Differentially Expressed Salivary Proteins in Dental Caries Patients

Dental caries is a multifactorial disease mainly caused by cariogenic bacteria commonly found in the oral cavity. Dental caries may cause demineralization of the tooth, cavitation, hypersensitivity, pulp inflammation, and even tooth loss if left untreated. Saliva secreted in the oral cavity can serve as a tool for identification of biomarkers for early detection of diseases. In the present study, differential expression of salivary proteins from 33 dental caries patients was compared with 10 control subjects. The unstimulated saliva was analyzed by 12% SDS-PAGE and two-dimensional gel electrophoresis. Gelatin and casein zymography was performed to check for protease activity. Also, salivary IgAs from both groups were compared by sandwich ELISA technique. Dental caries patient’s saliva showed decreased caseinolytic and increased gelatinolytic activity probably due to metalloproteases and cathepsins. Mean salivary levels of sIgA were also significantly higher ( p < 0.018 ) in dental caries saliva samples. The 2D electrophoresis profile of both the groups showed regions on gel with visually detectable alterations in protein expression. The present study is among the few initial studies in the locality for identification of protein differences in saliva from dental caries patients and has demonstrated a good potential to identify alterations. However, a large population-based analysis is required to validate these findings to be translated as a tool for indicative applications.

Identification of Pseudomonas jessenii and Pseudomonas gessardii as the most proteolytic Pseudomonas isolates in Iranian raw milk and their impact on stability of sterilized milk during storage

Identification of the most proteolytic Pseudomonas strains that can produce heat-resistant proteases and contribute to the Ultra High Temperature (UHT) milk destabilization is of great interest. In the present study, among the 146 Pseudomonas isolates that encoded the aprX gene, five isolates with the highest proteolytic activity were selected and identified based on 16S rRNA, rpoD and gyrB gene sequences data. The identification results were confirmed by phylogenetic analysis based on multilocus sequence analysis and identified the representative isolates as P. jessenii (two isolates) and P. gessardii (three isolates). Casein zymography demonstrated the ability of these species to produce heat-resistant enzymes, AprX, with molecular mass of about 48 kDa during storage at 7° C for 72 h. In sterilized milk samples, the residual activity of AprX caused a considerable enhancement in the degree of protein hydrolysis, non-protein nitrogen and non-casein nitrogen contents of the samples during a two-month storage. This enhancement was slightly higher in samples containing enzyme produced by P. jessenii compared to P. gessardii ones, resulting in earlier onset of sterilized milk destabilization. Hence, this study revealed that P. jessenii and P. gessardii can play a considerable role in deterioration of Iranian commercial long-life milk.

CALPAIN ACTIVITY UNDER EXPERIMENTAL INCREASING OF DOPAMINE LEVEL

The aim of our study was to identify the activity of calpains under conditions of an experimental increase in the level of dopamine. The work was performed at three levels: in vivo, in situ, in vitro. An in situ study was carried on a model of isolated nerve endings - synaptosomes. Using casein zymography in solution with FITC-casein, it was shown that incubation of synaptosomes dopamine leads to calpains secretion into the synaptosomal medium. The dopamine ability to directly activate calpain was demonstrated by casein zymography in a gel. Incubation in an activation buffer containing dopamine instead of the classical activator, calcium chloride, led to the activation of calpain-2. An in vivo experiment was performed on Wistar rats. The experimental group was orally administered the drug L-dopa (100 mg/kg), the control group - saline was injected in the same way.

The proteolytic activity of Listeria monocytogenes HtrA

Background High temperature requirement A (HtrA) is a widely expressed chaperone and serine protease in bacteria. HtrA proteases assemble and hydrolyze misfolded proteins to enhance bacterial survival under stress conditions. Listeria monocytogenes (L. monocytogenes) is a foodborne pathogen that induces listeriosis in humans. In previous studies, it was shown that deletion of htrA in the genome of L. monocytogenes increased the susceptibility to cellular stress and attenuated virulence. However, expression and protease activity of listerial HtrA (LmHtrA) were never analyzed in detail. Results In this study, we cloned LmHtrA wildtype (LmHtrAwt) and generated a proteolytic inactive LmHtrASA mutant. Recombinant LmHtrAwt and LmHtrASA were purified and the proteolytic activity was analyzed in casein zymography and in vitro cleavage assays. LmHtrA activity could be efficiently blocked by a small molecule inhibitor targeting bacterial HtrA proteases. The expression of LmHtrA was enhanced in the stationary growth phase of L. monocytogenes and significantly contributed to bacterial survival at high temperatures. Conclusions Our data show that LmHtrA is a highly active caseinolytic protease and provide a deeper insight into the function and mechanism, which could lead to medical and biotechnological applications in the future.

Abstract 032: Soluble fms-like Tyrosine Kinase-1 (sFlt-1)/Placental Growth Factor (PlGF) Imbalance Disrupts Vascular and Uteroplacental Gelatinolytic Matrix Metalloproteinase MMP-2 and -9 and Collagenolytic MMP-1 and -7 Balance in Hypertensive Pregnancy

Preeclampsia is a complication of pregnancy manifested as hypertension-in-pregnancy (HTN-Preg) and fetal growth restriction. Placental ischemia could be an initiating event, but the linking mechanisms and tissue targets are unclear. Placental ischemia is associated with increased sFlt-1/PlGF ratio, and we have shown changes in MMPs in pregnant rat model of reduced uterine perfusion pressure (RUPP). To test the hypothesis that sFlt-1/PlGF imbalance could target vascular and uteroplacental MMPs, we tested if raising sFlt-1/PlGF ratio by infusing sFlt-1 (10 μg/kg/day) in Preg rats would increase BP and alter MMPs, and if correcting sFlt-1/PlGF ratio by infusing PlGF (20 μg/kg/day) in RUPP rats improves BP and reverses the changes in MMPs. On day 19, BP was recorded and the aorta, placenta, uterus and uterine artery were isolated for measuring MMP activity using gelatin and casein zymography and protein levels of MMPs and collagen I and IV using Western blots. BP was in Preg+sFlt 121±3 > Preg 93±7 and in RUPP+PlGF 97±4 < RUPP 118±4 mmHg. Litter size and pup weight were in Preg+sFlt < Preg and in RUPP+PlGF > RUPP. MMP-2 levels were in aorta of Preg+sFlt 3.30±0.05 < Preg 7.75±0.13, and in RUPP+PlGF 6.70±0.19 > RUPP 2.62±0.12. Similarly, MMP-2 in placenta, uterus and uterine artery and MMP-9 in all tissues were in Preg+sFlt < Preg and in RUPP+PlGF > RUPP. In contrast, MMP-1 levels were in aorta of Preg+sFlt 1.73±0.33 > Preg 0.55±0.49, and in RUPP+PlGF 0.53±0.18 < RUPP 4.04±0.30. Also, MMP-1 in placenta, uterus and uterine artery and MMP-7 in all tissues were in Preg+sFlt > Preg and in RUPP+PlGF < RUPP. The MMP-2 and -9 substrate collagen IV was in aorta of Preg+sFlt 0.16±0.00 > Preg 0.06±0.01, and in RUPP+PlGF 0.01±0.00 < RUPP 0.18±0.01. Similarly, collagen IV in placenta, uterus and uterine artery and the MMP-1 and -7 substrate collagen I in all tissues were in Preg+sFlt > Preg and in RUPP+PlGF < RUPP. Thus, sFlt-1/PlGF imbalance is a likely mechanism linking placental ischemia to decreased gelatinases, increased collagenases and improper vascular and uteroplacental remodeling. Correcting sFlt-1/PlGF balance by infusing PlGF could rectify the balance between gelatinolytic and collagenolytic MMPs and thereby restore proper vascular and uteroplacental remodeling in HTN-Preg.

Dioscorea nipponica Attenuates Migration and Invasion by Inhibition of Urokinase-Type Plasminogen Activator through Involving PI3K/Akt and Transcriptional Inhibition of NF-κB and SP-1 in Hepatocellular Carcinoma

High mortality and morbidity rates for hepatocellular carcinoma (HCC) in Taiwan primarily result from uncontrolled tumor metastasis. In our previous studies, we have reported that Dioscorea nipponica Makino extract (DNE) has anti-metastasis effects on human oral cancer cells. However, the effect of DNE on hepatoma metastasis have not been thoroughly investigated and remains poorly understood. To determine the effects of DNE on the migration and invasion in HCC cells we used a wound healing model, Boyden chamber assays, gelatin/casein zymography and Western blotting. Transcriptional levels of matrix metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator (u-PA) were detected by real-time PCR and promoter assays. In this study, DNE treatment significantly inhibited the migration/invasion capacities of Huh7 cell lines. The results of gelatin/casein zymography and Western blotting revealed that the activities and protein levels of the MMP-9 and u-PA were inhibited by DNE. Tests of the mRNA levels, real-time PCR, and promoter assays evaluated the inhibitory effects of DNE on u-PA expression in human hepatoma cells. A chromatin immunoprecipitation (ChIP) assay showed not only that DNE inhibits u-PA expression, but also the inhibitory effects were associated with the down-regulation of the transcription factors of NF-[Formula: see text]B and SP-1 signaling pathways. Western blot analysis also showed that DNE inhibits PI3K and phosphorylation of Akt. In conclusion, these results show that u-PA expression may be a potent therapeutic target in the DNE-mediated suppression of HCC invasion/migration. DNE may have potential use as a chemo-preventive agent against liver cancer metastasis.