Real-time electro-mechano-acoustic imaging for monitoring interactions between trypsin and different inhibitors in articular cartilage.

The purpose of this study was to observe the real-time interactions between trypsin and various inhibitors in articular cartilage in vitro using a novel electro-mechano-acoustic imaging method. Monitored in real-time, articular cartilage specimens from bovine patellae were first treated with trypsin to reach half proteoglycan depletion (Phase I), then the trypsin solution was replaced with (i) physiological saline buffer (PS), (ii) fetal bovine serum (FBS), (iii) protease inhibitor cocktail (PI) and (iv) 10% formalin (F), respectively, to observe their effects on residual digestion (Phase II). Ultrasound radio frequency signals from the articular cartilage were used to form a M-mode image, where the interface between trypsin digested and intact cartilage tissues could be observed with an additional echo generated. The inhibition time, the digestion depth and digestion fraction were measured for each specimen. The results showed that the dilution of trypsin using saline solution was not sufficient to stop the enzyme action instantly. Although groups FBS and PI had a similar inhibition time of approximately 1.5 h, their digestion depth was obviously different (0.25±0.03 and 0.06±0.06 mm, respectively). In contrast, formalin only took <30 min to stop the trypsin digestion with almost no further digestion. The results demonstrated that the current system was capable of monitoring the trypsin digestion and inhibition process in real time. Also, different chemicals affected the residual trypsin digestion to different degrees.

Pain Controlling and Cytokine-regulating Effects of Lyprinol, a Lipid Extract of Perna Canaliculus, in a Rat Adjuvant-induced Arthritis Model.

Using an adjuvant-induced arthritis rat model, we investigated the effects of a lipid extract of Perna canaliculus (Lyprinol(R)) on pain. Radiological examinations, as well as levels of pro- and anti-inflammatory (AI) cytokines, were measured aiming to provide independent objective data to the pain controlling investigation. We confirmed the ability of Lyprinol(R) to control pain at the initial phase of its administration; with similar efficacy to that observed with Naproxen. The pain scores slowly increased again in the group of rats treated with Lyprinol(R) after day 9-14. The Naproxen-treated rats remained pain-free while treated. Both Naproxen and Lyprinol(R) decreased the levels of the pro-inflammatory cytokines TNF-alpha and IFN-gamma, and increased that of IL-10. Extra-virgin olive oil was ineffective on cytokine secretion. Rats treated with Lyprinol(R) were apparently cured after 1 year. This study confirms the AI efficacy of this lipid extract of P. canaliculus, its initial analgesic effect, its perfect tolerance and its long-term healing properties.

Detecting nitrated proteins by proteomic technologies.

Nitration is a posttranslational modification of tyrosine residues of proteins mediated by peroxynitrite (ONOO(-)). It commonly occurs in neurological and pathological disorders, which involve nitric oxide (NO)-mediated oxidative stress. Nitration of tyrosine or tyrosyl groups of a protein modulates protein function and initiates signal transduction pathways, which lead to alternation of cellular metabolism and functions. Because of its apparent significance, there is an increasing urge to identify nitrated proteins as a bridge to expand our understanding of their involvement in different biological processes. This chapter describes strategies that could be used for rapid screening and detection of nitrated proteins, subsequent resolution, and identification of nitrated proteins and peptides using proteomic technologies. These include two-dimensional gel electrophoresis coupled with Western blotting and matrix-assisted laser desorption/ionization time of flight mass spectrometry, as well as liquid chromatography-linked tandem mass spectrometry.

Sodium nitroprusside affects the level of photosynthetic enzymes and glucose metabolism in Phaseolus aureus (mung bean).

Nitric oxide (NO) is an important signaling molecule in plants. The present study aims to investigate the downstream signaling pathways of NO in plants using a proteomic approach. Phaseolus aureus (mung bean) leaf was treated with sodium nitroprusside (SNP), which releases nitric oxide in the form of nitrosonium cation (NO+) upon light irradiation. Changes in protein expression profiles of the SNP treated mung bean leaf were analyzed by two-dimensional gel electrophoresis (2-DE). Comparison of 2-DE electropherograms revealed seven down-regulated and two up-regulated proteins after treatment with 0.5 mM SNP for 6 h. The identities of these proteins were analyzed by a combination of peptide mass fingerprinting and post-source decay using a matrix-assisted-laser-desorption-ionisation-time-of-flight (MALDI-TOF) mass spectrometer. Six out of these nine proteins found are involved in either photosynthesis or cellular metabolism. We have taken our investigation further by studying the effect of NO+ on glucose contents in mung bean leaves. Our results clearly demonstrated that NO+ rapidly and drastically decrease the amount of glucose in mung bean leaves. Moreover, four out of nine of these proteins are chloroplastic isoforms. These results suggested that chloroplasts might be one of the main sub-cellular targets of NO in plants.

Identification of the heterogeneous nuclear ribonucleoprotein A2/B1 as the antigen for the gastrointestinal cancer specific monoclonal antibody MG7.

MG7 is an early gastrointestinal cancer specific monoclonal antibody. It can detect gastric cancer with high sensitivity and specificity. However, the target antigen for MG7 has not been identified. Western blot analysis revealed that the MG7 antibody reproducibly recognized two approximately 35 kDa proteins in the total cell lysates of human gastric carcinoma cell lines KATO III and MKN-45. Using a proteomic approach, we identified these MG7 immunoreactive proteins as the human heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1). Western blot analysis of nuclear and cytosolic fraction of KATO III cells using either MG7 or hnRNP A2/B1 antibodies confirmed that the target antigen is located exclusively in the nucleus. With the use of archival samples, we also found that the level of hnRNP A2/B1 protein was increased in gastric cancer tissues (4 out of 5 patients), when compared to their corresponding matching normal stomach tissue.

Proteomic identification of plant proteins probed by mammalian nitric oxide synthase antibodies.

Several studies suggest that a mammalian-like nitric oxide synthase (NOS) exists in plants. Researchers have attempted to verify its presence using two approaches: (i) determination of NOS functional activity and (ii) probing with mammalian NOS antibodies. However, up to now, neither a NOS-like gene nor a protein has been found in plants. While there is still some controversy over whether the NOS functional activity seen is due to nitrate reductase, using the mammalian NOS antibodies in western blot analysis, several groups have reported the presence of immunoreactive protein bands in plant homogenates. Based on these results, immunohistochemical studies using these antibodies have also been used to localize NOS in plant tissues. However, plant NOS has never been positively identified or characterized. Thus, we used a proteomic approach to verify the identities of plant proteins that cross-reacted with the mammalian NOS antibodies. Proteins extracted from maize (Zea mays L.) embryonic axes were separated by two-dimensional gel electrophoresis and subjected to western blot analysis with the mammalian neuronal NOS and inducible NOS antibodies. Twenty immunoreactive protein spots recognized on a corresponding Coomassie blue-stained two-dimensional gel were subjected to tryptic digestion, followed by identification using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Fifteen proteins were successfully identified and they have described functions that are unrelated to NO metabolism. The remaining five proteins could not be identified. The amino acid sequences of these identified proteins and those used to raise the antibodies were aligned. However, no homologous region could be found. Our results demonstrate that the mammalian NOS antibodies recognize many NOS-unrelated plant proteins. Therefore, it is inappropriate to infer the presence of plant NOS using this immunological technique.