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Abstract. Tobacco usage kills more than 8 million people a year. Approximately 7 million of those deaths are the result of direct tobacco use, while approximately 1.2 million are the result of non-smokers being exposed to second-hand smoke. About 200 million of the world’s one billion smokers are women and usage among women is increasing in some countries. Nicotine from smoking tobacco, specifically its metabolite cotinine, has negative effects on human health causing lung cancer, COPD and non-respiratory problems. Over a billion people worldwide are Vitamin D deficient or insufficient, which is prevalent across all age-groups, geographic regions, and sunlight. With the discovery of Vitamin D in 1919, a new chapter in the prevention of rickets was introduced opening the door to its therapeutic properties for other diseases. Since 1919, there have been many clinical and epidemiolocal studies performed globally on the effect of the vitamin on prevention of other diseases, including but not limited to, cancer, autoimmune disorders, cardiovascular diseases, and osteoporosis. Exposure to environmental tobacco smoke has been associated with reduced levels of Vitamin D in the blood stream and sinus tissues. Manavi et al. (2015) demonstrated that among three smoking categories (heavy, light, non-smokers), black female heavy smokers have lower vitamin D (13.374 ng/ml), than Hispanic (19.213 ng/ml) or white (24.929 ng/ml) females correlating to higher levels of cotinine. Therefore, blood serum concentrations of cotinine contribute to decreased Vitamin D concentrations in addition to other factors such as gender and ethnicity. Further information is provided in the text.

We have undertaken a new and more detailed Fourier-transform infrared (FTIR) spectroscopic study of alpha-lactalbumin (in D2O solution) aimed at correlating its secondary structures to observed Amide I' infrared bands. The spectra reported here were interpreted in light of the recently determined crystal structure of alpha-lactalbumin and by comparison with the spectra and structure of the homologous protein lysozyme. Of particular importance is the new evidence supporting the assignment of the band at 1639 cm-1 to 3(10)-helices. This assignment is in excellent agreement with one based on theoretical and experimental studies of 3(10)-helical polypeptides. The frequency observed for 3(10)-helices is distinctly different from that at which alpha-helices are typically found (viz., around 1655 cm-1). In the present study, two bands are clearly resolved in the latter region at 1651 and 1659 cm-1. Both are apparently associated with alpha-helices. These results suggest that for D2O solutions of globular proteins. FTIR spectroscopy can be a facile method for detecting the presence of these two different types of helical conformation and distinguishing between them. This provides a distinct advantage over ultraviolet circular dichroism spectroscopy (UV-CD). This work also provides a basis for future studies of alpha-lactalbumin which examine the effects of environment (e.g., pH, temperature) and ligands (e.g., Ca2+, Mn2+) on its conformation.

To investigate the effect of blood serum concentration of cotinine among non-smokers, passive/light smokers and active smoker females in the United States population as it compares to vitamin D blood serum concentrations.National Health and Nutrition Examination Survey (NHANES) that is designed to assess the health and nutritional status of adults and children in the United States by Centers for Disease Control and Prevention (n=22,196).The analyses demonstrated that among all three smoking categories, black female active smokers have lower vitamin D (13.374 ng/ml), than hispanic (19.213 ng/ml) or white (24.929 ng/ml) females. It was demonstrated that the active smoker black females have the highest percentage of vitamin D deficiency and inadequacy in the population compared to other ethnic females.The cotinine blood serum concentrations can also affect vitamin D concentrations in addition to other factors such as gender, ethnicity, dietary supplement intake and sun exposure.

Because of low energy reserves at birth, piglets are dependent on the sow’s milk for survival. Breed differences can influence milk composition. The objective of this study was to examine litter characteristics, milk composition including calcium and the water holding capacity of casein, solvation, between the highly prolific Meishan breed and lean crossbred pigs. Samples were collected by hand-milking at early (days 3–5) and late (days 20–21) lactation. Milk fat percentage for Meishans was higher than for the crossbred, especially during early lactation (10.72±0.5>8.91±0.4 (crossbred); P 6.49±0.39 (crossbred); P>0.05. No differences were observed between breeds for lactose or protein at either sampling time. Calcium concentration (mg/dl) was higher in Meishan milk than in crossbred milk at early lactation (0.204±0.01>0.178±0.01; P 11.1±0.7; P

A growth inhibitory protein, mammary inhibitory activity (MIA), was purified to apparent homogeneity from human milk. At concentrations of 5 to 10 ng/ml, the factor inhibited the growth of mammary epithelial cells by 30–80% and also inhibited the growth of normal rat kidney cells. Whereas the cell division of normal human mammary epithelium in primary culture was inhibited by MIA, cell division by fibroblasts from the same tissues was unresponsive. Inhibition was dose and time dependent and readily reversed when MIA was removed. MIA also inhibited growth in culture for three cell lines. The growth inhibitory protein migrated as a 14 kDa protein under reducing conditions on polyacrylamide gels in the presence of sodium dodecyl sulfate. The apparent isoelectric point was pI 5.0. The amino acid composition of MIA resembled that of α-lactalbumin, and sequence analysis of the N-terminal region comprising residues 1–24 and an isolated peptide were identical with the N-terminal and residues 66–81 of human α-lactalbumin. In addition, MIA was active in the lactose synthase system. The results strongly suggest that MIA and α-lactalbumin are identical proteins. Consistent with these results, α-lactalbumin preparations from several mammalian species, including human, goat, cow and camel, were all found to be growth inhibitory for cultured mammary epithelial cells. The inhibitory activity associated with human α-lactalbumin was destroyed by digestion with pepsin or chymotrypsin, by carboxymethylation of cysteine, or by cleavage of methionine 90 following cyanogen bromide treatment. The results raise the possibility that during lactation α-lactalbumin, a product of mammary cell differentiation, could be a physiologically relevant feed-back inhibitor of mammary cell growth and perhaps of other cell types as well.

Whole native caseinate (WNC) and casein (CN) fractions from preparative DE-52 cellulose urea columns were chromatographed using C-8 reverse-phase (RP) and DEAE-type anion-exchange (AEx) HPLC systems. With RP, αS2-CN and κ-CN eluted first as several small peaks; αS1-CN eluted later as two peaks, followed by β-CN peaks. With AEx, κ-CN eluted early as a group of peaks, β-CN eluted next, and αS1-CN and αS2-CN coeluted last. Standard curves were prepared for αS1-CN and β-CN using RP-PHLC and showed correlation coefficients of 0.99 and 0.98, respectively. The caseins in WNC, nonfat dry milk casein, commercial casein(ates) and caseins from milks of individual cows were determined.

The strong calcium-binding site of alpha-lactalbumin comprises the carboxylate side chains of aspartic acid 82, 87, and 88 and the carbonyl oxygens of residues 79 and 84. A single methionine residue was selectively modified by controlled CNBr cleavage to yield homoserine at position 90. The CNBr-cleaved alpha-lactalbumin lost the ability to bind calcium strongly as monitored by intrinsic fluorescence, electrophoretic mobility, atomic absorption, and x-ray fluorescence. Remarkably, the modified protein was still competent in lactose biosynthesis, although activity was reduced to 1/40th that of the native form of the protein. Although the strong calcium-binding site was destroyed as a result of the cleavage of the calcium-binding loop, a secondary calcium site was retained that directly affects a rate enhancement of lactose biosynthesis when saturated, resulting in approximately a two- to threefold increase in rate at 1 mM CaCl2 with an activation equilibrium constant of 350 +/- 40 microM.

Potato acid phosphatase was immobilized on succinamidopropyl-derivatized glass beads for use in removing phosphate groups from bovine whole casein. Controlled pore glass beads (with and without glyceryl coating) were silanized and succinylated. The enzyme was covalently bound to derivatized glass beads through carbodiimide coupling. The pH optima for the immobilized enzyme remained at pH 5.5 for p -nitrophenyl phosphate and pH 6.0 to 6.5 for casein substrates. Activities of acid phosphatase (pH 5.5, p -nitrophenyl phosphate substrate) immobilized on the plain and glyceryl-coated controlled pore glass beads were 14 and 4%, respectively, of the free enzyme. When whole casein was used as substrate, the 15 to 20min enzyme activity of free enzyme was 16% of its activity on p -nitrophenyl phosphate and after 4h, 14% of its original rate on casein. After 18h of incubation, the activity of the immobilized enzyme was 36 to 40% of its rate at 4h. A 70% dephosphorylation of whole casein was attained using a concentration of 2.6mg enzyme controlled pore glass beads complex per milligram of whole casein at pH 6.5, 37°C, for 18h.

Textural differences between Cheddar and Cheshire cheeses were examined rheologically to provide a means of distinguishing these two English cheeses. Body breakdown of a 60-wk Cheshire sample occurred at a lower strain than did a 20-wk sample, whereas a 60-wk Cheddar sample did not break down under the same conditions. All cheeses followed the Arrhenius equation, and the energy of activation obtained varied with age and type of cheese. Other analytical techniques showed differences between the samples but were not suitable for distinguishing one type of cheese from the other.

Reduction of Cheddar cheese making time was investigated by regulating the amount of lactic starter culture initially added to the milk and preparing the cheese at 37.8°C. Laboratory trials showed that in conventional cheese making at 31.1°C, an initial cell count of 10 7 cfu/ml was necessary to achieve a 10 9 cfu/g population density and desirable acidity (pH 5.3 to 5.4) at the milling stage. In the modified process, cheese curd was prepared by prewarming milk to 37.8°C and adding starter culture at 10 8 cfu/ml and milk coagulant (veal rennet) simultaneously. This method shortened preparation time by as much as 90min, depending on the acid production of the starter culture. Cheeses made in six trials were stored at 4.4°C for 1 yr. Ratings by an expert panel showed no significant differences in flavor, body, and texture characteristics that could be attributed to the method of preparation. On a 5-point intensity scale, increase in sharpness correlated significantly (P⩽.05) with storage time with slightly greater increase observed in the experimental cheese. No difference in sharpness was significant between samples made by conventional and experimental methods. Decline in curdiness with storage time showed similar correlations. The results suggested that reduction of make time by increasing starter culture inoculum to 10 8 cfu/ml and renneting at the time of setting at a higher temperature (37.8°C) improved aging characteristics of the finished cheese over the storage time studied.

β -Lactoglobulin and α -Lactalbumin were isolated and crystallized by the classical method of Aschaffenburg and Drewry, and the purity of the crystals was assessed by native PAGE and SDS-PAGE. Although β -lactoglobulin was free of contaminants, α -lactalbumin was still contaminated after five recrystallization steps. Yields of crystals in this study were lower than reported by Aschaffenburg and Drewry. Further, it was observed that α -lactalbumin undergoes a Ca 2+ /Ca 2+ -free, dependent electrophoretic shift characteristic of many calcium-binding proteins.

Bovine mammary gland calmodulin, purified by conventional fractionation procedures, was compared with similarly purified bovine brain calmodulin. Affinity chromatography on W-7 agarose of the crude fractions from mammary gland and brain yielded pure proteins containing one trimethyllysine residue per 16,800 daltons with essentially identical amino acid compositions. Kinetic parameters of these two proteins with respect to their ability to activate phosphodiesterase were determined. The constants for half maximum activation were .39 and .44 nM for bovine brain and bovine mammary gland calmodulins, respectively; both proteins gave similar maximum velocities. Based on the amino acid composition and kinetic data, it is concluded that the two proteins are essentially identical.

With a simple, sensitive, and rapid SDS-PAGE procedure, developed during the course of this study, calmodulin was not found in cows' milk. The absence of calmodulin at the level of detection provided by this procedure (.4 μ g) was unexpected. Because milk is a repository for many protein and enzyme systems, and because the mammary gland is known to contain calmodulin, it was conjectured that at least some "leakage" of the protein into milk would occur, especially into colostrum.

Calmodulins (CaM) were isolated and characterized from two well-known latex producing plants, Papaver somniferum and Euphorbia lathyris. The molecular weights of both were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 17,000 comparable to that of bovine brain CaM. Amino acid compositions also compared similarly with those of known CaMs, with regard to the presence of trimethyllysine and the ratio of phenylalanine to tyrosine. The Cornish-Bowden equation (SDeltan) revealed strong statistical correlations of P. somniferum and E. lathyris CaM with those of other plants and animals, although their amino acid compositions were not identical. Both plant CaM stimulated CaM dependent cAMP phosphodiesterase: for Papaver somniferum the K(a) was found to be 1.09 nanomolar and for Euphorbia lathyris, 2.01 nanomolar.

α s1 -Casein has been observed to differ from α s1 -B by its solubility in CaCl 2 solutions at 1 and 37C, and by its stabilization profile in the presence of κ-casein and calcium ions. The different characteristics of the protein α s1 -A have been attributed to the deletion of a segment of nonpolar amino acids resulting in decreased hydrophobic interactions among α s1 -A molecules. The deletion also has impaired the formation of α s1 -κ-casein micelles under conditions of normal formation.

Milk protein polymorphism has been studied in depth since Aschaffenburg's discovery of genetically distinct forms of β-lactoglobulin in 1955. The significance of these various protein polymorphisms, as related to the synthesis and secretion of milk, is yet to be elaborated fully. However, certain areas in milk protein polymorphism warrant particular attention. These include: assignment of mRNA triplet codons to known amino acid substitutions; biosynthesis and primary structure of polymorphs; observations of micelle formation in vivo through electron microscopy; mode of inheritance, breed specificity, and linkage of these genes; and, finally, biological function of the milk protein variants.

1. 1. β-Caseins B ( Bos taurus ), B Z and D ( Bos indicus ) were examined for amino acid composition. 2. 2. B Z differs from B in content of several neutral amino acids and possibly glutamic acid, as well as in tryptic “fingerprints”. 3. 3. β-D differs from B Z notably in content of arginine (+1), lysine (+1) and histidine (−1), but also in one additional residue each of threonine, serine and alanine. 4. 4. β-D (like β-C, Bos taurus ) lacks 1–2 residues of phosphorus as compared with B. 5. 5. It is likely that β-C ( Bos taurus ), itself, occurs as polymorphs.

The three known genetic variants of β -casein (A, B, and C) have been purified by column chromatography in 3.3m urea, and their purity verified by polyacrylamide-gel electrophoresis. Qualitative determination of the amino terminal amino acid by two independent chemical methods revealed arginine for each of the three variants. During reaction with carboxypeptidase A, valine was released most rapidly and was closely followed by isoleucine. The data indicate either an ileu-ileu-val or an ileu-val-ileu C-terminal sequence in each variant. Molecular weights of about 25,000 for β -caseins A, B, and C were calculated from the amount of valine released.

Isolated α s5 -casein (zone 0.86) is converted by treatment with 2-mercaptoethanol into two components with electrophoretic mobilities identical to those of α s3 -casein (zone 1.04) and of α s4 -casein (zone 1.00). The molar ratios of amino acids of α s3 -, α s4 -, and α s5 -caseins are similar. The data suggest that α s5 -casein is a molecule of α s3 -casein united to a molecule of α s4 -casein by at least one intermolecular disulfide bond. Moreover, a close relationship between α s3 -casein and α s4 -casein is revealed by the similarity of their amino acid molar ratios.

κ -Casein, after reduction of the disulfide bond(s) by 2-inercaptoethanol, can be separated from α s1 - and β -casein variants, as well as from whole casein, by DEAE-cellulose-urea chromatography. When κ -easein is in the reduced form it elutes from DEAE-cellulose at 0.05m NaCl. Use of a reduction step simplifies purification of the major casein components ( α s1 -, β ) by chromatography.

The Nomenclature Committee has given particular attention to genetic polymorphism in the proteins of cow's milk, and has adopted a nomenclature scheme which embraces genetic designations. The β-lactoglobulins are to be referred to as β-lactoglobulins A, B, and C. Three genetic forms of β-casein have been reported to occur either singly or in pairs in the milk of individual cows, and are appropriately referred to as β-caseins A, B, and C. The term αs1-casein has been recommended and refers to those components of the α-casein complex that are precipitated by calcium and stabilized by κ-casein against precipitation by calcium. They constitute the major proportion of the αs-caseins, and have been reported to exist in three genetic forms, A, B, and C. The genetic forms of αs1- and β-caseins can be conveniently identified by their relative mobilities by zonal electrophoresis.

1. 1. The major casein of cow's milk (αs1− occurs in four known polymorphic forms, A,B, C and D. 2. 2. αs1− Casein A is devoid of eight amino acid residues (apparently in sequence) which are present in the more common variants, B and C. 3. 3. αs1− A is soluble in calcium chloride solutions above 0·10 M at 1°C while the B and C variants are insoluble. 4. 4. The increased solubility of the A variant appears to have arisen as a result of either less extensive hydrophobic bonding and/or a conformational change of the protein brought about by the deletion of the nonpolar amino acids alanine, valine, two phenylalanines and three leucines and one polar amino acid, arginine.

Three genetically determined variants of αs1-casein have been prepared, and the purity of each has been verified by starch-gel-urea electrophoresis. The amino-terminal end group was determined qualitatively by two independent chemical methods. Arginine was the only amino-terminal amino acid revealed by each method for all three variants. The results of the action of carboxypeptidase A can be interpreted most simply to infer a leucyl-tryptophan carboxyl-terminal sequence for all three proteins. The kinetics of amino acid release by carboxypeptidase was similar for the three αs1-caseins. A molecular weight of ~31,000 was determined from the carboxypeptidase data, assuming a single polypeptide chain. The results indicate that all amino acid differences among the variants must occur within the polypeptide chain, if the latter assumption is valid.

A linear relationship (r=.89) was found between casein-pellet-solvation and heat stability at 135C for individual cow milks. Casein-pellet-solvation was determined by centrifugation of whole milk at 68,000 g for 35min at 37C.

alpha-Lactalbumin, the modifier protein of galactosyl transferase in the synthesis of lactose by the mammary gland, has been shown to undergo a Ca2+-dependent electrophoretic shift. Such shifts, characteristic of most calcium modulated proteins, are related to gross conformational changes upon binding calcium when detected in the presence of detergent (SDS-PAGE). However, we detected the calcium shift for alpha-lactalbumin using non-denaturing PAGE (ND-PAGE) where electrical charge changes are observed upon binding calcium. In order for a shift to be observed between the apo and calcium bound protein, calcium ion binding to proteins must have minimal dissociation constants (Kdiss) of 10(-7) M; alpha-lactalbumin is reported to bind calcium at Kdiss = 10(-10) to 10(-12) M. The electrophoretic shift identifies alpha-lactalbumin in complex milk whey patterns of many species of mammals.

α-Lactalbumin undergoes a calcium-dependent electrophoretic shift at pH 8.3. When Ca 2+ is removed by a chelator, the mobility of the protein increases, reflecting the exposure of negative electrical charges. The shift, however, is not observed by electrophoresis in the presence of SDS, which demonstrates that α-lactalbumin does not undergo a measurable conformational change upon debinding of Ca 2+ . Relative electrophoretic mobilities vary from 1.0 (no shift) to 1.4 among α-lactalbumins of different orders of mammals. The differences suggest a variable number of gram atoms of Ca 2+ bound to α-lactalbumin or substitution of amino acid Ca 2+ ligands in the calcium-binding loop.

The tapped latex of Euphorbia lathyris can convert acetate to several structurally different triterpenols (TOH) and their fatty acid esters (TE) (1). Simple low speed centrifugation of the latex affords a pellet that utilizes mevalonic acid as a triterpenoid precursor (2). Biosynthetic activity in the latex pellet is absolutely dependent upon the presence of an osmoticum (0.4 M sorbitol), indicating that biosynthesis occurs in an osmotically sensitive organelle. Calcium constitutes about 7.5% of dry latex weight, and the addition of Ca2+ to an EGTA-treated latex pellet stimulates triterpene biosynthesis. This study was undertaken to investigate whether the calcium binding protein, calmodulin, mediates Ca2+ effects upon triterpenoid biosynthesis.

Calcium-induced changes in protein solubility play a role in a variety of important biological processes including the deposition of bone and dentin and the secretion of milk. The phenomena of salt-induced (calcium) precipitation of proteins (salting-out), and the resolubilization of these proteins at higher salt concentrations (salting-in) have been studied and quantitated using an approach based on the concepts of Wyman's thermodynamic linkage. Salting-out has been described by a salt-binding constant, k1, the number of moles of salt bound per mole of protein, n, and S1, the fraction soluble at saturation of n; salting-in has been described by corresponding constants k2, m, and S2. Analysis of salt-induced solubility profiles was performed using nonlinear regression analysis. Results of calcium-induced solubility profiles of two genetic variants of alpha s1-casein (alpha s1-A), (alpha s1-B), and beta-casein C (beta-C) at 37 degrees C, where hydrophobic interactions are maximized, showed no salting-in behavior and for salting-out, yielded k1 values of 157, 186, and 156 liters.mol-1 and n values of 8, 8, and 4, respectively. The values of k1 can be correlated with the apparent association constant for calcium binding to casein, while the values of n can be correlated with the number of calcium binding sites of the respective caseins. At 1 degree C, where hydrophobic interactions are minimized, nominally only hydrophilic and electrostatic interactions can be linked to the salt-induced solubility profiles; here beta-C is totally soluble at all calcium concentrations and alpha s1-B and alpha s1-A were now found to have salting-in parameters, k2 and m, of 2.5 liters.mol-1 and 4, and 11 liters.mol-1 and 8, respectively. alpha s1-A is more readily salted-in and studies on the variation of S1 with added KCl for this protein at 1 degree C indicated that salting-in is also mainly electrostatic in nature and may result from competition between K+ and Ca2+ for binding sites rather than from solute-solvent interactions as previously proposed. Comparison of k1 and k2 values between the two genetic variants, coupled with the known sequence differences (the A variant is a linear deletion of 13 amino acids) suggest the existence of a hydrophobically stabilized ion pair in alpha s1-B which is deleted in alpha s1-A; it is speculated that such bonds may play a role in other calcium-induced changes in protein solubility.

A variety of methods for phenotyping whey proteins and the caseins is summarized. The writer suggests what he believes are the best methods for phenotyping genetic variants of the milk protein system depending upon the availability of apparatus. No single method has yet been devised to simultaneously type the variants of α s1 - and β -caseins

α s1 -Casein A, a rare genetic polymorph of the α s1 -casein fraction of cow's milk, probably arose through the sequential deletion of eight amino acid residues. The α s1 -A allele was thought to occur only in American Holstein cattle; however, it has been reported to occur in low frequency in Red Danish cattle. No ancestral relationship can be found between Red Danish cattle and the Holstein breed, hence further confirmation of the identity of these two polymorphs was sought. The proteins were isolated by DEAE-cellulose chromatography and the fingerprints of their chynotryptic digests compared. The α s1 -A variant from the Red Danish breed proved to be identical with that of the Holstein breed where α s1 -A was first observed. Therefore, it is proposed that this mutant form of α s1 -casein is of more ancient ancestry, since no relationship between the Red Danish and the American Holstein breeds can be documented.

The genetic variants, A, B, and C, of αs1-casein from cow's milk were digested under controlled conditions with trypsin, chymotrypsin, and pepsin. The resulting peptides were examined by a mapping procedure and the patterns were compared within each set of digests. The B and C variants are known to differ in composition by a single amino acid substitution—a glutamic acid residue in B being replaced by a glycine in C. Probable difference peptides containing this substitution were observed in each set of proteolytic digestions. The results of specific staining for arginine, histidine, methionine, tyrosine, and tryptophan residues suggest that the presumed difference peptides are related and indicate the presence of several of these amino acids in the vicinity of the amino acid substitution. The αs1-casein A variant, which differs from the other two proteins quite substantially in amino acid composition, revealed peptide patterns almost identical to those of B and C, with the main difference being the absence of one or two major spots in A. The hypothesis is proposed that αs1-casein A, which represents an unusual type of genetic variant, is devoid of a portion of the molecule, with the remaining sequence similar to the B protein.

The amino acid composition of highly purified preparations of the genetically controlled variants of α s1 -casein, A, B, and C has been determined. B and C are virtually identical in composition, the only significant difference being that C contains one more glycine residue per mole than B. The data suggest that in B the missing glycine might be replaced by glutamic acid. The differences between A and the other variants are numerous and more substantial and, at present, cannot be accounted for in terms of single amino acid substitutions.

Milk is an unusually stable colloidal system; the stability of this system is due primarily to the formation of micelles by the major milk proteins, the caseins. Numerous models for the structure of casein micelles have been proposed; these models have been formulated on the basis of in vitro studies. Synthetic casein micelles (i.e., those formed by mixing the purified αsl- and k-caseins with Ca2+ in appropriate ratios) are dissimilar to those from freshly-drawn milks in (i) size distribution, (ii) ratio of Ca/P, and (iii) solvation (g. water/g. protein). Evidently, in vivo organization of the caseins into the micellar form occurs in-a manner which is not identical to the in vitro mode of formation.