the-determination-protease-specificity-mouse-tissue-extracts-maldi

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

Nihon Pharmaceutical University

We previously developed novel liposomal nanobubbles (Bubble liposomes [BL]) that oscillate and collapse in an ultrasound field, generating heat and shock waves. We aimed to investigate the feasibility of cancer therapy using the combination of BL and ultrasound. In addition, we investigated the anti-tumor mechanism of this cancer therapy. Colon-26 cells were inoculated into the flank of BALB/c mice to induce tumors. After 8 days, BL or saline was intratumorally injected, followed by transdermal ultrasound exposure of tumor tissue (1 MHz, 0-4 W/cm2 , 2 min). The anti-tumor effects were evaluated by histology (necrosis) and tumor growth. In vivo cell depletion assays were performed to identify the immune cells responsible for anti-tumor effects. Tumor temperatures were significantly higher when treated with BL + ultrasound than ultrasound alone. Intratumoral BL caused extensive tissue necrosis at 3-4 W/cm2 of ultrasound exposure. In addition, BL + ultrasound significantly suppressed tumor growth at 2-4 W/cm2 . In vivo depletion of CD8+ T cells (not NK or CD4+ T cells) completely blocked the effect of BL + ultrasound on tumor growth. These data suggest that CD8+ T cells play a critical role in tumor growth suppression. Finally, we concluded that BL + ultrasound, which can prime the anti-tumor cellular immune system, may be an effective hyperthermia strategy for cancer treatment.

Tumor growth suppression by the combination of nanobubbles and ultrasound.

Recombinant tissue-type plasminogen activator (rt-PA) is effective and widely used in the treatment of acute ischemic stroke (AIS). However, symptomatic intracranial hemorrhage (ICH), an adverse reaction of rt-PA, is known to occur depending on underlying diseases and rt-PA doses, and to occur more frequently with a greater delay from stroke onset until initiation of rt-PA. Therefore, limitations on the use of rt-PA, such as having to be started within 4.5 h of stroke onset, mean that rt-PA is only indicated in some stroke patients. However, the number of patients in whom rt-PA is indicated could increase if symptomatic ICH induced by rt-PA could be reduced. Therefore, we believe that, if the incidence of adverse reactions such as ICH could be reduced by using lower rt-PA doses together with ultrasound (US), the number of patients eligible for rt-PA treatment would increase. In other words, we hypothesized that, if thrombolysis can be accelerated by US, then recanalization rates similar to currently used doses of rt-PA can be achieved at reduced rt-PA doses. Therefore, to investigate to what extent US enhances the thrombolytic efficacy of rt-PA, the relationship between acceleration of rt-PA thrombolysis and US acoustic intensity was quantitatively evaluated in an in vitro bovine thrombus model. It was found that, within a range of US output that is noninvasive in humans, the combined use of US can increase thrombolytic activity up to 2.5 times more than with rt-PA alone. These findings suggest that US can greatly reduce the required doses of rt-PA.

Ultrasound Acceleration of rt-PA Thrombolysis Depends on Acoustic Intensity.

For improved thrombolysis therapy based on ultrasound irradiation, researchers and practitioners would strongly benefit from an easy and efficient in vitro assay system of thrombolysis activity involving irradiated ultrasound. For the present study, we designed a new in vitro sonothrombolysis assay system using a sheet-type clot.

An In Vitro Assay for Sonothrombolysis Based on the Spectrophotometric Measurement of Clot Thickness.

Proteolysis constitutes a major post-translational modification. For example, proteases regulate the activation or inactivation of various proteins, such as enzymes, growth factors, and peptide hormones. Proteases have substrate specificity, and protease expression regulates the specific and regional activation or inactivation of several functional proteins.

Article	Year
Tumor growth suppression by the combination of nanobubbles and ultrasound. Cancer science\| PubMed ID: 26707839	2016
Ultrasound Acceleration of rt-PA Thrombolysis Depends on Acoustic Intensity. Biological & pharmaceutical bulletin\| PubMed ID: 27773879	2017
An In Vitro Assay for Sonothrombolysis Based on the Spectrophotometric Measurement of Clot Thickness. Journal of ultrasound in medicine : official journal of the American Institute of Ultrasound in Medicine\| PubMed ID: 28150461	2017
Identification of Protease Specificity Using Biotin-Labeled Substrates. The open biochemistry journal\| PubMed ID: 28567123	2017

Yoshikazu Sawaguchi

Biography

JoVE Journal Publications

Other Publications