1. Physiological roles of milk enzymes: an evolving picture

2. Nissim Silanikove Department of Food Science Agricultural Research Organization, The Volcani Center, Israel

7. Milk phases

8. Role Of Milk In Regulation Of Milk Secretion: Negative Feed Back Mechanism Induced By Milk Components

9. Occasional changes in gland emptying

10. Daily changes in breast volume

11. Daily changes in breast milk synthesis

12. Plasminogen/Plasmin System

13. Milk plasminogen and plasmin concentrations (throughout lactation)

14. Milk plasmin x Milk yield x bST (throughout lactation)

15. Stress and the plasmin system Silanikove et al, Life Sci., 2000 TreatmentControl *17.3±0.211.8±0.1PA (units/ml) *8.75±0.85.42±0.9Plasmin (units/ml) 24.8±2.127.3±1.4Plasminogen (units/ml) *2.83±0.95.03±0.8Plasminogen/Plasmin The effect of dexamethasone on the PPS system in cows Values are mean ± SE; *P < 0.05 by t-test

16. The Negative Feedback Mechanism Flow-Chart: The ARO View. Blue arrows denote flow of signal along the feedback loop, red arrows denote positive effect and black arrows denote suppressive effect

17. Milk yield (half) of sheep or goat infected with CNS specie in one gland and the contra-lateral being free. Leitner et al., JDS, 2004 Sheep – open bars Goats – hatched bars

18. PL activity, units/mL Plasmin activity: sheep or goat with one gland infected with CNS specie and the contra-lateral being free Sheep + 73.7%, P < 0.0007 Goat + 195%, P < 0.0003 Leitner et al, JDS, 2004

19. P-p, g/L Proteose-peptone concentration: sheep or goat with one gland infected with CNS specie and the contra-lateral being free Sheep + 247%, P < 0.0001 Goat +151%, P < 0.0001 Leitner et al, JDS, 2004

20. Conclusions The basal level of PL activity is higher in sheep than in goats, which explains the higher basal level of proteose-peptoneThe basal level of PL activity is higher in sheep than in goats, which explains the higher basal level of proteose-peptone PL activity in infected glands is higher in sheep than in goats, which explains the higher increase in proteose-peptone PL activity in infected glands is higher in sheep than in goats, which explains the higher increase in proteose-peptone The higher increase in proteose-peptone concentration in sheep than in goats explains the more acute reduction in milk yield in sheep The higher increase in proteose-peptone concentration in sheep than in goats explains the more acute reduction in milk yield in sheep

21. Role Of The Plasmin System In Induction Of Active Involution

22. What happens in case of surplus?

23. Involution Definition

24. Involution

25. The model: Each goat or cow were Injected with casein hydrolyzate in the experimental gland (+) whereas the control gland (-) was treated with intact casein + -

26. CNH in Goats:Multiple treatments Silanikove et al, Life Sci., 2002

27. CNH treated glands. CNH treated glands. Silanikove et al, unpublished data Histology of section in the alveolus Most of the cells stained in the CNH treated gland are leukocytes control

28. Milk Enzymes As Components Of The Innate Immune System: Formation Of Free Radicals And Bacterocidic Bacteristatic Environment During Active Involution

29. Or 2H 2 O 2 Nitrate Nitrite SOD The many faces of XOR

30. Reaction of Lactoperoxidase with Hydrogen peroxide and Nitrite 1. LPO compound I + H 2 O 2 LPO compound 1 2. LPO compound I + NO 2 - LPO compound II + ● NO 2 3. LPO compound I + NO 2 - LPO + ● NO 2

31. Silanikove et al, FRBM, 2005

32. Silanikove et al, FRBM, 2005

35. Scenario of NO cycling and metabolism in mammary secretion

36. LPO Supply to the young * NO Nitrite Glutathione cycle in milk 2 2 *

37. Question Number 1 1.In the mammary gland, XOR has an essential, non- enzymatic, structural role in fat secretion (Vorbach et al. Genes Dev 2002, 16:3223) 2. It is well established that XOR associated with fat secretion is located within the inner side of MFGM ) e.g. J. Physiol 2002, 545:567) Do we have sufficient XO to support its role in innate immunity? (Free radicals biol Med 2005, 38: 1139 )

38. Distribution of xanthine oxidase, alkaline phosphatase and acid phosphatase in milk fractions Xanthine oxidase Alkaline phosphatase Acid phosphatase % of total % of total % of total Whoe milk 100 100 100 Fat* (MFGM) 33 45 48 WMP 21 39 34 Phos.lipids 54 84 82 Casein 3 - - Truly soluble 43 16 18

39. a. Physiologically inactive XD in the inner side of MFGM and vesicles, and b. Physiologically active in the form of XO, on the outside surface of the vesicles. XOR is mostly associated with milk serum as XO, though its activity is highest on membranes and where it is distributed between

40. Question Number 2 As mature fresh milk do not contains measurable amount of xanthine, but contains uric acid in the range of 30 40 -micro-molar, it is important to know whether it derived from milk xanthine or secreted as uric acid? Fresh milk (i.e., milk secreted into the alveoli within 5 to 10 min before sampling) was obtained at the end of noon-milking following injection of oxytocine.

41. Xanthine + hypoxanthine and uric acid concentration in oxytocin-induced and mature milk

42. Disappearance of hypoxanthine and appearance of uric acid in fresh milk

44. Scenario of NO cycling and metabolism in mammary secretion

45. Question Number 3 Does Catalase really plays a critical role in protecting the gland from oxidative stress by converting active nitrite to less active nitrate?

46. Xanthine dose-dependently enhance the conversion of nitrite to nitrate. Silanikove et al, unpublished data Under the experimental conditions, approximately 40 mM of xanthine are converted to urate via XO within 4 h

48. Catalase inhibitor, amino triazole, prevent the conversion of nitrite to nitrate. Silanikove et al, unpublished data reaction conditions Nitrite-50 mM, Xanthine-200 mM, incubation time - 30 min.

50. Relative changes in lipid oxidation in milk Silanikove et al, unpublished data milk stored for 6 hours in the dark at 40C (A), Effects of catalase inhibitor (B), nitrite (10 mM) (C) and nitrite + catalase (D) inhibitor

52. Conclusions Regarding the Control of Oxidative Stability in Milk Formation of nitric dioxide is a key process in oxidative stress in milk. Thus, controlling this process should improve milk oxidative stability. The function of catalase is rate limited by hydrogen peroxide availability. The supply of the latter at required physiological rate may be provided from dissolved oxygen via XO by using electrons gained during the conversion of xanthine to urate. XO and catalase works interactively as an antioxidant system.

53. Question Number 4 Does XO-LPO derived oxidative stress play a role in sub-clinical mastitis; i.e., under conditions that do not elicit an apparent classical inflammatory symptoms

54. + - The model: Each cow tested had at least one uninfected quarter (NBF) and one of the other quarters infected with one of the following bacteria: NumberBacteria 33 NBF 23 Streptococci 11 CNS 8 E. Coli 9 S. aureus Cork 2005

55. Uric acid and nitrate in sub-clinically infected glands Nitrate (micro-molar) Uric Acid (micro- molar) Bacteria 19 ± 9 a 35 ± 13 a NBF 38 ± 12 b 72 ± 14 b Strep. DG 17 ± 11 a 38 ± 14 a CNS 42 ± 12 b 85 ±15 b E. coli 20 ± 11 a 39 ± 19 a S. aureus BOLFA 2006

56. Clotting time and curd firmness Curd firmness (V) Clotting time (sec) Bacteria 6.58±0.2 650±63 NBF 1.02±0.3 2490±340 Strep. 3.80±0.8 1255±468 CNS 0.92±0.32590±370 E. coli 3.28±0.7 1078±193 S. aureus Cork 2005

59. Question Number 5 Does XO-LPO derived oxidative stress play a role in clinical mastitis; i.e., under conditions that elicit an apparent classical inflammatory symptoms

60. The model: Each cow tested was infused in one quarter once with Casein hydrolyzate, lipopolysaccharide, or saline, and samples from each gland were sampled for two days post-treatment BOLFA 2006

61. Effect of infusion of CNH and LPS into the mammary gland on the immune cell population Treatment SCC (×1000) PMN (%) CD4+ (% CD8+ (%) CD14+ (%) Control 116±20 a 29±3.3 a 3.1±0.9 a 5.7±1.6 a 5.5±1.8 a CNH 3146±324 b 57±7 b 3.3±1.1 a 10.5±2.0 b 12.6±2.2 b LPS 4960±793 c 90±9.1 c 1.8±2.2 b 4.4±4.0 a 6.6±4.4 a c

62. Caseinolysis (proteose peptone formation) in CNH and LPS treated glands

63. Uric acid in CNH / LPS treated glands

64. Major conclusions Our data suggest that XO is post- transcriptional regulated through allocation of substrate (xanthine) availability. Together with lactic peroxidase they involve in the oxidative (mostly nitrosative) stress in certain type of sub-clinical mastitis. This system is the main driving force of oxidative/nitosative stress in E.Coli/LPS driven mastitis.

65. The Jekyll and Hyde sides of uric acid Uric acid is a major anti-oxidant in blood plasma and milk However, uric acid is also a danger signal that alerts the immune system to dying cells (Nature 425: 516, 2003). In hyperuricemia, crystals of uric acid can precipitate in joins, where they cause gout and/or in other tissues causing inflammation. Does XO-depended gouty inflammation involve in the pathogenesis induced by E. coli/LPS in the mammary gland ?

66. Concluding Remarks Milk enzymes have an important biological role and are involved in control of milk secretion, developmental stage (involution), gland innate immune system and preventing oxidative damage to its essential nutrients. For that purpose milk congregate many enzymes which constantly consume metabolites, produce free- radicals and modify its composition if needed. Milk enzyme along with other components (e.g., cytokines, enzyme inhibitors) form complex metabolic pathways.

67. Thank you: I hope that this lecture will contribute to our ability to raise healthier cows and produce better dairy products BOLFA 2006