Silk fibroin has a protective effect against high glucose induced apoptosis in HIT-T15 cells

High glucose levels induce cell death in many cell types, including pancreatic β-cells. Although protective agents against glucotoxicity have been searched for extensively, so far none have been found. In this report, we tested silk fibroin (SF) as a candidate material for antiglucotoxicity in the pancreatic β-cell (HIT-T15 cell) line. Approximately 50% of cells were killed after treatment with 80 mg/mL glucose. This reduction of cell number was recovered by the addition of SF at 50 mg/mL. SF treatment also decreased cellular reactive oxygen species (ROS) and increased proliferating cellular nuclear antigen (PCNA) immunoreactivity. In addition, TUNEL assays demonstrated that SF protects against glucose-induced apoptosis of HIT-T15 cells, suggesting that SF might protect cells from cell death by lowering cellular ROS levels. SF also induced expression of the insulin-like growth factor-1 (IGF-1) gene, and IGF-1 expression may be the cause of SF-induced protection against glucose toxicity. Taken together, these results suggest that SF could serve as a potential therapeutic agent to treat the hyperglycemia-induced death of pancreatic β-cells.     

Generation of fusion genes carrying drug resistance, green fluorescent protein, and herpes simplex virus thymidine kinase genes in a single cistron

We generated new fusion genes carrying positive- and negative-selection markers, and a reporter gene in a single reading frame. The new genes were constructed by sequentially linking the coding sequences of drug-resistance genes (hygro, or puro), a green fluorescence protein (GFP) gene (gfp), and the thymidine kinase gene (tk). The new synthetic genes (hygro/gfp/tk and puro/ gfp/tk) were inserted into retroviral vectors to test their usefulness as selective markers and reporters. The genes were functional in a positive selection in the presence of hygromycin (hygro/gfp/tk) or puromycin (puro/gfp/ tk). In addition, cells expressing the new fusion genes were clearly identifiable by their green fluorescence emitted from GFP. At the same time, these cells were sensitive to a gancyclovir treatment, allowing efficient removal of the transduced cells. The presently described synthetic genes will be valuable tools in both gene therapy and basic gene transfer studies, where positive selection of the transduced cells, monitoring gene expression, and negative selection of the transduced cells are simultaneously required.     

Highly stable enzyme precipitate coatings and their electrochemical applications

This paper describes highly stable enzyme precipitate coatings (EPCs) on electrospun polymer nanofibers and carbon nanotubes (CNTs), and their potential applications in the development of highly sensitive biosensors and high-powered biofuel cells. EPCs of glucose oxidase (GOx) were prepared by precipitating GOx molecules in the presence of ammonium sulfate, then cross-linking the precipitated GOx aggregates on covalently attached enzyme molecules on the surface of nanomaterials. EPCs-GOx not only improved enzyme loading, but also retained high enzyme stability. For example, EPC-GOx on CNTs showed a 50 times higher activity per unit weight of CNTs than the conventional approach of covalent attachment, and its initial activity was maintained with negligible loss for 200 days. EPC-GOx on CNTs was entrapped by Nafion to prepare enzyme electrodes for glucose sensors and biofuel cells. The EPC-GOx electrode showed a higher sensitivity and a lower detection limit than an electrode prepared with covalently attached GOx (CA-GOx). The CA-GOx electrode showed an 80% drop in sensitivity after thermal treatment at 50°C for 4 h, while the EPC-GOx electrode maintained its high sensitivity with negligible decrease under the same conditions. The use of EPC-GOx as the anode of a biofuel cell improved the power density, which was also stable even after thermal treatment of the enzyme anode at 50°C. The excellent stability of the EPC-GOx electrode together with its high current output create new potential for the practical applications of enzyme-based glucose sensors and biofuel cells.     

Clinical Effect of Antioxidant Glasses Containing Extracts of Medicinal Plants in Patients with Dry Eye Disease: A Multi-Center,Prospective, Randomized,Double-Blind,Placebo-Controlled Trial

Purpose

To investigate the clinical efficacy and safety of wearable antioxidant glasses containing extracts of medicinal plants in patients with mild dry eye disease (DED).

Methods

Fifty patients with mild DED were randomly assigned to wear either extracts of antioxidant medicinal plants containing (N = 25) or placebo glasses (N = 25). Patients wore the glasses for 15 min three times daily. The ocular surface disease index (OSDI) score, tear film break up time (BUT), and Schirmer’s test were evaluated and compared within the group and between the groups at baseline, 4 weeks, and 8 weeks after treatment.

Results

OSDI score and tear film BUT were significantly improved in the treatment group at 4 and 8 weeks after wearing glasses (all P < 0.001). Compared to the placebo group, the OSDI scores were significantly lower in the treatment group at 8 weeks (P = 0.007). The results of the Schirmer’s test showed significant improvement in the treatment group at 4 weeks (P = 0.035), however there were no significant differences between the other groups or within the groups. No adverse events were reported during the study.

Conclusions

Antioxidant glasses containing extracts of medicinal plants were effective in improving in DED both subjectively and objectively. Wearing antioxidants glasses might be a safe and adjunctive therapeutic option for DED.

Trial Registration

ISRCTN registry 71217488     

Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8

Alternative splicing plays an important role in gene expression by producing different proteins from a gene. Caspase-2 pre-mRNA produces anti-apoptotic Casp-2S and pro-apoptotic Casp-2L proteins through exon 9 inclusion or skipping. However, the molecular mechanisms of exon 9 splicing are not well understood. Here we show that knockdown of SRSF3 (also known as SRp20) with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRSF3 promoted exon 9 skipping. Thus we conclude that SRSF3 promotes exon 9 skipping. In order to understand the functional target of SRSF3 on caspase-2 pre-mRNA, we performed substitution and deletion mutagenesis on the potential SRSF3 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRSF3 binding site on exon 8 severely disrupted the effects of SRSF3 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRSF3 interacts with the potential SRSF3 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26 nt RNA from 5′ end of exon 8, a 33 nt RNA from 3′ end of exon 10 and a 2225 nt RNA from intron 9 did not compromise the function of SRSF3 on exon 9 splicing. Therefore we conclude that SRSF3 promotes exon 9 skipping of caspase-2 pre-mRNA by interacting with exon 8. Our results reveal a novel mechanism of caspase-2 pre-mRNA splicing.     

Autoreactive memory CD4+ T lymphocytes that mediate chronic uveitis reside in the bone marrow through STAT3-dependent mechanisms

Organ-specific autoimmune diseases are usually characterized by repeated cycles of remission and recurrent inflammation. However, where the autoreactive memory T cells reside in between episodes of recurrent inflammation is largely unknown. In this study, we have established a mouse model of chronic uveitis characterized by progressive photoreceptor cell loss, retinal degeneration, focal retinitis, retinal vasculitis, multifocal choroiditis, and choroidal neovascularization, providing for the first time to our knowledge a useful model for studying long-term pathological consequences of chronic inflammation of the neuroretina. We show that several months after inception of acute uveitis, autoreactive memory T cells specific to retinal autoantigen, interphotoreceptor retinoid-binding protein (IRBP), relocated to bone marrow (BM). The IRBP-specific memory T cells (IL-7Rα(High)Ly6C(High)CD4(+)) resided in BM in resting state but upon restimulation converted to IL-17/IFN-γ-expressing effectors (IL-7Rα(Low)Ly6C(Low)CD4(+)) that mediated uveitis. We further show that T cells from STAT3-deficient (CD4-STAT3KO) mice are defective in α4β1 and osteopontin expression, defects that correlated with inability of IRBP-specific memory CD4-STAT3KO T cells to traffic into BM. We adoptively transferred uveitis to naive mice using BM cells from wild-type mice with chronic uveitis but not BM cells from CD4-STAT3KO, providing direct evidence that memory T cells that mediate uveitis reside in BM and that STAT3-dependent mechanism may be required for migration into and retention of memory T cells in BM. Identifying BM as a survival niche for T cells that cause uveitis suggests that BM stromal cells that provide survival signals to autoreactive memory T cells and STAT3-dependent mechanisms that mediate their relocation into BM are attractive therapeutic targets that can be exploited to selectively deplete memory T cells that drive chronic inflammation.     

Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana

Four mutants that show the delayed leaf senescence phenotype were isolated from Arabidopsis thaliana . Genetic analyses revealed that they are all monogenic recessive mutations and fall into three complementation groups, identifying three genetic loci controlling leaf senescence in Arabidopsis . Mutations in these loci cause delay in all senescence parameters examined, including chlorophyll content, photochemical efficiency of photosystem II, relative amount of the large subunit of Rubisco, and RNase and peroxidase activity. Delay of the senescence symptoms was observed during both age-dependent in planta senescence and dark-induced artificial senescence in all of the mutant plants. The results indicate that the three genes defined by the mutations are key genetic elements controlling functional leaf senescence and provide decisive genetic evidence that leaf senescence is a genetically programmed phenomenon controlled by several monogenic loci in Arabidopsis . The results further suggest that the three genes function at a common step of age-dependent and dark-induced senescence processes. It is further shown that one of the mutations is allelic to ein2-1 , an ethylene-insensitive mutation, confirming the role of ethylene signal transduction pathway in leaf senescence of Arabidopsis .     

Calcium/calmodulin inhibition of the Arabidopsis BRASSINOSTEROID-INSENSITIVE 1 receptor kinase provides a possible link between calcium and brassinosteroid signalling

The receptor kinase BRI1 (BRASSINOSTEROID-INSENSITIVE 1) is a key component in BR (brassinosteroid) perception and signal transduction, and has a broad impact on plant growth and development. In the present study, we demonstrate that Arabidopsis CaM (calmodulin) binds to the recombinant cytoplasmic domain of BRI1 in a Ca2+-dependent manner in vitro. In silico analysis predicted binding to Helix E of the BRI1 kinase subdomain VIa and a synthetic peptide based on this sequence interacted with Ca2+/CaM. Co-expression of CaM with the cytoplasmic domain of BRI1 in Escherichia coli strongly reduced autophosphorylation of BRI1, in particular on tyrosine residues, and also reduced the BRI1-mediated transphosphorylation of E. coli proteins on tyrosine, threonine and presumably serine residues. Several isoforms of CaM and CMLs (CaM-like proteins) were more effective (AtCaM6, AtCaM7 and AtCML8, where At is Arabidopsis thaliana) than others (AtCaM2, AtCaM4 and AtCML11) when co-expressed with BRI1 in E. coli. These results establish a novel assay for recombinant BRI1 transphosphorylation activity and collectively uncover a possible new link between Ca2+ and BR signalling.     

Turnover characteristics in continuous l-lysine fermentation

The turnover characteristics of a microbial bioreactor were comparatively investigated as a closed (batch) and a continuous stirred tank reactor (CSTR) open system, using a 2-l fermentor. Corynebacterium glutamicum (ATCC 21544) was chosen as the microorganism since it has the ability to produce l-lysine. Parameters measured were l-lysine production rates, glucose consumption rates and biomass production rates as a function of dilution rate, bioreactor volume and biomass concentration. The modes of microbial cell behaviour under steady-state and transition-state conditions were examined. Investigations on scaling properties of the CSTR system were also aimed at comparing scaling or allometry of metabolic rates in organisms that are also open energy dissipative systems.This investigation was first presented at the 10th Dechema-Jahrestagung der Biotechnologen, 1–3 June 1992, Karlsruhe, Germany