BOLFA by xiaoyounan

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									  Distribution of Xanthine Dehydrogenase
  and Xanthine Oxidase in milk fractions :
      Evidence for post-transcriptional
regulation of Xanthine Oxidase in the frame
  of the mammary innate immune defense
                   system




Nissim Silanikove, Fira Shapiro, Adi Rauch & Gabriel
                       Leitner
          Nitrite
Nitrate
  NAD     NADH




                    Or 2H2O2
                      SOD
        Reaction of Lactoperoxidase
        with Hydrogen peroxide and
                  Nitrite
          Nitric Oxide is a Free Radical
                    N=O

1. LPO + H2O2         LPO compound 1

2. LPO compound I + NO2-      LPO compound II + ●NO2

3. LPO compound II + NO2-     LPO + ●NO2
Scenario of NO cycling and metabolism in
 mammary secretion (Free radicals Biol Med, 2005)
             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 )
Xanthine + hypoxanthine and uric acid
concentration in oxytocin-induced and
             mature milk
               50
               45
               40
               35
 Micro-molar




               30
               25
               20
               15
               10
               5
               0

                      Oxytocin Induced    Mature milk

                    Xanthin+Hypoxanthin
                    Uric Acid
          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.
  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
Distribution of enzymes inside and
   outside of WMP membranes

         XO (U/ml) XD (U/ ml) XD/XO Total XD/XO Inside AlP (U/ml) AcP (U/ml)


Intact    1.11         -            -           -          114       16.2



Total     1.55        1.10       0.71        2.5           118       15.1
Distribution of enzymes in MFGM
     with and without plasmin
           deactivation
            With plasmin deactivation   Without plasmin deactivation
XO (U/g)            2.4 ± 0.4                      3.7 ± 0.6
XD (U/g)            1.7 ± 0.5                          -

XD/XO Total         0.7 ± 0.08                        -

XD/XO inside        4.25 ± 0.5                       -
AlP (U/g)           135 ± 10                       142 ± 15
AcP (U/g)            39 ± 7                         42 ± 9
  Distribution of protein in milk
             fractions

Total protein     Casein          Protein in WMP Protein in MFGM
g/l % of total   g/l % of total    g/Kg % of total  g/Kg % of total
29.2 100         22.7   78         44.3   0.094     40.3   0.095
Lipid composition (as % of dry matter)
 in whey membrane particles (WMP)
   and milk fat globule membranes
              (MFGM)
               WMP         MFGM
            Average SD     Average    SD
Total lipid 25.6     2.7     29.7     3.1
Lipid P     0.530 0.09      0.621    0.11
Phos.lipids 13.25 2.25     15.53     2.75
         Question Number 3

Does XO-derived oxidative stress play a
 role in sub-clinical mastitis; i.e., under
 conditions that do not elicit an apparent
    classical inflammatory symptoms
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:
                        Bacteria       Number
                        NBF             33
                        Streptococci    23
                        CNS             11
      +     -           E. Coli          3
                        S. aureus        9
                                        Cork 2005
  Uric acid and nitrate in sub-clinically
             infected glands
Bacteria    Uric Acid (micro-      Nitrate
                 molar)         (micro-molar)
NBF          35 ± 13a           19 ± 9a
Strep. DG    72 ± 14b           38 ± 12b
CNS          38 ± 14a           17 ± 11a
E. coli      85 ±15b            42 ± 12b

S. aureus   39 ± 19a            20 ± 11a

                                           BOLFA 2006
      Clotting time and curd firmness

Bacteria      Clotting time    Curd firmness
                  (sec)              (V)
NBF           650±63            6.58±0.2
Strep.       2490±340           1.02±0.3
CNS          1255±468           3.80±0.8
E. coli     2590±370          0.92±0.3

S. aureus    1078±193          3.28±0.7

                                          Cork 2005
log Somatic Cell Count




                         1000




                                Strep. dysgalactiae             S. aureus


                          100
                                 0     1      2      3      4          5   6
                                       Curd firmness (A30 Optigraph)
         Question Number 4

Does XO-derived oxidative stress play a
   role in clinical mastitis; i.e., under
conditions that elicit an apparent classical
        inflammatory symptoms
        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
   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±20a       29±3.3a   3.1±0.9a    5.7±1.6a    5.5±1.8a

CNH         3146±324b     57±7b     3.3±1.1a   10.5±2.0b    12.6±2.2b

LPS         4960±793c     90±9.1c   1.8±2.2b    4.4±4.0a    6.6±4.4a
  Caseinolysis (proteose peptone
formation) in CNH and LPS treated
              glands

                      6,000
                              LPS
                      5,000
                              CNH
    prot-pept ug/ml




                      4,000   CONTROL

                      3,000

                      2,000

                      1,000

                          -
                               0                    +24                +48
                                        Tim e relative to treatm ent
Uric acid in CNH / LPS treated glands

                       400
                             LPS
                       350
                             CNH
                       300
                             CONTROL
      Uric acid unol




                       250
                       200
                       150
                       100
                       50
                         -
                              0                   +24               +48
                                       Time relative to treatment
Nitrate in CNH and LPS treated glands

                   160
                         LPS
                   140
                         CNH
                   120
                         CONTROL
    Nitrate unol




                   100
                   80
                   60
                   40
                   20
                     -
                               0               +24                +48
                                   Tim e relative to treatm ent
             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.
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 ?
Thank you: I hope that this lecture
 will contribute to our ability to
 raise healthier cows and produce
       better dairy products




                                BOLFA 2006

								
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