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The high temperatures used in the production of milk may induce modifications in proteins structure. Due to occurrence of the Maillard reaction, lactose binds lysine residues in proteins, affecting the nutritional value. Milk is also an important source of allergenic proteins (i.e., caseins, β-lactoglobulin and α-lactalbumin). Thus, this modification may also affect the allergenicity of these proteins. Focusing on milk whey proteins, a screening on different Ultra High Temperatures (UHT) and pasteurized milk samples was performed to identify lactosylation sites, in particular in protein known epitopes, and to verify the correlation between lactosylation and the harshness of the treatment. Whey proteins were extracted from milk samples after caseins precipitations at pH 4.6 and, after chymotryptic and tryptic in solution digestion, peptides were analysed by UPLC-MS and LTQ-Orbitrap. Results show the presence of lactosylated lysine residues in several known epitopes. Then, a β-lactoglobulin epitope was selected and synthesized by solid phase synthesis followed by in solution lactosylation, obtaining high reaction yields and purities. The synthesis of lactosylated allergenic epitopes, described here for the first time, is a useful tool for further studies on the technological impacts on food allergenicity.

In the last years, donkey milk had evidenced a renewed interest as a potential functional food and a breast milk substitute. In this light, the study of the protein composition assumes an important role. In particular, β-lactoglobulin (β-LG), which is considered as one of the main allergenic milk protein, in donkey species consists of two molecular forms, namely β-LG I and β-LG II. In the present research, a genetic analysis coupled with a proteomic approach showed the presence of a new allele, here named F, which is apparently associated with a null or a severely reduced expression of β-LG II protein. The new β-LG II F genetic variant shows a theoretical average mass (M av ) of 18,310.64 Da, a value practically corresponding with that of the variant D (∆ mass  < 0.07 Da), but differs from β-LG II D for two amino acid substitutions: Thr 100 (variant F) → Ala 100 (variant D) and Thr 118 (variant F) → Met 118 (variant D). Proteomic investigation of the whey protein fraction of an individual milk sample, homozygous FF at β-LG II locus, allowed to identify, as very minor component, the new β-LG II F genetic variant. By MS/MS analysis of enzymatic digests, the sequence of the β-LG II F was characterized, and the predicted genomic data confirmed.

Functional recombinant bovine β-lactoglobulin has been produced by expression in E. coli using an engineered protein gene and purified to homogeneity by applying a new protocol. Mutations L1A/I2S introduced into the protein sequence greatly facilitate in vivo cleavage of the N-terminal methionine, allowing correctly folded and soluble protein suitable for biochemical, biophysical and structural studies to be obtained. The use of gel filtration on Sephadex G75 at the last purification step enables protein without endogenous ligand to be obtained. The physicochemical properties of recombinant β-lactoglobulin such as CD spectra, ligand binding (n, K a, ΔH, TΔS, ΔG), chemical and thermal stability (ΔG D, C mid) and crystal structure confirmed that the protein obtained is almost identical to the natural one. The substitutions of N-terminal residues did not influence the binding properties of the recombinant protein so that the lactoglobulin produced and purified according to our protocol is a good candidate for further engineering and potential use in pharmacology and medicine.

The objective of this study was to characterize the genetic architecture underlying the absolute concentrations of 2 important milk proteins, κ-casein (κ-CN) and β-lactoglobulin (β-LG), in a backcross population of (Holstein × Jersey) × Holstein cattle. A genome-wide association analysis was performed using a selective DNA pooling strategy and the Illumina BovineHD BeadChip assay [777,000 (777K) SNP markers; Illumina Inc., San Diego, CA]. After correction for multiple testing, 25 single nucleotide polymorphisms were found to be associated with κ-CN and 36 single nucleotide polymorphisms were associated with β-LG. A pathway association analysis revealed 15 Gene Ontology (GO) terms associated with the κ-CN trait and 28 GO terms associated with β-LG. In addition, several GO terms were associated with both milk proteins. Further analysis revealed that κ-CN and β-LG production is regulated by both kinase and phosphatase activity, including mechanisms regulating the extracellular matrix. These results are in concordance with the complex multihormonal process controlling the expression of milk proteins and interactions between mammary epithelial cells and extracellular matrix components. Although κ-CN and β-LG milk proteins are expressed by single genes, the results from this study showed that many loci are involved in the regulation of the concentration of these 2 proteins., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: The aim of the study was to investigate bioactive whey protein concentrations in cow's milk collected in late pasture (LP) and early indoor feeding (EIF) season from conventional and organic farms in Poland., Results: Results showed that in the LP somatic cell count (SCC) was higher under organic farming conditions. However, percentages of protein and fat were higher under conventional farming conditions. In EIF, milk from conventional dairy farms had a higher percentage of fat and lactose and a lower concentration of protein and SCC in comparison to milk from organic farms. Organic milk in LP had higher concentrations of beneficial whey proteins than conventional milk, including β-lactoglobulin (β-Lg, 4.12 vs. 2.68 g L⁻¹), lactoferrin (Lf, 334.99 vs. 188.02 mg L⁻¹), and lysozyme (Lz, 15.68 vs. 12.56 µg L⁻¹). However, conventional milk in EIF had higher concentrations of bovine serum albumin (146.47 vs. 118.65 mg L⁻¹), Lf (49 vs. 185.27 mg L⁻¹), and Lz (16.63 vs. 13.22 µg L⁻¹)., Conclusions: The results show significant differences in the investigated parameters between organic milk and milk from conventional system during EIF and LP. Moreover, extending the pasture season during EIF in organic farms decreases concentration of bioactive compounds of milk., (Copyright © 2012 Society of Chemical Industry.)

The production of soluble and correctly folded eukaryotic proteins in prokaryotic systems has always been hampered by the difference in or lack of cell machinery responsible for folding, post-translation modification and secretion of the proteins involved. In the case of bovine beta-lactoglobulin (BLG), a major cow's milk allergen and a protein widely used for protein folding studies, a eukaryotic yeast expression system has been the preferred choice of many researchers, particularly for the production of isotopically labeled protein required for NMR studies. Although this system yields high amounts of recombinant protein, the BLG produced is usually associated with extracellular polysaccharides, which is problematic for NMR analysis. In our study we show that when co-expressed with the signal-sequence-less disulfide bond isomerase (Delta ssDsbC) in the dual expression vector, pETDUET-1, both BLG A and BLG B can be reproducibly produced in a soluble form. Expression was carried out in Escherichia coli Origami(DE3), a trxB/gor mutant for thioredoxin- and glutathione reductase, which allows for proper formation of disulfide bonds in the cytoplasm. The protein was purified by anion exchange chromatography followed by salting-out at low pH and size exclusion chromatography. Our expression system is able to consistently produce milligram quantities of correctly folded BLG A and B with no additional amino acid residues at the N-terminus, except for a methionine. (15)N-labeled BLG A and B, prepared and purified using this method, produced HSQC spectra typical of native bovine BLG., ((c) 2010. Published by Elsevier Inc.)

Beta-lactoglobulin (beta-LG) is the major whey protein in cow's milk. It is well established that the predominant 2 genetic variants, beta-LG A and B, are differentially expressed. Extensive investigation of the genetic variation in the promoter region of the BLG gene revealed the existence of specific haplotypes associated with the A and B variants, respectively. However, the genetic basis for the differential expression of BLG A and B alleles is still elusive. We have previously reported a quantitative beta-LG B variant, characterized by a very low beta-LG protein expression level. Here, we report that the corresponding BLG allele (BLG B*) shows a correspondingly low mRNA expression level. Comparative DNA sequencing of 7,670 bp of the BLG B* allele and the established BLG B allele revealed a unique difference of a C to A transversion at position 215 bp upstream of the translation initiation site (g.-215C>A). This mutation segregated perfectly with the differential phenotypic expression in a paternal half-sib family and could be confirmed in 2 independent cases. The sequence of the BLG B allele in the region of the mutation is highly conserved among 4 related ruminant species. The site of the mutation corresponds to a putative consensus-binding sequence for the transcription factors c-Rel and Elk-1 as predicted by searching the TRANSFAC database. The beta-LG B* site might be relevant in the natural production of milk of low beta-LG content.

The role of amino acids (AA) on translational regulation in mammary epithelial cells cultured under lactogenic conditions was studied. The rates of total protein synthesis and beta-lactoglobulin (BLG) synthesis in mouse CID-9 cells were 2.1- or 3.1-fold higher, respectively, than in their bovine L-1 counterparts. Total AA deprivation or selective deprivation of Leu had a negative protein-specific effect on BLG synthesis that was more pronounced in bovine cells than in murine cells. Dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and S6 kinase (S6K1) on Thr(389) but not on Ser(411) was also more prominent in bovine cells. Noteably, deprivation of Leu had a less marked effect on BLG synthesis and 4E-BP1 or S6K1 phosphorylation than deprivation of all AA. In AA-deprived CID-9 cells, Leu specifically restored BLG synthesis from pre-existing mRNA whereas AA also restored total protein synthesis. This restoration was associated with a more pronounced effect on 4E-BP1 and S6K1 phosphorylation in bovine versus murine cells. Rapamycin specifically reduced Leu- and AA-stimulated BLG translation initiation in a dose-dependent manner. A further reduction was observed for Leu-treated cells in the presence of LY294002, a PI3K (phosphatidylinositol 3-kinase) inhibitor, which also reduced total protein synthesis. These findings suggest that direct signaling from AA to the translational machinery is involved in determining the rates of milk protein synthesis in mammary epithelial cells., ((c) 2006 Wiley-Liss, Inc.)

Background: The use of probiotics such as Lactococcus lactis and other lactic acid bacteria (LAB) has been proposed for the management of food allergy. However, no experimental study has clearly demonstrated any preventive or therapeutic inhibition of an allergen-specific IgE response., Objective: We aimed to study the immunomodulatory effect of recombinant L. lactis expressing bovine beta-lactoglobulin (BLG), a major cow's milk allergen, in a validated mouse model of allergy., Methods: Six-week-old female Balb/c mice received five repeated doses of BLG, of L. lactis plus BLG, or of recombinant L. lactis by gavage. Different recombinant strains were inoculated, which corresponded to BLG doses ranging from 4 to 70 microg/mice. Mice were then sensitized by intra-peritoneal injection of BLG emulsified in incomplete Freund's adjuvant to induce high IgE concentrations., Results: Pre-treatment with natural L. lactis plus BLG allowed induction of BLG-specific T-helper type 1 (Th1) response, and abrogated the oral tolerance induced by BLG alone, demonstrating the adjuvant effect of this non-colonizing LAB. Moreover, pre-treatment with some of the recombinant strains favoured the development of a Th1 response inhibiting the Th2 one: it induced a significant decrease of specific IgE response, and an intense increase of specific IgG2a and IFN-gamma productions. The most efficient strains that inhibited the IgE response were those producing the highest amounts of the BLG protein., Conclusion: Oral administration of some recombinant L. lactis was demonstrated to induce a specific Th1 response down-regulating a further Th2 one. Prophylaxis protocols will thus be evaluated using the most efficient strains.

The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine beta-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (approximately 10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at approximately 8 microg/ml (approximately 2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.

Bovine and porcine beta-lactoglobulins were cloned and expressed in host cells with the aim of developing the tools necessary for their structural, functional and conformational characterisation by NMR techniques. Both lipocalins were expressed in Pichia pastoris, where the use of a constitutive promoter turned out to allow the highest productivity. The yield of recombinant proteins was further improved through multiple integration of the encoding genes and by increasing aeration of the transformed cultures. Both proteins were obtained in the culture medium at the concentration of 200 microg/ml. Recombinant lipocalins were purified by ion-exchange chromatography from the culture medium. A preliminary NMR characterisation showed that both proteins were correctly folded.

5' and 3' flanking region of ovine BLG were amplified from sheep genomic DNA according to the published whole sequence of ovine BLG and cloned to pGEM-T vector correspondently. By partially sequencing, the sequences of BLG 5' and 3' flanking were the same as that of publication completely. The recombinant structure used to direct exogenous gene especially to express in mammary gland was constructed by joining 4.2 kb 5' flanking with 2.1 kb 3' flanking. In order to assess the efficiency of BLG regulatory elements, green fluorescent protein (GFP) gene as a reporter was fused with BLG construct and transfected the mammary epithelial cells (TD47). Through observation under UV microscope and detection by fluorometer, it is demonstrated that the GFP has been successfully expressed in TD47 cell line. By virtue of direct observation and quantitative analysis, the BLG-GFP construct can be served as a model for the quick assessment of mammary gland expression construct.

Background: Mucinous carcinoma of the skin (MCS) is a rare epithelial tumor which arises primarily in the skin. Metastatic MC from extracutaneous sites, especially breast or colon, mimics MCS and cannot be differentiated from MCS by routine histology alone., Methods: Nine cases of MCS were analyzed immunohistochemically using monoclonal antibodies against cytokeratins (CKs) and human milk fat globulin 1 (HMFG) in order to clarify their nature and compare the immunophenotypes with those of other MCs studied in the literature., Results: Expression of simple epithelial CKs in most of the tumor cells of all cases studied and co-expression of simple and stratified epithelial CKs in some tumor cells of two cases were recognized. CK 20 expression could not detected in any tumor cells. Focal HMFG expression in the luminal or outer surface of the nests was observed in three cases., Conclusion: From CKs expression, MCS was speculated to differentiated mainly toward the secretory cells of the sweat glands, and some tumor cells toward the transient portion between the dermal duct and the secretory portion. Focal HMFG expression suggested either a consequence of malignant transformation or apocrine differentiation. No expression of CK 20 in MCS suggests that we may exclude the diagnosis of metastatic colorectal MC which expressed CK 20.

In this paper we quantify and characterize the expression of recombinant beta-lactoglobulin (rBLG) in prokaryote and eukaryote cells. In Escherichia coli we used the pET26 vector, which permits the secretion of rBLG in periplasm. We studied the expression of rBLG in COS-7 cells and in vivo in mouse tibialis muscle. The expression of rBLG was measured by two immunoassays specific, respectively, for BLG in its native and denatured conformation. We observed that rBLG was essentially expressed in a denatured form in E. coli even in the periplasm, whereas rBLG in eukaryote cells was found in its native conformation., (Copyright 1999 Academic Press.)

By targeting the expression of sequences encoding non-milk proteins to the mammary gland of transgenic farm animals, the organ could serve as a 'bioreactor' for producing pharmacologically active proteins on a large scale. Here we report the generation of transgenic sheep bearing a fusion gene construct with the human blood clotting factor VIII (hFVIII) cDNA under the transcriptional control of a 2.2 kb fragment of the mammary gland specific promoter of the ovine beta-Lactoglobulin (beta-Lac) gene. Six founder animals were generated bearing a hFVIII cDNA construct with the introns of the murine metallothionein (MtI) gene (beta-Lac/hFVIII-MtI). Founders transmitted the transgene in a Mendelian fashion and two transgenic lines were generated. Ten out of 12 transgenic F1-females expressed rhFVIII mRNA in exfoliated mammary epithelial cells isolated from the milk. But only in transgenic F1 ewes 4010 and 603 hFVIII clotting activity estimated at 4-6 ng/ml was detected in defatted milk. Furthermore, the presence of rhFVIII-protein in ovine milk was demonstrated by a specific band at approximately 190 kD following immunoprecipitation and immunoblotting. Transgenic founder 395 expressed rhFVIII mRNA in biopsied mammary gland tissue, in exfoliated mammary cells as well as ectopically in brain, heart, spleen, kidney and salivary gland, suggesting that the employed beta-Lac promoter fragment lacks essential sequences for directing expression exclusively to the mammary gland. A rhFVIII standard preparation (rhFVIIIstd) was rapidly sequestered in a saturable fashion in ovine milk, thus rendering it largely inaccessible to immunoprecipitation although its biological activity was retained. Recovery of hFVIIIstd was dependent on milk donor, storage temperature and dilution of milk sample.

A dramatic uncoupling of the expression of chimaeric beta-lactoglobulin (BLG)/human serum albumin (HSA) gene constructs at the RNA and protein levels was observed in cultured mammary explants of virgin transgenic mice. Upon explantation, both HSA RNA and protein were expressed at high levels. However, when the explants were grown in hormone-free medium. HSA RNA continued to accumulate, whereas the synthesis of the corresponding protein was dependent on the presence of insulin and prolactin with a minor contribution of hydrocortisone. The untranslated HSA RNA was indistinguishable from its translatable counterpart in its mobility on agarose gels, was transported normally from the nucleus to the cytoplasm and was translated efficiently in rabbit reticulocyte lysate. In the presence of cycloheximide, HSA RNA rapidly disappeared suggesting a dependency on ongoing protein synthesis. Its estimated half-life of 5-6 h in hormone-free medium increased significantly in the presence of insulin, hydrocortisone and prolactin and was comparable to that of beta-casein RNA. The uncoupling of the expression of the BLG/HSA transgenes at the RNA and protein levels was also confirmed by in situ hybridization and immunohystochemistry on sections from virgin mammary explants. HSA synthesis was initiated within 13 h of the addition of insulin and prolactin in explants that had accumulated untranslated HSA RNA and was fourfold higher than that observed with insulin alone. Addition of hydrocortisone contributed to an additional 20% in HSA synthesis. We believe this is the first demonstration of translational control of exogenous milk protein gene expression in the mammary gland of transgenic animals.

The regulation of milk constituents, synthesis and secretion in tissue cultures of the bovine mammary gland was altered by a whey fraction of bovine milk. alpha-Casein gene expression, casein secretion and fatty acid synthesis were inhibited by the whey fraction in a dose-dependent manner. The whey fraction inhibited the enhancement activity of prolactin on alpha-casein gene expression and fatty acid synthesis, and also inhibited casein secretion to the medium, in explants cultured in a medium with or without prolactin. No effect on the expression of the beta-lactoglobulin gene was found.

Eccrine hidrocystomas and apocrine cystadenomas are morphologically related cystic sweat gland tumors. To elucidate their cellular differentiation we examined by immunohistochemistry the expression of keratins and of human milk fat globulin 1 in 12 of each of these tumors, diagnosed using established conventional histological criteria. All tumors diagnosed as apocrine cystadenomas by these criteria were characterized by a keratin pattern of secretory type. In addition, they expressed human milk fat globulin 1. Tumors diagnosed as eccrine hidrocystomas expressed a keratin pattern of excretory type. A part of the tumors with an excretory keratin pattern expressed human milk fat globulin, while others did not. Some presumed eccrine hidrocystomas expressed the very same antigens as apocrine cystadenomas. Thus, our study reveals three distinct types of tumors, in contrast to the conventional distinction of only eccrine hidrocystomas and apocrine cystadenomas. Apocrine cystadenomas differentiate towards the secretory coil of apocrine sweat glands. Presumed eccrine hidrocystomas may represent cystic tumors of the eccrine sweat duct, or they may represent cystic tumors of the apocrine duct. Thus, the name hidrocystoma should be used without further specification of an eccrine or apocrine nature, unless certainty is reached by immunohistochemical characterization. Also, hidrocystomas often prove to be histologically misdiagnosed apocrine cystadenomas because of a flattened cyst wall secondary to increased intraluminal pressure.

Many transgenes, particularly those comprising cDNA sequences fail to be expressed when they are introduced into transgenic mice. We have previously shown that this problem can be overcome in the mammary gland by co-integrating a poorly expressed cDNA transgene, comprising the sheep beta-lactoglobulin promoter, with the efficiently expressed, unmodified beta-lactoglobulin gene. In this report we demonstrate that the transcription of the beta-lactoglobulin gene is associated with this effect because co-integration with a non-transcribed beta-lactoglobulin gene fails to rescue expression. By contrast, co-integration with a translationally inactivated beta-lactoglobulin transgene does rescue the expression of the second gene, but without the co-production of beta-lactoglobulin protein.

The major milk whey protein of ruminants is beta-lactoglobulin. Ovine beta-lactoglobulin-encoding gene expression is restricted to the sheep mammary gland. This report describes the expression profile of a truncated beta-lactoglobulin transgene which, although not expressed in the mammary gland, is expressed in the kidney in the majority of lines generated. The high frequency of ectopic kidney expression may relate to the ability of the larger beta-lactoglobulin transgenes to be expressed in a position-independent manner in the mammary gland of transgenic mice.

Mammary epithelial cell cultures from transgenic mice carrying the human serum albumin (HSA) gene or minigenes behind the regulatory sequences of the ovine beta-lactoglobulin gene were analyzed. Previously, we demonstrated that non-HSA-secreting transgenic strains retain the potential to express the HSA transgene in vitro and that mammary epithelial cell cultures from non-HSA-secreting strains express higher levels of HSA when grown on tissue culture plastic than they do when grown on collagen. In this study we studied the expression of BLG/HSA fusion genes in epithelial cell cultures of additional transgenic strains and additional substrata. Our results show that: (1) The BLG/HSA fusion gene in only one of seven HSA-secreting or nonsecreting transgenic strains tested accurately responded to signals from the EHS matrix; (2) HSA DNA sequences dominantly affected the activity of BLG as well as the whey acidic protein promoters; and (3) HGF/SF induced both milk proteins and HSA gene expression. These results suggest that the response to the extra cellular matrix (ECM) plays a key role in the expression of BLG/HSA fusion genes and that the function of the regulatory elements within the promoter regions of milk protein genes involved in response to the ECM, in developmental and in tissue specificity, very much depend on the downstream gene sequences.

The immunological characteristics of a recombinant beta-lactoglobulin were studied using monoclonal antibodies, polyclonal antiserum and sera from allergic patients. Recombinant beta-lactoglobulin (rBLG) was expressed in Escherichia coli strain DH5alpha and purified as described previously [Cho et al. (1994) J. Biol. Chem. 269, 11 102-11 107]. The method has been modified by adding an immunoaffinity purification step. A quantity of 5-10mg of purified rBLG per liter of medium culture can be produced. rBLG shared the same molecular weight as the natural BLG (nBLG) and also possessed at least one intrachain disulfide bridge. In HPLC, rBLG appeared as a single peak, and the purity was estimated to be greater than 95%. All the monoclonal antibodies (mAbs) used in this study recognized different epitopes of the BLG and presented compatible binding. No differences could be detected between rBLG and nBLG when tested in a Western blot with rabbit polyclonal antiserum or with three mAbs that bound preferentially the reduced and S-carboxymethylated form of BLG. In a competitive enzyme immunoassay (EIA) using either a rabbit polyclonal antiserum or four mAbs that recognized conformational epitopes, we could not distinguish between rBLG or nBLG. In direct ELISA using nBLG or rBLG as the immobilized allergen, we measured a similar concentration of specific anti-BLG IgE in five sera from allergic patients. The results of this study indicate that we have obtained a rBLG with biochemical and immunological properties very similar to nBLG.

Eukaryotic pre-mRNAs undergo a variety of post-transcriptional modifications, including the removal of intronic sequences by splicing, leading to creation of a functional mRNA. We have compared the processing of transcripts generated from ovine beta-lactoglobulin gene constructs in stably transfected cells and in transgenic mice. In both the in vitro and in vivo model systems the removal of the middle two introns resulted in the inefficient splicing of the downstream, terminal intron. This intron-containing transcript was detected in the cytoplasmic RNA fraction. Thus, the initial in vitro analysis in cell lines of minigene constructs destined for expression in transgenic animals may provide a rapid and reliable indicator of the processing efficiency of the pre-mRNA produced by the construct in vivo. This is in contrast to the apparent limitations of in vitro systems in the analysis of transcription regulatory elements required for transgene expression.

The major milk whey protein of ruminants is beta-lactoglobulin. Transgenic mice which carry genomic fragments of ovine beta-lactoglobulin express the transgene at high levels in the mammary gland. Using DNaseI as a probe for transcription complex formation in chromatin, the temporal induction pattern of beta-lactoglobulin in transgenic mice has been addressed and compared to the known hormonal profiles during pregnancy. Prior to the 9th day of pregnancy no obvious hypersensitivity to DNaseI digestion at the beta-lactoglobulin promoter was evident. From the 9th day of pregnancy through to lactation, the beta-lactoglobulin promoter displays DNaseI hypersensitivity. These results support the hypothesis that placental lactogens are the major lactogenic influence from mid-pregnancy to parturition.

Mammary explants and epithelial cell cultures from transgenic mice carrying the human serum albumin (HSA) gene or minigenes behind the regulatory sequences of the ovine beta-lactoglobulin gene were analyzed. Previously we demonstrated that mammary explants from virgin female transgenic mice synthesize and secrete high levels of HSA during the first day in culture. Here we present a detailed analysis of endogenous and transgene expression during the first 20 h of mammary explant cultures. We show that HSA genes as well as endogenous milk protein genes are rapidly induced upon explantation. Unexpectedly, HSA was synthesized also in mammary explants from strains that do not secrete HSA into the milk, indicating the existence of a cryptic potential to express the transgene. Histological examination revealed that some luminal epithelial cells detached from the underlying extracellular matrix (ECM) soon after explantation. Epithelial cell cultures from nonsecreting strains grown on plastic rapidly induced transgene expression and secreted higher levels of HSA into the medium compared to cells grown on collagen. These results suggest that tissue organization and most likely the interaction of epithelial cells with the ECM are intimately involved in the control of HSA transgene expression.

To evaluate whether estrogen hastened involution of mammary tissue, Holstein cows were injected with 4 ml of ethanol excipient (n = 21) or 15 mg of estradiol-17 beta (n = 23) on each of the 4 d that preceded final milk removal. Dates of final milk removal (d 0) were designated as 60 d prior to expected dates of calving. Milk volumes were recorded, and samples were collected prior to the first and fourth injections. During the dry period, each mammary quarter within the cow was sampled once to collect secretions on dates that corresponded to d 0, 3, 11, and 25 or 1, 7, 18, and 30 of the dry period. Milk synthesis and secretion declined abruptly because of treatment. The decreased concentrations of alpha-lactalbumin, lactose, citrate, and potassium in secretions of controls, as well as the increased somatic cells, protein, lactoferrin, and sodium, occurred earlier in secretions from treated cows. These shifts of approximately 6 d, relative to days dry, suggested that exogenous estradiol increased the involution rate of mammary tissue.

High expression and secretion of recombinant ovine beta-lactoglobulin has been achieved in the yeast Kluyveromyces lactis. The yield of beta-lactoglobulin is 40-50 mg per litre of culture supernatant and accounts for approx. 72% of the total secreted protein. Constitutive expression is under the control of the Saccharomyces cerevisiae phosphoglycerate kinase promoter from an intronless version of the beta-lactoglobulin gene. Secretion is specified by the ovine protein's own signal sequence. this system, coupled to an efficient and novel recovery protocol, allows 30 mg of pure protein to be isolated from a typical 1 litre culture. The protein is virtually indistinguishable from beta-lactoglobulin conventionally purified from sheep milk by its behaviour in native PAGE and SDS/PAGE, reactivity to antibodies, CD, fluorescence spectroscopy and N-terminal sequencing. Attempts to achieve a similar expression and secretion system in the yeast S. cerevisiae met with only limited success, although it was found that heat-shock treatment modestly increased the yield up to approx. 3-4 mg per litre of culture supernatant. Site-directed mutagenesis showed that secretion in S. cerevisiae depended upon correct formation of the two disulphide bonds present in beta-lactoglobulin.

Transcription of tissue-specific genes in mammary gland requires signals from both prolactin and basement membrane. Here we address the mechanism by which this specialized extracellular matrix regulates transcription. Using mammary cell cultures derived from transgenic mice harboring the ovine beta-lactoglobulin gene, we show that either a basement membrane extract, or purified laminin-1, induced high levels of beta-lactoglobulin synthesis. It is known that prolactin signals through Stat5 (signal transducer and activator of transcription). This transcription factor interacts with gamma-interferon activation site-related motifs within the beta-lactoglobulin promoter, which we show are required for matrix dependence of beta-lactoglobulin expression. The DNA binding activity of Stat5 was present only in extracts of mammary cells cultured on basement membrane, indicating that the activation state of Stat5 is regulated by the type of substratum the cell encounters. Thus, basement membrane controls transcription of milk protein genes through the Stat5-mediated prolactin signaling pathway, providing a molecular explanation for previous studies implicating extracellular matrix in the control of mammary differentiation.

To explore the possibility that genes might compete for expression, we have studied transgenic mice producing high levels of the sheep milk protein, beta-lactoglobulin (BLG), in the mammary gland. Mice carrying one or more transgene loci expressed BLG in milk at levels ranging from 7 to 33 mg/ml. The effects of BLG synthesis on the levels of endogenous milk gene expression were examined. No significant increase in total milk protein concentration was recorded even in mice expressing the largest amounts of BLG. Measurement of individual milk proteins showed that transgene protein was manufactured at the expense of host protein synthesized in the gland. Whey acidic protein production was more suppressed than casein production. Suppression of endogenous proteins was matched by a reduction in the corresponding steady-state mRNA levels; in double-transgenic mice, which expressed the largest amounts of BLG, beta-casein and whey acidic protein mRNA populations were reduced to 75 and 56% of control levels respectively. We demonstrate that an exogenous gene competes effectively for expression with endogenous genes. Possible mechanisms of competition are discussed.

In the lactating mammary gland, expression of the ovine beta-lactoglobulin gene correlates with the presence of a strong DNaseI hypersensitive site encompassing the promoter region. At this stage of lactogenesis, prolactin is required for optimal expression. Using DNaseI as a probe for formation of a transcription complex on the beta-lactoglobulin promoter, the temporal pattern of ovine beta-lactoglobulin expression during pregnancy has been addressed. The appearance of nuclease hypersensitivity during pregnancy correlates with rising levels of placental lactogen which suggests that this hormone may be the stimulus for expression during the first stage of lactogenesis. This raises the possibility that a different signalling pathway to that present in the lactation functions during pregnancy.

Genomic constructs comprising the ovine beta-lactoglobulin gene are expressed in a position-independent manner in the mammary gland of transgenic mice. In some lines however, constitutive low-level transgene expression was detected in all other tissues. This ectopic expression presumably represents a position-dependent phenomenon since it was observed in only a proportion (40%) of the lines generated. Different lines of BLG transgenic mice displayed similar temporal patterns of ectopic expression. This pattern differed from that of BLG in the mammary gland. These data imply that the DNA elements that direct position-independent expression of beta-lactoglobulin transgenes in the mammary gland do not have the ability to insulate them from position effects in other tissues. Furthermore, the relatively high frequency and constitutive nature of ectopic expression suggests that transgene integration may not be totally random.

We compared the developmental pattern of expression of the sheep beta-lactoglobulin (BLG), the chimeric BLG/human serum albumin (HSA), and the endogenous murine beta-casein genes in the mammary gland of virgin, pregnant and lactating transgenic mice, both at the RNA (expression) and protein (synthesis and secretion) levels. The BLG and casein genes were expressed at very low levels in virgin animals and during early stages of pregnancy. The increase in the expression of these genes started at the second half of pregnancy and reached a peak between the end of pregnancy and day 10 of lactation. The accumulation of their RNA coincided with that of the corresponding proteins, indicating a transcriptional control of expression of these genes. The expression and secretion patterns of the endogenous casein gene in transgenic and nontransgenic mice were indistinguishable. The hybrid BLG/HSA gene constructs displayed distinct patterns of expression in virgin animals and at early stage of pregnancy, from that of the BLG transgene or the endogenous mouse milk protein gene. High levels of expression (17-60% of that on day 18 of pregnancy) were detected in the mammary gland of virgin animals. At day 5 of pregnancy there was a dramatic decrease in HSA synthesis and secretion in all transgenic strains tested. The down-regulation, revealed by immunoprecipitation and immunohistochemical studies, demonstrated that at that stage of pregnancy only 10-18% of ductal structures contained HSA expressing cells in contrast to the majority of ducts expressing HSA in virgin animals. These morphological studies also demonstrated that the down-regulation in HSA synthesis and secretion was correlated with the transition from ducts comprised of a single layer of epithelial cells (characteristic of the virgin state) to ducts composed of multilayers of such cells. In two of the three transgenic strains tested, the down-regulation at the protein level was associated with a similar decrease in HSA transcripts. In the exceptional strain no. 23, HSA transcripts continued accumulating even at this stage. The differences in the control of expression at the RNA level between these transgenic strains were also confirmed by in situ hybridization. Our results suggest the involvement of at least two regulatory mechanisms effective at early stages of gestation in the control of expression/secretion of the HSA transgene targeted for expression in the mammary gland by the BLG milk protein promoter. These putative mechanisms may play key roles in the interplay between normal mammogenesis and lactogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

The ovine beta-lactoglobulin gene is expressed abundantly in the mammary gland. This study determines whether the ovine beta-lactoglobulin promoter is sufficient for targeting tissue-specific expression in transgenic mice. To address this, the expression profile of an ovine beta-lactoglobulin promoter driven bacterial chloramphenicol acetyltransferase reporter construct was analysed. Comparison of the expression frequency of this hybrid transgene to that of a genomic beta-lactoglobulin transgene indicates that additional sequences, downstream of the promoter, are required for position-independent expression in transgenic mice. Nevertheless, the hybrid transgene was expressed specifically in the mammary gland. Furthermore, the hybrid transgene was expressed in the appropriate temporal pattern during pregnancy and lactation. Thus, the proximal promoter of the ovine beta-lactoglobulin gene contains sufficient sequence information to target expression to the mammary. This construct constitutes the basis for a compact mammary expression vector.

The production of transgenic animals from ungulate species is an inefficient and expensive procedure. The development of selection methods to identify the small number of transgenic preimplantation embryos produced following DNA microinjection of one-cell embryos would greatly reduce both the cost and effort of these procedures. This study has examined the fate of the ovine beta-lactoglobulin-human alpha 1-antitrypsin (AATB) minigene construct or a subfragment of this following microinjection into one-cell mouse embryos. It has examined two PCR-based methods that were designed to identify a biochemical difference between microinjected DNA constructs to select preimplantation stage embryos in which chromosomal integration of exogenous DNA has occurred. The two methods involved the modification of the AATB DNA construct either by dam methylation or the substitution of dTTP by dUTP. The dam-sensitive DNA endonuclease DpnI, that was used to digest nonintegrated AATB sequences at sites located between PCR oligonucleotide sequences, was found to interfere with the activity of the subsequent PCR reaction. Analyses of the fate of dUTP-DNA indicated that either repair or replication of microinjected DNA interfered with the ability to distinguish between integrated and nonintegrated DNA constructs in the mid-preimplantation stage embryo. The distribution of microinjected AATB DNA between the blastomeres of individual four and eight-cell stage embryos was also examined by the PCR reaction. Microinjected DNA was not found to be evenly distributed between all the blastomeres of individual embryos.

Polypeptide and steroid hormones regulate the transcription of milk protein genes in the mammary gland. The promoter sequence motifs and factors through which these hormones mediate their effects in vivo are not clearly defined. Milk protein binding factor (MPBF) is a factor that has recognition sites in the promoters of many milk protein genes including three sites in the promoter of the sheep beta-lactoglobulin (BLG) gene. Mutagenesis of these sites reduced expression of the BLG gene in lactating mammary glands of transgenic mice but did not affect the tissue specificity of the transgene. Furthermore, mutation of all three sites abolished the response of the BLG gene to lactogenic hormones in HC11 mammary cells. Together these results indicate that MPBF mediates the effects of lactogenic hormones in the mammary gland but does not play a role in determining mammary specificity. The similarity between the MPBF binding site and the gamma-interferon activating site suggests that MPBF is related to the STAT family of cytokine-induced transcription factors.

We produced transgenic mice carrying the native sheep beta-lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. In contrast, transgenic females carrying the BLG/HSA fusion constructs also expressed the HSA RNA ectopically in skeletal muscle, kidney, brain, spleen, salivary gland and skin. Ectopic expression of HSA RNA was detected only in strains that express the transgene in the mammary gland. There was no obvious correlation between the level of the HSA RNA expressed in the mammary gland and that found ectopically. In three transgenic strains analysed, the expression of HSA RNA in kidney and skeletal muscle increased during pregnancy and lactation, whereas in the brain HSA expression decreased during lactation in one of the strains. HSA protein was synthesized in skeletal muscle and skin of strain #23 and its level was higher in lactating mice compared with virgin mice. Expression of HSA was also analysed in males and was found to be more stringently controlled than in females of the same strains. In situ hybridization analyses localized the expressed transgene in the skin, kidney, brain and salivary glands of various transgenic strains. Distinct strain-specific and cell-type specific HSA expression patterns were observed in the skin. This is in contrast to the exclusive expression of the HSA transgene in epithelial cells surrounding the alveoli of the mammary gland. Taken together, these results suggest that the absence of sufficient mammary-specific regulatory elements in the BLG promoter sequences and/or the juxtaposition of the BLG promoter with the HSA coding sequences leads to novel tissue- and cell-specific expression in ectopic tissues of transgenic mice.

The retinol-binding site of beta-lactoglobulin has been located by selective modification of amino acid residues which reside in the two putative binding sites. Based upon two separate crystallographic analyses of bovine beta-lactoglobulin, different binding sites for retinol have been proposed: one proposal favors an interior cavity, the other a surface cleft. To discriminate between these two models, we have made four separate site-directed mutations introducing a W19A or a K70M in the interior pocket and a F136A or K141M in the surface pocket. The K70M beta-lactoglobulin exhibited a marked decrease in its binding of retinoic acid compared to the F136A, K141M, and wild-type proteins. Retinyldenepropylamine, a retinyl Schiff base analog of retinol, was synthesized and its absorption spectrum when bound to the wild-type, K70M, and K141M proteins was examined to probe its interaction with the respective lysine residues. The retinylidenepropylamine bound in the K70M beta-lactoglobulin exhibited a kinetic red shift as distinct from the blue shift observed when it is bound to either the K141M or wild-type beta-lactoglobulins. The blue shift indicates protonation of the Schiff base. The resulting tagged peptide was isolated after cyanogen bromide cleavage and found to be the Ala25-Met107 peptide, consistent with the Lys70 being the residue which interacts with the bound retinol. These results support the proposal that retinol binds to an evolutionarily conserved interior cavity rather than the surface pocket.

Mammary tissue from pigs on days 60, 80, 90, 100 and 100+ (days 106-111) of pregnancy has been cultured in vitro as explants. The total accumulation in tissue and culture medium of the whey proteins alpha-lactalbumin and beta-lactoglobulin has been measured using specific radioimmunoassays. The control, uncultured tissue showed progressive morphological development from sparse, non-secretory epithelial tissue on day 60 to full lobulo-alveolar development with some accumulated secretion from day 100. In uncultured explants beta-lactoglobulin could be detected consistently from day 90 (13 +/- 12 ng/micrograms DNA, n = 4) and alpha-lactalbumin from day 100 (1.3 +/- 0.5 ng/micrograms DNA, n = 11). At all stages of pregnancy, both whey proteins increased markedly during the period of culture (up to 7 d). Stimulation of alpha-lactalbumin appeared to be primarily under prolactin control. Prolactin increased alpha-lactalbumin accumulation to a similar extent alone, or in the presence of insulin and/or corticosterone. The response to prolactin was dose-dependent over the range 0.4-20 nM (10-500 ng/ml). Porcine prolactin was more potent than ovine prolactin. There was no effect of porcine growth hormone and no synergism detected between prolactin and tri-iodothyronine. By contrast, no specific hormonal requirements were established for accumulation of beta-lactoglobulin, which appeared to increase in vitro if tissue remained viable in various combinations of insulin, corticosterone and prolactin. It was not stimulated by growth hormone. There was some indication of a prolactin-sensitive component in longer term cultures after day 4.

Transgenic mice were produced, carrying hybrid genes comprised of the ovine beta-lactoglobulin (BLG) milk protein gene promoter and human serum albumin (HSA) coding sequences. In situ hybridization revealed high levels of BLG/HSA hybrid mRNA, confined to the epithelial cells of the lactating mammary gland with a several hundred fold lower concentration in virgin mammary glands. During the first 24 h in culture, exceptionally high levels of HSA were secreted from explants of virgin mice, independent of hormonal control. HSA secretion was reduced considerably during subsequent days in culture and became dependent on the presence of insulin, hydrocortisone and prolactin. This temporal and hormonal pattern of regulation of HSA was different than that found for the secretion of caseins. In contrast to the vast difference in the mRNA content, the amount of HSA secreted from explants derived from lactating mice during the first 24 h in culture was only 2- to 5-fold higher than that found with explants from virgin transgenic mice, suggesting post-transcriptional control of HSA synthesis. The high-level synthesis and secretion of HSA in mammary explants of lactating mice was also dependent on the presence of insulin, hydrocortisone and prolactin. This study confirms previous suggestion that mammary explants from virgin transgenics may serve as a powerful tool for screening the potential of transgenic animals to secrete foreign proteins in their milk.

The major milk whey protein of sheep, beta-lactoglobulin (BLG), is expressed specifically in the mammary gland in a developmentally regulated pattern. To identify the cis-acting DNA regions involved in the regulation of BLG expression, resected gene constructs were analysed in transgenic mice. BLG transgenes which contain at least the proximal 406 bp of the 5' flanking region were expressed in all mice analysed, at levels related to transgene copy number, and thus were expressed in a position-independent manner. Expression was restricted to the mammary gland, except in a few lines where low-level expression was also detected in the salivary gland. In these mice, BLG transgenes were expressed during pregnancy and lactation in the appropriate temporal pattern. Further resection of the 5' proximal region to -146 bp resulted in a dramatically reduced frequency of expression, without affecting tissue specificity, while a construct which retained only 79 bp of 5' flanking region was not expressed. Chromatin analysis of isolated sheep nuclei showed that the promoter resides within a DNAaseI-hypersensitive region in the mammary gland but not in the liver. A BLG transgene displayed a similar tissue-specific pattern of DNAaseI hypersensitivity in mice. These data demonstrate an essential role of the proximal DNAaseI-hypersensitive sequences for position-independent expression of the BLG gene.

Mammary development and milk secretion were studied in transgenic mice which exhibited mammary tissue-specific expression of the sheep beta-lactoglobulin gene, and secreted significant quantities of the foreign protein in milk. Mammary development was unaffected by transgenesis. Tissue DNA content and the activities of several key enzyme markers of cell differentiation were similar in transgenic mice and non-transgenic controls. Milk yield, whether estimated by pup weight gain or measured by a 3H2O-dilution method, was unchanged by foreign gene expression. Gross milk composition, including milk protein concentration, was also similar in transgenic and non-transgenic animals, even though beta-lactoglobulin accounted for 29% of total milk protein. Therefore the foreign gene product was synthesized at the expense of endogenous milk proteins. However, transgenic mammary tissue in vitro exhibited a significantly higher rate of total protein synthesis than did control tissue. This suggested that a factor limiting milk protein synthesis or secretion in transgenic mice in vivo may have been removed by short-term explant culture of mammary tissue. The results emphasize that the use of transgenesis for manipulating milk composition may depend not only on high-level mammary-specific expression of the foreign gene, but also on the biosynthetic capacity of the mammary gland itself.

Five month-old transgenic female pigs from three lines carrying the mouse whey acidic protein (WAP) gene and nontransgenic female littermates were implanted with slow-release estrogen and progesterone pellets. Histological analysis of biopsies taken at the time of implantation and 4 weeks later revealed that mammary alveolar development had occurred upon hormonal stimulation in vivo. beta-Casein and beta-lactoglobulin mRNA was found in all induced animals, and WAP mRNA was detected in two of the three transgenic pigs. Differential hormonal regulation between the transgenes in the three lines and also between endogenous milk protein genes was observed in induced mammary tissue cultured in vitro. In the presence of insulin, hydrocortisone, and PRL, beta-casein and WAP mRNA levels increased in all transgenic pigs. In contrast, beta-lactoglobulin mRNA had reached or exceeded lactational levels in response to the in vivo induction, and no further increase was observed in vitro. This suggests that the regulation of the beta-lactoglobulin gene is distinct from that of beta-casein and WAP. Differences were also observed during pregnancy; whereas beta-lactoglobulin gene expression was induced in early pregnancy, a time when PRL levels are low, WAP mRNA levels increased sharply around parturition. Finally, the observation that hormonal regulation of WAP transgenes greatly differed between the three lines suggests that chromatin surrounding the integration site can modify the response of transcription elements.

We are studying the tissue-specific expression of the sheep milk-whey protein gene, beta-lactoglobulin. We have used sequences derived from this gene to target the expression of biomedical proteins into milk with the intention to exploit this technology in transgenic sheep as a means of protein production. In the present study, a series of beta-lactoglobulin hybrid genes and beta-lactoglobulin minigenes were evaluated for expression in the mammary gland of transgenic mice. In particular, we have assessed whether there is a requirement for introns for efficient transgene expression in the mammary gland, since the coding sequences of many candidate proteins are available only as cDNAs. The results suggest that the inclusion of natural introns in constructs can enhance the efficiency of transgene expression. Thus, a hybrid construct comprising 4.3 kb of the immediate 5' flanking sequences of beta-lactoglobulin fused to a genomic minigene encoding human alpha-antitrypsin (alpha 1AT) was expressed much more efficiently than an alpha 1AT-cDNA construct containing the same beta-lactoglobulin segment. Similarly, the intact beta-lactoglobulin gene was expressed more efficiently than the corresponding intronless beta-lactoglobulin minigene. This effect was not seen in transient expression experiments in baby hamster kidney cells when beta-lactoglobulin-alpha 1AT constructs were driven by SV40 enhancer sequences. The effect cannot be explained by a simple requirement for splicing, since the inclusion of the first beta-lactoglobulin intron into cDNA constructs encoding human alpha 1AT or beta-lactoglobulin itself failed to enhance the efficiency of transgene expression. It is concluded that sequence elements within introns may interact with the upstream 5' flanking sequences of beta-lactoglobulin and enable the latter to function efficiently in the mammary gland of transgenic mice.

A bovine beta-lactoglobulin (beta-LG) was expressed in Saccharomyces cerevisiae carrying bovine pre-beta-LG cDNA and secreted into its growth medium. The expression plasmid was constructed by inserting the whole coding region of the cDNA encoding pre-beta-LG between the promoter and terminator of the yeast glyceraldehyde 3-phosphate dehydrogenase gene of pYG100, a yeast expression vector. In the supernatant of the yeast growth medium, beta-LG with a native conformation was detected by sandwich ELISA, and its amount was estimated to be 1.1 mg/l. A Western-immunoblotting analysis revealed that beta-LG secrected in the growth medium had the same mobility as that of authentic bovine beta-LG. The N-terminal sequence was also identical with that of authentic mature bovine beta-LG.

The release of tumour necrosis factor (TNF), lactoferrin (LF) and cathepsin C (CC) into plasma and production of thromboplastin (TPL) in monocytes were studied in lipopolysaccharide (LPS) stimulated heparinized whole blood from 10 healthy donors. The influence of dextran 70, haemaccel and methylprednisolone on levels of these parameters were examined. TNF concentration in plasma 5 min after the addition of LPS (0 h) was 250 pg/ml (median), 520 pg/ml after 1 h and 1300 pg/ml after 3 h. The addition of dextran 70 to the blood in addition to LPS at the same intervals gave significantly higher values of 740 pg/ml and 1800 pg/ml after 1 h and 3 h respectively. Unstimulated cells had no TPL but after 1 h with LPS, the TPL activity in incubated cells was 2.3 mU/10(6) monocytes and after 3 h, 2.7 mU/10(6) monocytes. LPS induced the secretion of LF from granulocytes (PMN) and the levels 5 min after the addition of LPS (0 h) were 2.1 mg/l (control 0.2 mg/l) and after 1 h, 5.3 mg/l (control 1.3 mg/l) in plasma after LPS stimulation. Haemaccel enhanced the LPS-induced generation of TPL in monocytes and production of CC. The LPS-induced secretion of LF was, to a small extent, influenced by the three reagents tested. Methylprednisolone (1 mmol/l) reduced the production and appearance of TNF in plasma and the generation of TPL activity in monocytes. This model for stimulating heparinized whole blood is suitable for examination of the production and appearance of cellular factors and the influence of drugs on this production.

Bovine beta-lactoglobulin A was expressed in Escherichia coli in its mature form. The gene was constructed using a cDNA clone which coded for amino acid residues Leu-11 to Ile-162 and a synthetic oligonucleotide coding for the initial 10 amino acids preceded by a translational start. The met-beta-lactoglobulin was expressed using a tac promoter vector, pTTQ18, and accounted for approximately 15% of the total cellular protein. The recombinant met-beta-lactoglobulin migrated with the same molecular weight as native beta-lactoglobulin A on SDS-PAGE. The majority of the met-beta-lactoglobulin produced was found in an insoluble form but could be solubilized using guanidine-HCl. The renatured preparation was greater than 80% pure and migrated similarly to purified beta-lactoglobulin A under nondenaturing conditions.

Synthesis of beta-lactoglobulin and alpha-lactalbumin by explants of ovine mammary gland and evolution of concentration of these proteins in cow and sheep colostrum and milk throughout early lactation have been studied. The evolution of both proteins was similar in cow and sheep species. The highest concentration was found in the first milking (19 and 2 mg/ml for beta-lactoglobulin and alpha-lactalbumin, respectively). Then, levels of beta-lactoglobulin decreased sharply and those of alpha-lactalbumin slowly during the first days of lactation, reaching stable values during the second week postpartum (4 and 1.5 mg/ml). The concentration ratio beta-lactoglobulin/alpha-lactalbumin was four times greater in colostrum than in mature milk. On the other hand, synthesis of these proteins represented about 25-30% of the synthesized total soluble proteins. The synthesis ratio beta-lactoglobulin/alpha-lactalbumin in explants obtained at 12 and 30 hours postpartum was found to be 3.5 and 1.7. These results indicate that the synthesis and secretion of beta-lactoglobulin are comparatively greater than those of alpha-lactalbumin during colostral period, suggesting that beta-lactoglobulin could have some specific function during this period.

The processing and intracellular transport of lactoferrin of the neutrophil specific granules was investigated by biosynthetic labeling with (14C)leucine of bone marrow cells from healthy individuals and patients with chronic myeloid leukemia. Lactoferrin was precipitated with antilactoferrin serum and the immunoprecipitates were analyzed by sodium dodecyl sulfate (SDS), polyacrylamide gel electrophoresis (PAGE) followed by fluorography. In contrast to myeloperoxidase of azurophil granules, lactoferrin was not synthesized as a larger precursor, and it was not found to be phosphorylated. The transfer to granules of newly synthesized lactoferrin was demonstrated in pulse-chase labeling experiments followed by centrifugation of cell homogenate in a Percoll gradient. Monensin, which exchanges protons for Na+ and NH4+ cation, blocked the transfer completely, indicating a need for acidification mechanisms. Unlike myeloperoxidase, newly synthesized lactoferrin rapidly became resistant to endoglycosidase H, indicating a transport through the medial and transcisternae of the Golgi apparatus with conversion of "high mannose" to "complex" oligosaccharide side chains. Intracellular transfer of some major neutrophil azurophil and specific granule constituents is obviously regulated differently. Lactoferrin seems to be processed like proteins destined for secretion, while myeloperoxidase is processed more or less like lysosomal enzymes.