Penetration of Cow Milk Angiogenin into the Blood of Mice after Peroral Introduction

The cow milk angiogenin consumed by mice perorally penetrated from the gastrointestinal tract in the blood flow. In the experimental animals, the blood level of exogenous angiogenin first increased to reach a maximum, and then gradually decreased to zero. The dynamics of this process depends on the age of animals. The data obtained suggest that the cycle of perorally introduced cow milk angiogenin in blood is realized in the infantile-juvenile mice at a higher rate than in the adult-presenile mice.     

Milk Ultrafiltrate as a Promising Source of Angiogenin

The use of membrane technologies in the production of curd (soft cheese for children) is associated with the appearance of up to 80% of angiogenin in the ultrafiltrate. An electrophoretically homogeneous preparation of angiogenin (MW 17 kDa) was obtained from milk ultrafiltrate by two-stage ion-exchange chromatography. The yield of the angiogenin was 60%, which corresponds to a 586-fold purification of the raw material. The obtained preparation retained stability in the course of lyophilization and could be stored at 4°C for a long time without decomposition.     

Absorption of bovine angiogenin into peripheral blood of rats orally administered milk basic protein

Bovine angiogenin is a major component of the bone resorption inhibitory activity of milk basic protein (MBP). The intestinal absorption of bovine angiogenin was investigated in a rat model, where it was detected in an intact form in the peripheral blood after the oral administration of MBP. This finding demonstrates that orally administered bovine angiogenin is absorbed without being degraded.     

High level production of bovine angiogenin in E. coli by an efficient refolding procedure

Recombinant bovine angiogenin (rbAng) was expressed in E. coli at up to 30% of total cell proteins but was produced as inclusion bodies. By investigating the effect of various factors on the refolding yield, we obtained about 60% refolding. After chromatographic purification, about 60 mg purified angiogenin was obtained from 1 l culture. The purified recombinant bovine angiogenin was identical to native bovine angiogenin (nbAng) obtained from cow's milk. Our approach is highly efficient and can be generally used for the production of various types of angiogenin for functional and structural studies as well as therapeutic purposes.     

High level production of bovine angiogenin in E.?coli by an efficient refolding procedure

Recombinant bovine angiogenin (rbAng) was expressed in E. coli at up to 30% of total cell proteins but was produced as inclusion bodies. By investigating the effect of various factors on the refolding yield, we obtained about 60% refolding. After chromatographic purification, about 60 mg purified angiogenin was obtained from 1 l culture. The purified recombinant bovine angiogenin was identical to native bovine angiogenin (nbAng) obtained from cow's milk. Our approach is highly efficient and can be generally used for the production of various types of angiogenin for functional and structural studies as well as therapeutic purposes.     

The complete amino acid sequence of bovine milk angiogenin

The amino acid sequence of angiogenin isolated from bovine milk was deduced by gas-phase sequencing of the protein and its fragments. The protein contains 125 residues and has a calculated molecular mass of 14,577 Da. The sequence is highly homologous (65% identity) to the sequence of human angiogenin, most of the differences being the result of conservative replacements. Like human angiogenin, the bovine protein is also homologous to bovine pancreatic RNase A (34% identity) and the three major active site residues known to be involved in the catalytic process, His-14, Lys-41 and His-115, are conserved. When tested against conventional substrates for RNase A activity, bovine angiogenin displays the same selective ribonucleolytic activity as human angiogenin. The sequence of bovine angiogenin contains the cell recognition tripeptide Arg-Gly-Asp which is not present in the human protein.     

Milk ultrafiltrate as a promising source of angiogenin

The use of membrane technologies in the production of soft cheese (children's food) is associated with the appearance of up to 80% of angiogenin in the ultrafiltrate. An electrophoretically homogeneous preparation of angiogenin (MW approximately 17 kDa) was obtained from milk ultrafiltrate by two-stage ion-exchange chromatography. The yield of the angiogenin was approximately 60%, which corresponds to a 586-fold purification of the raw material. The obtained preparation retained stability in the course of lyophilization and could be stored at 4 degrees C for a long time without decomposition.     

Detection of Clostridium tyrobutyricum spores using polyclonal antibodies and flow cytometry

Aims: The present work investigates the feasibility of using flow cytometry (FCM) combined with fluorescent‐labelled specific polyclonal antibodies for the detection and presumptive identification of Clostridium tyrobutyricum spores in bovine milk. Methods and Results: Two fluorescent molecules (fluorescein isothiocyanate and Alexa Fluor 488) were conjugated to antispores polyclonal antibodies. Side scatter and forward scatter profiles of the Cl. tyrobutyricum spores marked with fluorescent antibodies permitted the detection of spores and differentiated them from other related microbial species. The detection limit of this method was 10³ spores per 100 ml of milk, and results could be achieved in 2 h. Conclusions: FCM combined with fluorochrome‐conjugated antibodies, especially Alexa Fluor, could be an efficacious means to detect and provide presumptive identification of Cl. tyrobutyricum spores, as well as differentiation from other Clostridium species that can also cause late blowing in cheese. Significance and Impact of the Study: This study describes the basis for the development of a method suitable for analysis of milk destined for cheese manufacture that would permit the detection of Cl. tyrobutyricum spores in a short period. This would enable the industry to use contaminated milk for dairy products other than cheese where Cl. tyrobutyricum does not cause a problem.     

原核表达MurA蛋白并制备其多克隆抗体用于免疫磁珠快速检测单增李斯特菌

【目的】制备MurA多抗,结合免疫磁珠与选择平板进行单增李斯特菌的快速检测,建立单增李斯特菌的免疫磁珠快速检测方法。【方法】构建MurA的原核表达载体,转化大肠杆菌进行优化表达。镍柱纯化表达产物,质谱鉴定重组蛋白,再免疫小鼠,制备其多克隆抗体。用所获多抗制备免疫磁珠,建立单增李斯特菌免疫磁珠-选择性培养基检测方法,并对人工污染牛奶样品进行检测。【结果】在大肠杆菌中高效表达了分子量约为72 kD的可溶性融合蛋白,质谱鉴定其为MurA蛋白;免疫小鼠获得的抗血清效价达1:10 000,与伤寒沙门氏菌、副溶血弧菌、大肠杆菌及属内其它病原菌均无交叉;所建立的免疫磁珠-选择性培养基检测法可检出浓度为103 CFU/mL及以上的单增李斯特菌,仅与英诺克李斯特菌存在一定交叉反应;牛奶样品单次仅需9 h增菌就能被检出,较常规增菌时间缩短39 h;检测限为0.4 CFU/mL。【结论】表达并纯化得到高纯度的单增李斯特菌MurA蛋白,制备的鼠源多克隆抗体亲和力高,特异性好;建立了快速检测单增李斯特菌的免疫磁珠联合选择性培养基法,在灵敏度不变的情况下,实现24 h内成功对牛奶样品的检测,较国标法减少42 h以上。     

Expression of human lactoferrin in milk of transgenic mice

The expression of human lactoferrin (hLF) in the milk of transgenic mice is described. Regulatory sequences derived from the bovine S1-casein gene were fused to the coding sequence of the hLF cDNA and several lines of transgenic mice were generated. Human LF RNA was detected exclusively in the mammary gland of lactating females and only after the onset of lactation. No aberrant RNA products could be detected using northern blotting and primer extension analysis. The hLF concentrations in the milk ranged from less than 0.1 to 36 g ml^–1. Human LF thus expressed did not differ from human milk derived LF, with respect to molecular mass and immunoreactivity with monoclonal and polyclonal antibodies.     

Identification of the human beta-casein C-terminal fragments that specifically bind to purified antibodies to bovine beta-lactoglobulin

The presence of foreign proteins in human milk after the ingestion of bovine dairy products is thought to be one of the possible causes of allergic sensitization in exclusively breast-fed predisposed infants. The immunologic determination of bovine beta-lactoglobulin (LG) concentration in human milk has been reported by several researchers, but the results are conflicting. Moreover, a strong cross-reactivity between antibodies to bovine beta-LG and human milk proteins and peptides was reported, throwing doubt on the reliability of radioimmunoassay and enzyme-linked immunosorbent assay detection and quantification assays for bovine beta-LG in human milk. Thus, the goal of this study was to isolate human milk peptides with a molecular mass >or= 1,000 Da cross-reactive with antibodies to bovine beta-LG in order to identify possible common epitopes between human and bovine milk proteins. The proteins were first isolated by affinity chromatography with purified polyclonal antibodies to bovine beta-LG, followed by gel filtration fast phase liquid chromatography and reverse phase-high performance liquid chromatography purification of the components specifically bound in the affinity separation step. Affinity-bound peptides were identified by determining their amino acid sequence. All the sequenced peptides belonged to the C-terminal part of human beta-casein, which confirms the cross-reactivity of human milk proteins and peptides with antibodies to bovine beta-LG and allows the identification of possible common epitopes between the two proteins. No bovine beta-LG peptides with a molecular mass >or= 1,000 Da were found in our milk samples from healthy mothers on a diet rich in bovine milk and dairy products.     

Detection of procathepsin D in rat milk

The presence of procathepsin D, a zymogen of the soluble lysosomal aspartic proteinase cathepsin D, was detected in rat milk using Western blot analysis and assay of proteolytic activity in acidic buffers. No other forms of cathepsin D were found. Two different polyclonal anti-procathepsin D antibodies were used for immunochemical detection of procathepsin D. Both antibodies we found to recognize rat procathepsin D. Proteolytic activity in acidic buffers was detected using a fluorogenic substrate specific for cathepsin D and was abolished by pepstatin A, a specific inhibitor of aspartic proteinases. This study represents third demonstration of presence of procathepsin D in mammal breast milk. Potential sources and physiological functions are discussed.     

Genes of the RNASE5 pathway contain SNP associated with milk production traits in dairy cattle

Background

Identification of the processes and mutations responsible for the large genetic variation in milk production among dairy cattle has proved challenging. One approach is to identify a biological process potentially involved in milk production and to determine the genetic influence of all the genes included in the process or pathway. Angiogenin encoded by angiogenin, ribonuclease, RNase A family 5 (RNASE5) is relatively abundant in milk, and has been shown to regulate protein synthesis and act as a growth factor in epithelial cells in vitro. However, little is known about the role of angiogenin in the mammary gland or if the polymorphisms present in the bovine RNASE5 gene are associated with lactation and milk production traits in dairy cattle. Given the high economic value of increased protein in milk, we have tested the hypothesis that RNASE5 or genes in the RNASE5 pathway are associated with milk production traits. First, we constructed a “RNASE5 pathway” based on upstream and downstream interacting genes reported in the literature. We then tested SNP in close proximity to the genes of this pathway for association with milk production traits in a large dairy cattle dataset.

Results

The constructed RNASE5 pathway consisted of 11 genes. Association analysis between SNP in 1 Mb regions surrounding these genes and milk production traits revealed that more SNP than expected by chance were associated with milk protein percent (P < 0.05 significance). There was no significant association with other traits such as milk fat content or fertility.

Conclusions

These results support a role for the RNASE5 pathway in milk production, specifically milk protein percent, and indicate that polymorphisms in or near these genes explain a proportion of the variation for this trait. This method provides a novel way of understanding the underlying biology of lactation with implications for milk production and can be applied to any pathway or gene set to test whether they are responsible for the variation of complex traits.     

Prostate cancer-derived angiogenin stimulates the invasion of prostate fibroblasts

Prostate fibroblasts promote prostate cancer progression by secreting factors that enhance tumour growth and induce the migration and invasion of prostate cancer cells. Considering the role of fibroblasts in cancer progression, we hypothesized that prostate cancer cells recruit these cells to their vicinity, where they are most directly available to influence cancer cell behaviour. To test this hypothesis, we performed modified Boyden chamber assays assessing the migration and collagen I invasion of normal primary prostate fibroblasts (PrSCs) and prostate cancer-associated fibroblasts (PCAFs) in response to media conditioned by the metastatic prostate cancer cell lines PC-3, LNCaP and DU145. During 4-hr incubations, PrSCs and PCAFs migrated and invaded in response to the conditioned media. To identify candidate proteins in the conditioned media that produced these effects, we performed cytokine antibody arrays and detected angiogenin in all three media. Angiogenin-blocked PC-3-conditioned medium, obtained using an anti-angiogenin polyclonal antibody or angiogenin siRNA, significantly reduced PC-3-induced PrSC and PCAF collagen I invasion. Furthermore, angiogenin alone at 1, 2 and 5 ng/ml significantly stimulated PCAF collagen I invasion. These results suggest that PC-3-derived angiogenin stimulates the invasion of normal prostate fibroblasts and PCAFs and is sufficient for invasion of the latter. Because prostate fibroblasts play key roles in prostate cancer progression, targeting their invasion using an anti-angiogenin-based therapy may be a strategy for preventing or treating advanced prostate cancer.     

Preparation and immunological characterization of β-lactoglobulin-amylose conjugate

β-Lactoglobulin (BLG), a major allergen of cow's milk, was conjugated with the N-hydroxysuccinimide ester of the amylose-glycylglycine adduct (AG-ONSu) to reduce its immunogenicity, and the biochemical and immunological properties of the resulting conjugate (AG-BLG) were studied. The conjugate was prepared by modifying BLG with AG-ONSu, and was purified in a Sephadex G-100 column. The analytical data for AG-BLG indicated that 10.5 moles of AG-ONSu, with a mean molecular weight of 2,800, was covalently attached to the amino groups of the BLG molecule. Conjugation with AG-ONSu greatly decreased the reactivity of BLG with anti-BLG polyclonal antibodies owing to its shielding action for epitopes on the protein's surface. These findings suggest that AG-ONSu can be used advantageously to suppress the hypersensitivity mediated by IgG antibodies in milk allergy.     

Development and application of indirect competitive enzyme immunoassay for detection of neomycin in milk

As a result of immunization of rabbits with neomycin B (NM) conjugated to sodium periodate-oxidized (SP) transferrin, polyclonal antibodies were generated and used to develop an indirect competitive enzyme-linked immunosorbent assay (ELISA) of NM. Several heterologous conjugates, namely, glutaraldehyde (GA)-polymerized NM, gelatin-ribostamycin (sp), and gelatin-NM (ga) were used as coating antigens in different ELISA variants for quantification of NM in milk. These variants were characterized by different dynamic ranges and detection limits of 1.0, 0.1, and 0.01 ng/ml, respectively. Maximum residue level (MRL) of this antibiotic in milk accepted in the EU can be detected without any special pretreatment at a 100-fold sample dilution in the least sensitive assay variant. The mean recovery rate from NM-spiked milk containing 1.5–10% fat was 111.7% and ranged from 84 to 125.2%. We found that 57 of 106 tested milk samples contained NM at concentrations higher than 100 ng/ml. In ten percent of cases (11/106), the residual level of this antibiotic was greater than 500 ng/ml. In one case, the MRL was exceeded (1690 ng/ml). The assay developed in this study is specific shows no cross-reactivity with other veterinary aminoglycosides, has a good sensitivity reserve, and can serve as an effective tool to monitor the NM content in milk stuff.     

MUC1粘蛋白的分离纯化及其抗体的制备

经高速离心从正常人乳中获得人乳汁颗粒膜(HMFGM),产量约0．4g／L。经进一步破碎、脱脂及sepharose CL-4B柱纯化,获得含MUC1粘蛋白的组分,并经SDS—PAGE、Western—blot及ELISA鉴定后,免疫家兔制备多抗。结果表明,进一步凝胶过滤获得MUC1粘蛋白,行SDS—PAGE后经希夫试剂和考马斯亮蓝染色呈单一条带,表观相对分子质量大干205000。Western—blot及ELISA结果表明可与MUC1特异性抗体结合。制备获得的多抗经ELISA测定效价为1：64000～1：128000。表明建立了MUC1粘蛋白的纯化方法,获得的MUC1粘蛋白及其抗体可进一步用于MUC1检测及其功能的研究。     

An automated method for the detection of staphylococcal heat stable deoxyribonuclease in dairy products

An automated sandwich immunoassay with specific polyclonal antibodies for the detection of Staphylococcus aureus thermostable nuclease (DNase) is described. To evaluate this assay, different quantities of purified S. aureus nuclease were added to dairy products. Additionally, staphylococcal counts and nuclease activity of milk samples inoculated with S. aureus were determined. Different extraction procedures were performed and compared. The results indicated that the automated test was a reliable method for detecting DNase activity in milk products. The procedure was completed in 2 h and detected 1 ng of DNase ml-1. Detection of the DNase was especially useful in cheeses and could be used to confirm positive enterotoxin results.     

Lipid kinase activity of antibodies from milk of clinically healthy human mothers

We have shown recently that polyclonal human milk sIgA contains a subfraction of antibodies (Abs) tightly bound to unusual minor milk lipids containing sialic acid. Here, we show that a small subfraction of milk IgG is tightly bound to the similar or the same minor lipids. The ability of small fractions of sIgA and IgG from human milk to phosphorylate selectively two minor lipids in the presence of [gamma-(32)P]nucleoside triphosphates was shown here for the first time to be an intrinsic property of these antibodies. In contrast to known kinases, antibodies with lipid kinase activity can transfer phosphoryl group to lipids not only from ATP but also from other different nucleotides (dATP, GTP, dGTP, UTP, TTP) with comparable efficiencies (30-100%). To our knowledge, there are no examples of enzymes using orthophosphate as a substrate of phosphorylation reactions. An extremely unusual property of lipid kinase Abs is their high affinity for orthophosphate (K(m)=1.6-5.6 microM) and capability to phosphorylate minor lipids using [(32)P]orthophosphate as donor of phosphate group. The relative specific activity and affinity of abzymes for orthophosphate and ATP depend significantly on donor milk. However, the levels of Ab-dependent phosphorylation of lipids for all Abs in the case of ATP (100%) and orthophosphate (60-80%) as substrates are comparable. The first example of natural abzymes with synthetic activity was milk sIgA with protein kinase activity. Most probably, lipid kinase sIgA and IgG of human milk are the second example of Abs with synthetic activity.