The CD99 signal enhances Fas-mediated apoptosis in the human leukemic cell line, Jurkat

The CD99 antigen has been implicated in various cellular processes, including apoptosis in T cells. Previously, we reported two monoclonal antibodies that recognize different epitopes of the CD99 molecule, named DN16 and YG32. In this study, we investigated the role of each CD99 epitope in T cell apoptosis. Unlike the DN16 epitope, CD99 ligation via the YG32 epitope failed to induce T cell death. Surprisingly, however, the YG32 signal enhanced Fas-mediated apoptosis in Jurkat T cells. Augmentation of Fas-mediated apoptosis by YG32 ligation was inhibited by treatment with either of the caspase inhibitors z-VAD-fmk or z-IETD-fmk, and YG32 ligation appeared to induce Fas oligomerization. These results suggest that each CD99 epitope plays a distinct role in T cell biology, especially in T cell apoptosis.     

Inhibitory effect of chlorinated guaiacols on methanogenic activity of anaerobic digester sludge

Of four chlorinated guaiacols, tetrachloroguaiacol at 62 M inhibited acetate methanogenesis, the strongest decreasing activity by 50%. 4,5,6-Trichloroguaiacol, 4,5-dichloroguaiacol, and 4-chloroguaiacol showed 50% inhibition at 0.13, 0.32, and 1.50 mM, respectively. Degradation test results of volatile fatty acids (acetic, propionic, and butyric acid) by anaerobic digester sludge (stored 5 weeks) indicated that syntrophic butyrate degraders of this sludge were more sensitive to tetrachloroguaiacol than acetoclastic methanogens and syntrophic propionate degraders.     

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.     

Synthesis and Mechanistic Studies of a Novel Homoisoflavanone Inhibitor of Endothelial Cell Growth

Preventing pathological ocular angiogenesis is key to treating retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. At present there is no small molecule drug on the market to target this process and hence there is a pressing need for developing novel small molecules that can replace or complement the present surgical and biologic therapies for these neovascular eye diseases. Previously, an antiangiogenic homoisoflavanone was isolated from the bulb of a medicinal orchid, Cremastra appendiculata. In this study, we present the synthesis of a novel homoisoflavanone isomer of this compound. Our compound, SH-11052, has antiproliferative activity against human umbilical vein endothelial cells, and also against more ocular disease-relevant human retinal microvascular endothelial cells (HRECs). Tube formation and cell cycle progression of HRECs were inhibited by SH-11052, but the compound did not induce apoptosis at effective concentrations. SH-11052 also decreased TNF-α induced p38 MAPK phosphorylation in these cells. Intriguingly, SH-11052 blocked TNF-α induced IκB-α degradation, and therefore decreased NF-κB nuclear translocation. It decreased the expression of NF-κB target genes and the pro-angiogenic or pro-inflammatory markers VCAM-1, CCL2, IL8, and PTGS2. In addition SH-11052 inhibited VEGF induced activation of Akt but not VEGF receptor autophosphorylation. Based on these results we propose that SH-11052 inhibits inflammation induced angiogenesis by blocking both TNF-α and VEGF mediated pathways, two major pathways involved in pathological angiogenesis. Synthesis of this novel homoisoflavanone opens the door to structure-activity relationship studies of this class of compound and further evaluation of its mechanism and potential to complement existing antiangiogenic drugs.     

Effect of dietary Platycodon grandiflorum on the improvement of insulin resistance in obese Zucker rats

The effect of dietary Platycodon grandiflorum on the improvement of insulin resistance and lipid profile was investigated in lean (Fa/-) and obese (fa/fa) Zucker rats, a model for noninsulin dependent diabetes mellitus. Dietary Platycodon grandiflorum feeding for 4 weeks resulted in a significant decrease in the concentration of plasma triglyceride in both lean and obese Zucker rats. Furthermore, dietary Platycodon grandiflorum markedly decreased both plasma cholesterol and fasting plasma insulin levels, and significantly decreased the postprandial glucose level at 30 min during oral glucose tolerance test in obese Zucker rats. Although there was no statistical significance, the crude glucose transporter 4 protein level of obese rats fed Platycodon grandiflorum tended to increase when compared with that of obese control rats. Therefore, the present results suggested that dietary Platycodon grandiflorum may be useful in prevention and improvement of metabolic disorders characterized by hyperinsulinemia states such as noninsulin dependent diabetes mellitus, syndrome X, and coronary artery disease.     

PLANT U-BOX PROTEIN10 Regulates MYC2 Stability in Arabidopsis

MYC2 is an important regulator for jasmonic acid (JA) signaling, but little is known about its posttranslational regulation. Here, we show that the MYC2 C-terminal region interacted with the PLANT U-BOX PROTEIN10 (PUB10) armadillo repeats in vitro. MYC2 was efficiently polyubiquitinated by PUB10 with UBC8 as an E2 enzyme and the conserved C249 in PUB10 was required for activity. The inactive PUB10(C249A) mutant protein retained its ability to heterodimerize with PUB10, thus blocking PUB10 E3 activity as a dominant-negative mutant. Both MYC2 and PUB10 were nucleus localized and coimmunoprecipitation experiments confirmed their interaction in vivo. Although unstable in the wild type, MYC2 stability was enhanced in pub10, suggesting destabilization by PUB10. Moreover, MYC2 half-life was shortened or prolonged by induced expression of PUB10 or the dominant-negative PUB10(C249A) mutant, respectively. Root growth of pub10 seedlings phenocopied 35S:MYC2 seedlings and was hypersensitive to methyl jasmonate, whereas 35S:PUB10 and jin1-9 (myc2) seedlings were hyposensitive. In addition, the root phenotype conferred by MYC2 overexpression in double transgenic plants was reversed or enhanced by induced expression of PUB10 or PUB10(C249A), respectively. Similar results were obtained with three other JA-regulated genes, TAT, JR2, and PDF1.2. Collectively, our results show that MYC2 is targeted by PUB10 for degradation during JA responses.     

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.     

Estimation of effective population size of HIV-1 within a host: a pseudomaximum-likelihood approach

Using pseudomaximum-likelihood approaches to phylogenetic inference and coalescent theory, we develop a computationally tractable method of estimating effective population size from serially sampled viral data. We show that the variance of the maximum-likelihood estimator of effective population size depends on the serial sampling design only because internal node times on a coalescent genealogy can be better estimated with some designs than with others. Given the internal node times and the number of sequences sampled, the variance of the maximum-likelihood estimator is independent of the serial sampling design. We then estimate the effective size of the HIV-1 population within nine hosts. If we assume that the mutation rate is 2.5 x 10(-5) substitutions/generation and is the same in all patients, estimated generation lengths vary from 0.73 to 2.43 days/generation and the mean (1.47) is similar to the generation lengths estimated by other researchers. If we assume that generation length is 1.47 days and is the same in all patients, mutation rate estimates vary from 1.52 x 10(-5) to 5.02 x 10(-5). Our results indicate that effective viral population size and evolutionary rate per year are negatively correlated among HIV-1 patients.     

Chitosan microspheres containing Bordetella bronchiseptica antigens as novel vaccine against atrophic rhinitis in pigs

The immune-stimulating activities of Bordetella bronchiseptica antigens containing dermonecrotoxin (BBD) loaded in chitosan microspheres (CMs) have already been reported in vitro and in vivo with a mouse alveolar macrophage cell line (RAW264.7) and mice. Therefore, this study attempted to demonstrate the successful induction of mucosal immune responses after the intranasal administration of BBD loaded in CMs (BBD-CMs) in colostrum-deprived pigs. The BBD was introduced to the CMs using an ionic gelation process involving tripolyphosphate (TPP). Colostrum-deprived pigs were then directly immunized through intranasal administration of the BBD-CMs. A challenge with a field isolate of B. bronchiseptica was performed ten days following the final immunization. The BBD-specific IgG and IgA titers, evident in the nasal wash and serum from the vaccinated pigs, increased with time (p<0.05). Following the challenge, the clinical signs of infection were about 6-fold lower in the vaccinated pigs compared with the nonvaccinated pigs. The grades for gross morphological changes in the turbinate bones from the vaccinated pigs were also significantly lower than the grades recorded for the nonvaccinated pigs (p<0.001). Therefore, the mucosal and systemic immune responses induced in the current study would seem to indicate that the intranasal administration of BBD-CMs may be an effective vaccine against atrophic rhinitis in pigs.