Immunohistochemical analysis of paraoxonase-1,2 and 3 expression in normal mouse tissues
ABSTRACT The paraoxonase (PON) enzyme family, comprising PON1, PON2, and PON3, are antioxidant enzymes that degrade oxidised phospholipids. We describe the immunohistochemical localisation of the PON proteins in the normal mouse. Antibodies were obtained by inoculating rabbits with peptides derived from specific sequences of mature PONs. PON1 and PON3 were detected in the skin external epithelium, acini of the sebaceous glands, tongue epithelium, acini of the submandibular gland, surface epithelia of the stomach and the intestine, hepatocytes, exocrine pancreas acini, fibre tracts of the encephalon and the spinal cord, skeletal and cardiac muscle, eye lens epithelium and retinal layers, adipocytes, chondrocytes, epithelial cells of the trachea and bronchiole, ovary follicular fluid, seminiferous tubules, spermatozoa, and kidney proximal tubules. PON2 expression was weaker than that of PON1 and PON3, and was absent in some of the tissues studied, such as submandibular gland, nerve cells, and adipocytes. In muscle cells, PON2 expression was restricted to the endomysium. Apolipoprotein A-I did not colocalise with PONs, suggesting local synthesis. This study provides an experimental model to investigate the role played by these enzymes as antioxidants and their relationship with the development of a variety of diseases.
- SourceAvailable from: Bilal Al-Nawas
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- "The signal at 55 kDa (Tubulin) demonstrates that comparable amounts of protein were used. Since PON2 is known to be expressed regularly in nearly all organs and tissues in mice (except submandibular gland, nerve cells and adipocytes), a regular expression in human OSCC cells was anticipated (Marsillach et al. 2008). Hence, a quantitative analysis of the actual PON2 amount in the different cell lines normalized to Tubulin was of interest. "
ABSTRACT: Patients with oral squamous cell carcinomas (OSCC) often receive radiotherapy to preferentially induce apoptosis of cancer cells through generation of overwhelming DNA damage. This is amplified by generation of reactive oxygen species (ROS), thereby causing oxidative stress and cell death. However, tumors resist through different mechanisms, including upregulation of anti-apoptotic factors and enhanced ROS resistance. We recently reported that the antioxidative enzyme PON2 significantly enhances cellular stress resistance by attenuating mitochondrial ROS-mediated apoptosis. Further, PON2 is often upregulated in cancer. This prompted us to investigate its yet unknown role in the protection of OSCC against irradiation-induced cell death. PON2 expression was determined after 7 Gy singular irradiation in four OSCC cell lines (PCI-13, PCI-52, SCC-4, SCC-68) accompanied by the detection of caspase 3/7 activity. A direct role of PON2 was tested by siRNA-mediated knockdown. In vivo PON2 expression was tested in five patients with oral carcinoma and compared with healthy mucosa for the evaluation of clinical significance. PON2 is variably expressed in OSCC in vitro and in vivo. Compared with the other cell lines, SCC-4 cells showed twofold more basal PON2 (p ≤ 0.05) and the lowest caspase 3/7 activity after singular irradiation (p ≤ 0.05). Contrarily, irradiation led to 1.2-fold induction of PON2 in PCI-13 with no effect on SCC-4 (≤0.05), suggesting that PON2 levels reflect the cells' irradiation sensitivity. In agreement, PON2 knockdown resulted in significant higher apoptosis rates (p ≤ 0.05). Our findings give first evidence that upregulation of PON2 may protect OSCC against irradiation-induced apoptosis.Journal of Cancer Research and Clinical Oncology 02/2015; 141(10). DOI:10.1007/s00432-015-1941-2 · 3.01 Impact Factor
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- "As P1 has the highest sperm concentration while P3 has the lowest (Saravia et al., 2009), there seems to be a relationship between the sperm numbers and SP-PON-1 activity, which is difficult to demonstrated from the results of this study. However, that P1 have the highest levels of SP-PON-1 could indicate a testicular or epididymal origin of the enzyme, considering the P1 spermatozoa bathe in an epididymal tail-dominating fluid, barely mixed with prostatic secretions (Saravia et al., 2009) and by a high PON-1 synthesis was found in the seminiferous tubules in mice (Marsillach et al., 2008). Spermatozoa in P1 better sustain several biotechnological procedures, such as liquid storage and freezing–thawing, than those contained in the rest of SRF (P2) and post-spermatic fraction (P3) (Pe~ na et al., 2006; Saravia et al., 2010; Siqueira et al., 2011). "
ABSTRACT: Paraoxonase 1 (PON-1) is a hydrolytic enzyme present in body fluids, capable of protecting cells against oxidative stress. The hypothesis was hereby to test that PON-1, present in seminal plasma (SP), acts protecting boar spermatozoa when showing a reasonable high activity in the ejaculate. SP-PON-1 activity differed (p < 0.001) among boars (from 0.10 to 0.29 IU/mL). Intra-boar variability was also observed (p < 0.05), but only in two of the 15 boars. SP-PON-1 activity differed among ejaculate portions, showing the spermatozoa-peak portion of spermatozoa-rich ejaculate fraction the highest levels (0.35 ± 0.03 IU/mL, ranging from 0.12 to 0.69) and the post-sperm ejaculate fraction the lowest levels (0.12 ± 0.01 IU/mL, ranging from 0.03 to 0.21). SP-PON-1 activity was positively correlated with the percentage of spermatozoa with rapid and progressive movement (p < 0.01) and negatively correlated with the generation of intracellular reactive oxygen species (p < 0.01) in semen samples after 72 h of liquid storage. SP-PON-1 activity was highest (p < 0.01) in boars with highest farrowing rates. In conclusion, SP-PON-1 activity differed among boars and ejaculate fractions/portions. SP-PON-1 activity was positively correlated with sperm quality and functionality of liquid-stored semen samples and it evidenced a positive association with in vivo fertility.Andrology 01/2015; 3(2). DOI:10.1111/andr.309 · 3.37 Impact Factor
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- "Molecular Reproduction & Development Marsillach et al . ( 2008 ) first immunohistochemically localised the PON proteins ( including PON - 1 , - 2 , and - 3 ) in mouse testis to spermatogonia , spermatocytes , and round and elongated spermatids . We extended these findings , and found PON - 2 enriched in the post - acroso - mal region of ejaculated boar spermatozoa . PON - 2 is considered a cytoplas"
ABSTRACT: This study revealed and characterised the presence of the antioxidant enzymes paraoxonase (PON) type 1 (PON-1, extracellular) and type 2 (PON-2, intracellular) in boar semen. To evaluate PON-1, an entire ejaculate from each of ten boars was collected and the seminal plasma was harvested after double centrifugation (1,500g for 10 min). Seminal plasma was analysed for concentration as well as enzymatic activity of PON-1 and total cholesterol levels. Seminal-plasma PON-1 concentration ranged from 0.961 to 1.670 ng/ml while its enzymatic activity ranged from 0.056 to 0.400 IU/ml, which represent individual variance. Seminal-plasma PON-1 concentration and enzymatic activity were negatively correlated (r = −0.763; P < 0.01). The activity of seminal-plasma PON-1 negatively correlated with ejaculate volume (r = −0.726, P < 0.05), but positively correlated with sperm concentration (r = 0.654, P < 0.05). Total seminal-plasma cholesterol concentration positively correlated with PON-1 activity (r = 0.773; P < 0.01), but negatively correlated with PON-1 concentration (r = −0.709; P < 0.05). The presence of intracellular PON-2 was determined via immunocytochemistry in spermatozoa derived from artificial insemination. PON-2 localised to the post-acrosomal area of the sperm head and principal piece of the tail in membrane-intact spermatozoa. In summary, PON is present in boar semen, with PON-1 at low levels in seminal plasma and PON-2 within the spermatozoa. Further studies are needed to characterise the relationship between antioxidant PONs with sperm and other seminal-plasma parameters. Mol. Reprod. Dev. 2014. © 2014 Wiley Periodicals, Inc.Molecular Reproduction and Development 12/2014; 82(1). DOI:10.1002/mrd.22444 · 2.68 Impact Factor