Takashi Baba

Exogenous control of mammalian gene expression through modulation of RNA self-cleavage.

Recent studies on the control of specific metabolic pathways in bacteria have documented the existence of entirely RNA-based mechanisms for controlling gene expression. These mechanisms involve the modulation of translation, transcription termination or RNA self-cleavage through the direct interaction of specific intracellular metabolites and RNA sequences. Here we show that an analogous RNA-based gene regulation system can effectively be designed for mammalian cells via the incorporation of sequences encoding self-cleaving RNA motifs into the transcriptional unit of a gene or vector. When correctly positioned, the sequences lead to potent inhibition of gene or vector expression, owing to the spontaneous cleavage of the RNA transcript. Administration of either oligonucleotides complementary to regions of the self-cleaving motif or a specific small molecule results in the efficient induction of gene expression, owing to inhibition of self-cleavage of the messenger RNA. Efficient regulation of transgene expression is shown in a variety of mammalian cell lines and live animals. In conjunction with other emerging technologies, this methodology may be particularly applicable to the development of gene regulation systems tailored to any small inducer molecule, and provide a novel means of biological sensing in vivo that may have an important application in the regulated delivery of protein therapeutics.

Multivariate Analyses of Inflammatory Cytokines in Eyes with Branch Retinal Vein Occlusion: Relationships to Bevacizumab Treatment

PURPOSE To characterize the differential expression of intraocular inflammatory cytokines in eyes with branch retinal vein occlusion (BRVO) and to assess their roles as prognostic determinants of BRVO. METHODS A prospective cohort study of 38 eyes with BRVO. Aqueous humor samples were collected just before the intravitreal injection of bevacizumab and were assessed for 18 cytokines, chemokines, and growth factors. For control, aqueous humor was collected from 28 eyes before cataract surgery. RESULTS In the aqueous of eyes with BRVO, the IL-23, IL-8, IL-6, IL-15, IL-12, and IL-17 levels were significantly higher than that in control eyes. Pretreatment visual acuity was significantly correlated with the concentrations of IL-8, IL-10, IL-2, IL-1β, IL-5, IL-6, IL-23, IL-4, MCP-1, IL-1α, IL-12, IL-13, IFN-γ, and IL-15. The pretreatment nonperfused area (NPA) was significantly correlated with the concentrations of IL-8, IL-2, MCP-1, and IL-6. Logistic regression analyses revealed significant associations between the BRVO and the concentrations of IL-8, IL-23, IL-12, IL-15, IL-10, IL-1β, and IL-13. IL-8 had the highest odds ratio (OR) and was significantly associated with NPA, central retinal thickness (CRT), and visual acuity. Bevacizumab treatment significantly improved visual acuity and CRT after 1 month. Refractoriness to bevacizumab (defined as CRT recovery 1 month after treatment by <90%) was significantly associated with the IL-12 level. CONCLUSIONS Of the induced cytokines in eyes with BRVO, IL-8 was the most significantly associated with the disease parameters of BRVO. IL-12 is most likely a factor that blocks the effect of bevacizumab treatment. (www.umin.ac.jp/ctr number, UMIN000003854.).

Hepatocyte growth factor activator is a serum activator of single-chain precursor macrophage-stimulating protein

Macrophage‐stimulating protein (MSP) is a plasma protein that circulates as a single‐chain proform. It acquires biological activity after proteolytic cleavage at the Arg483–Val484 bond, a process in which serum and cell surface serine proteinases have been implicated. In this article, we report that hepatocyte growth factor activator (HGFA), a serum proteinase which activates hepatocyte growth factor in response to tissue injury, may have a critical role in the activation of pro‐MSP. In vitro analysis has revealed that human HGFA efficiently cleaves human pro‐MSP at the physiological activation site without further degradation, resulting in biologically active MSP, as measured by the chemotactic response and MSP‐induced morphological change of peritoneal macrophages. The processing of pro‐MSP by HGFA is 10‐fold more efficient than processing by factor XIa. To search for a role of HGFA in pro‐MSP activation, we analyzed the processing of mouse pro‐MSP in sera from HGFA‐knockout (HGFA−/−) mice. The proform of MSP was the predominant molecular form in the plasma of both wild‐type and HGFA−/− mice. In wild‐type sera, endogenous pro‐MSP was progressively converted to the mature two‐chain form during incubation at 37 °C. However, this conversion was significantly impaired in sera from HGFA−/− mice. The addition of recombinant HGFA to HGFA‐deficient serum restored pro‐MSP convertase activity in a dose‐dependent manner, and a neutralizing antibody to HGFA significantly reduced the conversion of pro‐MSP in wild‐type serum. Moreover, initial infiltration of macrophages into the site of mechanical skin injury was delayed in HGFA−/− mice. We suggest that HGFA is a major serum activator of pro‐MSP.

Ethyl esterification of docosahexaenoic acid in an organic solvent-free system with immobilized Candida antarctica lipase

Ethyl docosahexaenoate (EtDHA) is regarded as a potentially useful pharmaceutical substance on account of its beneficial physiological activities. We attempted the ethyl esterification of docosahexaenoic acid (DHA) in an organic solvent-free system using Candida antarctica lipase, which acts strongly on DHA and ethanol. Esterification of 88% was attained by shaking a mixture of DHA/ethanol (1:1, mol/mol) and 2 wt% immobilized C. antarctica lipase at 30 degrees C for 24 h. However, even in the presence of an excess amount of ethanol, the extent of esterification could not be raised above 90%. To attain a higher level of esterification, a two-step reaction was found to be effective. The first step was performed in a mixture of DHA/ethanol (1:1, mol/mol), and the reaction mixture was then dehydrated. In the second step, the resulting mixture was shaken at 30 degrees C for 24 h with 5 molar equivalents of ethanol against the remaining DHA using 2 wt% immobilized lipase. By means of this two-step procedure, 96% esterification was attained. Repetition of the first and second reactions showed that the immobilized lipase was reusable for at least 50 cycles. In addition, DHA remaining in the second-step reaction mixture was removed by a conventional alkali refining process, giving purified EtDHA with a high yield.

Membrane-Bound Serine Protease Inhibitor HAI-1 Is Required for Maintenance of Intestinal Epithelial Integrity

Hepatocyte growth factor activator inhibitor type 1 (HAI-1), encoded by the serine protease inhibitor Kunitz type 1 (SPINT1) gene, is a membrane-bound serine protease inhibitor expressed in epithelial tissues. Mutant mouse models revealed that HAI-1/SPINT1 is essential for placental labyrinth formation and is critically involved in regulating epidermal keratinization through interaction with its cognate cell surface protease, matriptase. HAI-1/SPINT1 is abundantly expressed in both human and mouse intestinal epithelium; therefore, we analyzed its role in intestinal function using mice with intestinal epithelial cell-specific deletion of Spint1 generated by interbreeding mice carrying Spint1(LoxP) homozygous alleles with transgenic mice carrying the Cre recombinase gene controlled by the intestine-specific Villin promoter. Although the resulting mice had normal development and appearance, crypts in the proximal aspect of the colon, including the cecum, exhibited histologic abnormalities and increased apoptosis and epithelial cell turnover accompanied by increased intestinal permeability. Distended endoplasmic reticula were observed ultrastructurally in some crypt epithelial cells, indicative of endoplasmic reticular stress. To study the role of HAI-1/SPINT1 in mucosal injury, we induced colitis by adding dextran sodium sulfate to the drinking water. After dextran sodium sulfate treatment, intestine-specific HAI-1/SPINT1-deficient mice had more severe symptoms and a significantly lower survival rate relative to control mice. These results suggest that HAI-1/SPINT1 plays an important role in maintaining colonic epithelium integrity.

Loss of membrane‐bound serine protease inhibitor HAI‐1 induces oral squamous cell carcinoma cells' invasiveness

A loss of balance between cell membrane‐associated proteases and their inhibitors may underlie cancer invasion and metastasis. We analysed the roles of a membrane‐ associated serine protease inhibitor, HAI‐1, in oral squamous cell carcinoma (OSCC). While membranous HAI‐1 was widely observed in cancer cells of human OSCC tissues, this was significantly reduced at the infiltrative invasion front. In vitro, HAI‐1 was detected in all eight OSCC cell lines examined, in which its cognate membrane protease, matriptase was also expressed. HAI‐1 expression knock‐down (KD) in OSCC lines, SAS and HSC‐3, reduced the growth of both lines in vitro but significantly enhanced SAS tumourigenicity in vivo, which was accompanied by histological changes suggestive of the epithelial‐mesenchymal transition. Both HAI‐1‐KD lines also exhibited significantly enhanced migratory capability, and membrane‐associated but not truncated HAI‐1 was required to rescue this phenotype. Other OSCC lines (HSC‐2, Sa3, Ca9‐22) also showed enhanced migration in response to HAI‐1 KD. The enhanced migration is partly attributed to dysregulation of matriptase, as simultaneous matriptase KD alleviated the migration of HAI‐1‐KD cells. HAI‐1 deficiency also altered the expression of CD24, S100A4, CCND2 and DUSP6, all of which are involved in tumour progression. While matriptase was involved in the increased CD24 expression associated with HAI‐1 deficiency, the protease appeared to be not responsible for the altered expression of other genes. Therefore, a matriptase‐independent mechanism for the invasiveness associated with HAI‐1 KD is also present. Together, these observations suggest that HAI‐1 has a crucial suppressive role in OSCC cell invasiveness. Copyright

Metabolism and calcium antagonism of sodium alginate oligosaccharides

Sodium alginate oligosaccharides (NaAOs) consisting of a mixture of eight oligosaccharides have previously been reported to lower blood pressure. We investigated in this study the excretion of NaAOs into the urine or feces, and attempted to elucidate the mechanism for lowering blood pressure by using isolated mesenteric arteries from the rabbit. The recovery rate of P8, which is the main component of NaAOs, was 5.2% and 58.9% over 48 hours in the urine and feces, respectively. The mechanism for lowering blood pressure appeared to be NaAOs having calcium antagonist activity, especially voltage-operated calcium channels. Our results suggest that NaAOs are substantially excreted into the feces, although some of them may be absorbed internally, exerting antagonist activity towards the calcium channels, especially voltage-operated calcium channels.

Autoxidation Kinetic Analysis of Docosahexaenoic Acid Ethyl Ester and Docosahexaenoic Triglyceride with Oxygen Sensor

The application of ω-3 polyunsaturated fatty acids (PUFAs) as food additives is restricted by their chemically quite reactive properties. However, quantitative analyses of the oxidative kinetics of PUFAs are very few compared to other studies on food chemistry. In this study, the autoxidation kinetics of ethyl docosahexaenoate (DHAEE), docosahexaenoic triglyceride (DHA oil), and emulsified DHA oil were investigated with an oxygen sensor. The autocatalytic reaction rate constants for DHAEE, DHA oil, and the emulsified DHA oil with 20%(w/v) GA, 20% SSPS, or 20% SSPS containing 5% soy protein were obtained at 35, 50, and 70°C. A plot of the natural logarithm of the frequency factor, ln ka0, vs. the activation energy, Ea, demonstrated that ln ka0 against Ea fitted well with a single straight line both for the data from this study and for other reported results. This implies that the chemical compensation relationship holds between k a0 and E a for PUFA and emulsified DHA oil.

Changes in the Airborne Bacterial Community in Outdoor Environments following Asian Dust Events

Bacterial abundance and community compositions have been examined in aeolian dust in order to clarify their possible impacts on public health and ecosystems. The influence of transcontinentally transported bacterial cells on microbial communities in the outdoor environments of downwind areas should be determined because the rapid influx of a large amount of bacterial cells can disturb indigenous microbial ecosystems. In the present study, we analyzed bacteria in air samples (approximately 100 m3 d−1) that were collected on both Asian dust days and non-Asian dust days over 2 years (between November 2010 and July 2012). Changes in bacterial abundance and community composition were investigated based on their 16S rRNA gene amount and sequence diversity. Seasonal monitoring revealed that airborne bacterial abundance was more than 10-fold higher on severe dust days, while moderate dust events did not affect airborne bacterial abundance. A comparison of bacterial community compositions revealed that bacteria in Asian dust did not immediately disturb the airborne microbial community in areas 3,000–5,000 km downwind of dust source regions, even when a large amount of bacterial cells were transported by the atmospheric event. However, microbes in aeolian dust may have a greater impact on indigenous microbial communities in downwind areas near the dust source. Continuous temporal and spatial analyses from dust source regions to downwind regions (e.g., from the Gobi desert to China, Korea, Japan, and North America) will assist in estimating the impact of atmospherically transported bacteria on indigenous microbial ecosystems in downwind areas.

Detection of Alginate Oligosaccharides from Mollusks

We attempted in this study to detect alginate oligosaccharides (AO) from mollusks. The samples used were digestive organs taken from turban shells and abalones which commonly ate brown algae. High-performance liquid chromatography (HPLC) and negative-ion electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) analyses were used to confirm the presence of AO. Samples spiked with AO resulted in observable peaks where the HPLC area was increased. The highest content was estimated to be 401.8 mg/100 g of digestive organ. The product-ion data derived from AO molecular weight were detected at a constant interval by Q-TOF MS/MS analysis. These findings indicate that AO was present in the digestive organs of mollusks.