The antioxidant haptoglobin (Hp) is an acute-phase protein responsive to infectious and inflammatory diseases. Hp and somatic cell counts (SCC) are sharply elevated in bovine milk following intramammary administration of endotoxin or bacteria. However, the sources of milk Hp responsible for such increases are not fully understood. The purpose of this study was to define the source of milk Hp from dairy cows with naturally occurring mastitis. Quarter milk samples selected from 50 dairy cows were separated into four groups according to SCC as group A: < 100 (n = 19); B: 100–200 (n = 10); C: 201–500 (n = 10); and D: > 500 × 103 (n = 11) cells/mL. Our results reveal that milk Hp concentrations were correlated with SCC (r = 0.742; P < 0.01), and concentrations in group D were ~10-fold higher than in group A. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicates that the milk somatic cells from group D were not only capable of synthesizing Hp but could also markedly increase Hp mRNA expression. Western blot, immunocytochemistry, double confocal immunofluorescence, and Hp releasing experiments demonstrate that neutrophils were associated with the biosynthesis and release of Hp in milk. It further shows that Hp was significantly elevated in the epithelium of mammary gland tissue with mastitis and was also expressed in the cultured mammary epithelial cells. We propose that neutrophils and epithelial cells may play an essential role in elevating milk Hp in addition to previous suggestions that Hp may be derived from mammary tissues and circulation.
<b><i>Background:</i></b> Pseudorabies virus (PRV) infection induces apoptosis in swine cells both in vitro and in vivo; however, the mechanism associated with host-cell signaling has not been studied. This study investigated the role of free radicals caused by cellular oxidative stress after viral infection and examined whether the DNA damage response plays an important role in PRV-induced apoptosis. <b><i>Methods:</i></b> Several apoptosis assays and western blotting confirmed PRV-induced apoptosis. PRV-mediated oxidative stress was evaluated by reactive oxygen species (ROS) assay. <b><i>Results:</i></b> Our results showed that PRV caused apoptosis in a porcine kidney cell line, PK15, and induced expressions of proapoptotic Bcl family proteins in a dose- and time-dependent manner. Expressions of specific DNA damage sensors and phosphorylation of histone H2AX were also significantly increased, which subsequently activated the expressions of checkpoint kinase 1/2 and proapoptotic <i>p</i>53. Caffeine, a known DNA damage inhibitor, was found to inhibit caspase-3 activation and protect cells from PRV-induced apoptosis. Additionally, the antioxidant <i>N</i>-acetyl-L-cysteine was shown to prevent the production of cellular ROS, protecting DNA from cleavage. <b><i>Conclusions:</i></b> Our results confirmed that oxidative stress and free radicals arising from PRV infection cause DNA damage, which consequently triggers apoptosis.
Haptoglobin (Hp) is an acute-phase protein (responsive to infection and inflammation) that is present in the plasma of all mammals [1][2][3][4]. A recent study has found that Hp also exists in lower vertebrates (bony fish) but not in frog and chicken [5]. The most frequently reported biological functions of the protein are to capture released hemoglobin during excessive hemolysis [6] and to scavenge free radicals during oxidative Similar to blood types, human plasma haptoglobin (Hp) is classified into three phenotypes: Hp 1-1, 2-1 and 2-2. They are genetically inherited from two alleles Hp 1 and Hp 2 (represented in bold), but only the Hp 1-1 phenotype is found in almost all animal species. The Hp 2-2 protein consists of complicated large polymers cross-linked by a2-b subunits or (a2-b) n (where n ‡ 3, up to 12 or more), and is associated with the risk of the development of diabetic, cardiovascular and inflammatory diseases. In the present study, we found that deer plasma Hp mimics human Hp 2, containing a tandem repeat over the a-chain based on our cloned cDNA sequence. Interestingly, the isolated deer Hp is homogeneous and tetrameric, i.e. (a-b) 4 , although the locations of )SH groups (responsible for the formation of polymers) are exactly identical to that of human. Denaturation of deer Hp using 6 m urea under reducing conditions (143 mm b-mercaptoethanol), followed by renaturation, sustained the formation of (a-b) 4 , suggesting that the Hp tetramers are not randomly assembled. Interestingly, an a-chain monoclonal antibody (W1), known to recognize both human and deer a-chains, only binds to intact human Hp polymers, but not to deer Hp tetramers. This implies that the epitope of the deer a-chain is no longer exposed on the surface when Hp tetramers are formed. We propose that steric hindrance plays a major role in determining the polymeric formation in human and deer polymers. Phylogenetic and immunochemical analyses revealed that the Hp 2 allele of deer might have arisen at least 25 million years ago. A mechanism involved in forming Hp tetramers is proposed and discussed, and the possibility is raised that the evolved tetrameric structure of deer Hp might confer a physiological advantage.Abbreviations Hp, haptoglobin; b-ME, b-mercaptoethanol.
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