like growth factor-I in explant cells; (b) somatotropin, insulin-like growth factor-I, and their combination in confluent primary cells; and (c) the combination of somatotropin and insulin-like growth factor in growing primary cells. Approximately 65% of explant and confluent primary cells were in the G0G1 or differentiated phase compared to 47% for the growing primary cells. Whey protein content and secretion were similar among cell types. Explant cells contained and secreted more beta-casein than primary cells but secretion trends for beta-casein and k-casein were similar after 48 h for both cell types. Results suggest that primary cell cultures are comparable to explant cultures when used to study mechanisms of DNA and milk protein synthesis and secretion.

Adipose Tissue↗
PubMed1997

Effect of somatotropin and insulin-like growth factor-I on milk lipid and protein synthesis in vitro.

The objective of this research was to test the hypothesis that bST stimulates milk secretion through the action of IGF-I. Cocultures of bovine mammary, adipose, and liver tissues were incubated with increasing concentrations (0 to 1000 ng/ml) of bST, IGF-I, prolactin, or bST plus prolactin. In addition, cocultures of mammary and adipose tissues without liver tissue were incubated with IGF-I. The synthesis of milk lipids and proteins and the concentration of cellular DNA were measured. The addition of liver tissue depressed DNA concentration and the synthesis of lipids and proteins in mammary tissue. In mammary tissue, increasing bST concentration resulted in greater synthesis of FFA, total lipids, and proteins than that produced by increasing concentrations of IGF-I, and the effect on protein synthesis was linear. Conversely, in adipose tissue, increasing the concentration of IGF-I in the presence of liver resulted in greater synthesis of FFA than that obtained by bST, and the effect was linear. The results do not support the hypothesis that bST acts through the actions of IGF-I on mammary cells. Rather, bST alters the delivery of nutrients to the mammary cells from other tissues and affects milk component synthesis through a mechanism that may not involve IGF-I.

Adipose Tissue↗