Antiapoptotic effects of Phe140Asn,a novel human granulocyte colony-stimulating factor mutant in H9c2 rat cardiomyocytes

Granulocyte colony-stimulating factor (G-CSF) is used for heart failure therapy and promotes myocardial regeneration by inducing mobilization of bone marrow stem cells to the injured heart after myocardial infarction; however, this treatment has one weakness in that its biological effect is transient. In our previous report, we generated 5 mutants harboring N-linked glycosylation to improve its antiapoptotic activities. Among them, one mutant (Phe140Asn) had higher cell viability than wild-type hG-CSF in rat cardiomyocytes, even after treatment with an apoptotic agent (H₂O₂). Cells treated with this mutant significantly upregulated the antiapoptotic proteins, and experienced reductions in caspase 3 activity and PARP cleavage. Moreover, the total number of apoptotic cells was dramatically lower in cultures treated with mutant hG-CSF. Taken together, these results suggest that the addition of an N-linked glycosylation was successful in improving the antiapoptotic activity of hG-CSF, and that this mutated product will be a feasible therapy for patients who have experienced heart failure. [BMB Reports 2012; 45(12): 742-747]     

Enhancement of anti-tumor activity of gamma-irradiated silk fibroin via immunomodulatory effects

Silk fibers have proven to be effective in many clinical applications as well as for clothing. In addition to the substantial effect of silk fibers, the present study was conducted to explore its importance in a new dimension to reinforce the effects of its physiological function regarding anti-tumor activity and immune response with gamma-irradiated silk fibroin (GISF). The cytotoxicity results showed that pre-treatment of GISF in the mouse peritoneal macrophages (MPM) indicated a higher proliferative effect than that of non-irradiated silk fibroin (NISF) in a concentration-dependent manner. Based on the cytotoxicity result of MPM, GISF (50 and 150 kGy) was selected for an ex vivo study in an animal (C57BL6) system and evaluated about whether the non-specific immune response was also related to GISF. GISF (50 and 150 kGy) augmented immune responsiveness via activation of NK cells, T lymphocytes proliferation, NO production, and cytokine level, such as IL-6, IL-2, IL-12, IFN-γ, TNF-α, as compared with NISF, which strongly suggested that GISF significantly augmented an important element of all aspects of the innate and adaptive immune system. Therefore, from these results, it seems likely that the GISF will play a potent role in eliciting the effect of the non-specific immune response and anti-tumor activity as a value-added product in the medical industry.     

Effect of the weak Ca(2+)-binding site of subtilisin J by site-directed mutagenesis on heat stability.

The functional role of the negatively charged amino acid residue in subtilisin J from Bacillus stearothermophilus has been investigated by site-directed mutagenesis. Glu-195 located at the weak Ca2+-binding site was replaced with Gln to examine the role of Glu-195 in the heat stability of subtilisin J. Mutant enzyme was expressed in Bacillus subtilis and was purified from the culture supernatant. When the mutant enzyme was expressed at 37 degrees C in the presence of 2mM calcium chloride, the pattern of enzyme production was quite different from that of wild-type. The purified Gln-195 mutant enzyme was analyzed with respect to optimal temperature, optimal pH, and heat stability. The mutation was found to decrease the heat stability but not catalytic efficiency (kcat/Km) and optimal pH. These results demonstrate the important role of the negatively charged side chains at the weak Ca(2+)-binding site in the heat stability of subtilisin.     

Regulation of proteins related to melanogenesis by heartwood extract of<Emphasis Type="Italic">Morus bombycis</Emphasis> using proteome analysis

The ability of heartwood extract ofMorus bombycis in controlling the melanogenesis in B16 melanoma was investigated. An inhibitory effect was observed when the dose concentration was less than 10 mg/L. When the dose was 10 mg/L, the extracellular melanin accumulated was only 5% of the control. Proteome analysis with 2-D PAGE and MALDI-TOF showed that there were various proteins involved in inhibitory melanogenesis. It seems that down-regulation of TRP2, vacuolar protontranslocating ATPase 100 kDa subunit isoform a1-I and V-ATPase E2 subunit were involved in melanosome development and regulation that could reduce melanin synthesis.     

A Skate Skin Hydrolysate Restores Cognitive Function in 5XFAD Alzheimer Disease Mice Model by Suppressing Amyloid-β Accumulation via Upregulation of ERK-CREB

International Journal of Peptide Research and Therapeutics - Alzheimer’s disease is a neurodegenerative disease hallmarked by gradual loss of neurons. The prevalence of such degenerative...     

Elicitation kinetics of taxane production in suspension cultures of Taxus baccata Pendula

Methyl jasmonate increased taxane production in suspension cultures of Taxus baccata Pendula. Time course changes of taxane production after methyl jasmonate addition were different from normal kinetics without elicitation. Baccatin III and 10-deacetyl baccatin III were detected first and paclitaxel, 10-deacetyl taxol and cephalomanine followed each other in sequence. Paclitaxel was not a dead-end metabolite.     

The leader sequence of streptokinase is responsible for its post-translational carboxyl-terminal cleavage

When the expression of streptokinase from two tac promoter-controlled expression vectors, one of these deleted a putative leader sequence of streptokinase and the other not, was compared, both normal and degraded streptokinase were detected in proteins expressed from the leader-contained vector, but only normal streptokinase was detected from the leader-deleted vector. These findings indicate that the characteristic carboxyl-terminal cleavage of streptokinase is correlated with its leader sequence and occurs during the defective secretion. The homogeneous preparation of streptokinase was facilitated by expressing from this leader-deleted vector.     

Purification and characterization of angiotensin I converting enzyme (ACE) inhibitory peptides from Alaska pollack (Theragra chalcogramma) skin

Proteolytic digestion of gelatin extracts from Alaska Pollack (Theragra chalcogramma) skin brings about a high angiotensin I converting enzyme (ACE) inhibitory activity. Gelatin extracts were hydrolyzed by serial protease-treatments in the order of Alcalase, pronase E, and collagenase using a three-step recycling membrane reactor. Fragments arising from the third step were composed of peptides ranging from 0.9 to 1.9 kDa and responsible for ACE inhibitory activity. Catalytically active two peptides were separated by the consecutive chromatographic methods including gel filtration, ion-exchange chromatography, and reverse-phase high performance liquid chromatography. The isolated peptides were composed of Gly-Pro-Leu and Gly-Pro-Met and showed IC₅₀ values of 2.6 and 17.13 μM, respectively. These results suggested that Gly-Pro-Leu would be useful as a new antihypertensive agent.     

<Emphasis Type="Italic">In Planta</Emphasis> Measurements of Na<Superscript>+</Superscript> Using Fluorescent Dye CoroNa Green

Fluorescent indicators of Na⁺ are valuable tools for nondestructive monitoring of its spatial and temporal distribution in plants. We tested whether CoroNa Green fluorescent dye, a newly developed sodium indicator, is suitable for measuring relative concentrations in planta. To determine the ideal conditions for its use, we incubated NaCl-pretreated Arabidopsis thaliana seedlings with different concentrations of CoroNa Green and visualized fluorescence in each organ with a fluorescein isothiocyanate filter. When 50 μM of dye was applied, fluorescence was distributed more uniformly and intensely in the root tips than in other tissues. Under those conditions, fluorescence gradually increased in the root tips when Na⁺ was bound to CoroNa Green for concentrations up to 100 mM NaCl. Confocal fluorescence microscopy revealed that when Arabidopsis seedlings were incubated with the same concentration of NaCl, the sos1 mutant had much stronger fluorescence than the wild type. This report is the first to describe the properties of CoroNa Green for measuring Na⁺ content in intact plants and demonstrates the usefulness of this technique for investigating the mechanism of Na⁺ homeostasis.