Immunostimulating Effects of Extract of Acanthopanax sessiliflorus

As malfunction/absence of immune cells causes a variety of immunosuppressive disorders and chemical synthetic drugs for curing these diseases have many adverse effects, vigorous studies are being conducted. The Acanthopanax family has been used as traditional medicines for gastric ulcer, diabetes, etc. and culinary materials in East-South Asia. In this study, the immunostimulating properties of A. sessiliflorus were evaluated. A. sessiliflorus increased not only the splenocyte number but also immune-related cytokines such as TNF-α. However, it could not upregulate the expressions of IFN-γ and IL-2. A. sessiliflorus increased the swimming time, and comparison of organ weights relative to body weights for immune-related organs such as the spleen and thymus after a forced swim test showed that it could recover the spleen and thymus weights. It also increased the expression of TNF-α and slightly increased the concentration of IFN-γ but not IL-2. From the results, we concluded that as A. sessiliflorus has not only a host defense effect but also a stress-ameliorating property, further study it will be a promising material of immunostimulating material.     

Traceless and Site-specific Attachment of Proteins onto Solid Supports

Many experimental approaches in biology and biophysics, as well as applications in diagnosis and drug discovery, require proteins to be immobilized on solid supports. Protein microarrays, for example, provide a high-throughput format to study biomolecular interactions. The technique employed for protein immobilization is a key to the success of these applications. Recent biochemical developments are allowing, for the first time, the selective and traceless immobilization of proteins generated by cell-free systems without the need for purification and/or reconcentration prior to the immobilization step.     

Localization of aquaporin-7 in rat and mouse kidney using RT-PCR, immunoblotting, and immunocytochemistry

To establish the segmental, cellular, and subcellular localization of AQP7 in rat and mouse kidney, we used RT-PCR, immunocytochemical, and immunoblotting approaches. RT-PCR of rat and mouse kidney zones revealed AQP7 mRNA in cortex and outer stripe of the outer medulla. RT-PCR on microdissected nephron segments revealed AQP7 mRNA in proximal convoluted and straight tubules. Immunoblotting using peptide-derived rabbit antibodies to either rat or mouse AQP7 revealed a 28-kDa band in kidney and testes from rat and mouse, respectively. Immunocytochemistry revealed strong AQP7 labeling of segment 3 proximal tubules and weaker labeling of proximal convoluted tubules in both rat and mouse kidneys. The labeling was almost exclusively confined to the brush border with no basolateral labeling. No labeling was observed of thin descending limbs or collecting duct. Immunolabeling controls were negative. The presence of AQP7 in the proximal tubule brush border indicates a role of AQP7 in proximal tubule water reabsorption.     

Acquisition of heat shock tolerance by regulation of intracellular redox states

In the yeast Saccharomyces cerevisiae, a mild heat treatment strongly induces Hsp104p which provides acquisition of thermotolerance. The mechanism by which Hsp104p protects cells from the severe heat shock has not yet been completely elucidated. In this study, a pivotal role of Hsp104p as an efficient scavenger of the reactive oxygen species (ROS) is investigated. In our previous study, we reported that Hsp104p acted as a regulator in the mitochondrial respiration pathway. In this report, the recombinant wild-type and hypersensitive ras mutants (ira2Delta) with the extrachromosomal plasmids wild-type and mutant hsp104 genes were studied. The resulting strains successfully expressed both wild-type and mutant Hsp104p and showed the thermotolerance phenotype in the strain with the functional wild-type Hsp104p expressed. Upon treatment with H2O2 and menadione, the strains with the functional Hsp104p expressed showed higher survival rates than the other mutants, indicating the protective role of Hsp104p from the oxidative stress. Fluorescence measurement of the oxidation-dependent probe, 2',7'-dichlorofluoroscein diacetate (H2DCFDA), also indicated that Hsp104p significantly reduced the amount of ROS. Resistance to the oxidative stress was independent of the amount of the glutathione in the hyperactivated ras mutants. Taken all together, this study confirms that Hsp104p plays a crucial role in keeping cells from being damaged by the oxidative stress, thus acting as a modulator of the intracellular redox state.     

Phospholipase C-delta1 rescues intracellular Ca2+ overload in ischemic heart and hypoxic neonatal cardiomyocytes

Ischemia and simulated ischemic conditions cause intracellular Ca2+ overload in the myocardium. The relationship between ischemia injury and Ca2+ overload has not been fully characterized. The aim of the present study was to investigate the expression and characteristics of PLC isozymes in myocardial infarction-induced cardiac remodeling and heart failure. In normal rat heart tissue, PLC-delta1 (about 44 ng/mg of heart tissue) was most abundant isozymes compared to PLC-gamma1 (6.8 ng/mg) and PLC-beta1 (0.4 ng/mg). In ischemic heart and hypoxic neonatal cardiomyocytes, PLC-delta1, but not PLC-beta1 and PLC-gamma1, was selectively degraded, a response that could be inhibited by the calpain inhibitor, calpastatin, and by the caspase inhibitor, zVAD-fmk. Overexpression of the PLC-delta1 in hypoxic neonatal cardiomyocytes rescued intracellular Ca2+ overload by ischemic conditions. In the border zone and scar region of infarcted myocardium, and in hypoxic neonatal cardiomyocytes, the selective degradation of PLC-delta1 by the calcium sensitive proteases may play important roles in intracellular Ca2+ regulations under the ischemic conditions. It is suggested that PLC isozyme-changes may contribute to the alterations in calcium homeostasis in myocardial ischemia.     

Efficacy and Safety of Sorafenib Therapy on Metastatic Renal Cell Carcinoma in Korean Patients: Results from a Retrospective Multicenter Study

Objective

To evaluate the efficacy and safety of sorafenib for Korean patients with metastatic renal cell carcinoma (mRCC).

Methods

A total of 177 mRCC patients using sorafenib as first- (N = 116), second- (N = 43), and third-line (N = 18) therapies were enrolled from 11 Korean centers between 2006 and 2012. The patient characteristics, therapy duration, tumor response, disease control rate, and tolerability were assessed at baseline and at routine follow-ups, and the progression-free survival (PFS) and overall survival (OS) times and rates were analyzed.

Results

Among all patients, 18 (10.2%) stopped sorafenib treatment for a median of 1.7 weeks, including 15 (8.5%) who discontinued the drug, while 40 (22.6%) and 12 (6.8%) patients required dose reductions and drug interruptions, respectively. Severe adverse events (AEs) or poor compliance was observed in 64 (36.2%) patients, with 118 (7.4%) ≥grade 3 AEs. During the treatment, one myocardial infarction was observed. The number of ≥grade 3 AEs in the first-line sorafenib group was 71 (6.8% of the total 1048 AEs). During a median follow-up of 17.2 months, the radiologically confirmed best objective response rate, disease control rate, median PFS, and median OS were 22.0%, 53.0%, 6.4 months (95% confidence interval [CI], 5.2–8.9), and 32.6 months (95% CI, 27.3–63.8) for the total 177 sorafenib-treated patients, respectively, and 23.2%, 56.0%, 7.4 months (95% CI, 5.5–10.5), and not reached yet (95% CI, 1.0–31.1) for the first-line sorafenib group, respectively.

Conclusions

Sorafenib produced tolerable safety, with a ≥grade 3 AE rate of 7.4% and an acceptable disease control rate (53.0%) in Korean mRCC patients.     

Enhancing the processivity of a family B-type DNA polymerase of Thermococcus onnurineus and application to long PCR

Mechanisms that allow replicative DNA polymerases to attain high processivity are often specific to a given polymerase and cannot be generalised to others. Amplification efficiency is lower in family B-type DNA polymerases than in family A-type (Taq) polymerases because of their strong 3′–5′ exonuclease-activity. Here, we have red the exonuclease domain of the Thermococcus onnurineus NA1 (TNA1) DNA polymerase, especially Asn210 to Asp215 residues in Exo II motif (NXXXFD), to improve the processivity. N213D mutant protein had higher processivity and extension rate than the wild-type TNA1 DNA polymerase, retaining a lower mutation frequency than recombinant Taq DNA polymerase. Consequently, the N213D mutant could amplify target DNA up to 13.5 kb in length from human genomic DNA and 16.2 kb in length from human mitochondrial DNA while wild-type TNA1 amplified target DNA of 2.7 kb in length from human genomic DNA.     

Catalytic Domain of AfsKav Modulates Both Secondary Metabolism and Morphologic Differentiation in Streptomyces avermitilis ATCC 31272

Genetic characterization of afsK-av (SAV3816) in Streptomyces avermitilis ATCC 31272 was performed to evaluate the role(s) of this eukaryotic-type serine–threonine protein kinase (STPK) in the regulation of morphologic differentiation and secondary metabolism. The afsK-av::neo mutant (SJW4001) was defective in sporulation, melanogenesis, and avermectin production. These phenotypic defects were complemented by introduction of either the intact afsK-av or the 900-nt catalytic domain region. The catalytic domain restored sporulation and melanogenesis to SJW4001 whereas it partially recovered avermectin production. This study reveals that AfsKav is a pleiotropic regulator and demonstrates in vivo that the C-region of AfsKav is not essential for its regulatory role in S. avermitilis differentiations.     

A survey of natural and ethyl methane sulfonate-induced variations of eIF4E using high-resolution melting analysis in Capsicum

Allele mining is a method used to find undiscovered natural variations or induced mutations in a plant, and has become increasingly important as more genomic information is available in plants. A high-throughput method is required to facilitate the identification of novel alleles in a large number of samples. In this paper we describe the application of a high-resolution melting (HRM) method to detect natural variations and ethyl methane sulfonate (EMS)-induced mutations in Capsicum. We have scanned single polymorphic mutations in the first exon of the eIF4E gene, wherein the mutations confer resistance to potyviruses. Sixteen allelic variations out of 248 germplasm collections were identified using HRM analysis, and one accession carrying an allelic variation (pvrHRM1 ¹³ ) was confirmed to be resistant to the TEV-HAT strain. In addition, five single polymorphic mutations in the eIF4E gene were identified in an EMS-induced mutant population. These results demonstrate that HRM allows for the rapid identification of new allelic variants in both natural and artificial mutant populations.