Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety of proteins. However, little is known about how CaM directly ...regulates transcription. Screening of an Arabidopsis cDNA expression library using horseradish peroxidase-conjugated calmodulin as a probe identified a calmodulin-binding NAC protein (CBNAC). Using gel overlay assays, a Ca2+-dependent CaM-binding domain was identified in the C terminus of this protein. Specific binding of CaM to CaM-binding domain was corroborated by site-directed mutagenesis and a split-ubiquitin assay. Using a PCR-mediated random binding site selection method, we identified a DNA-binding sequence (CBNACBS) for CBNAC, which consisted of a GCTT core sequence flanked on both sides by other frequently repeating sequences (TTGCTTANNNNNNAAG). CBNAC was able to bind to CBNACBS, which resulted in the repression of transcription in Arabidopsis protoplasts. Interestingly, the transcriptional repression mediated by CBNAC was enhanced by CaM. These results suggest that CBNAC may be a CaM-regulated transcriptional repressor in Arabidopsis.
Background
Nephrotoxicity is often referred to as a side effect of most drugs, but methods for effectively detecting nephrotoxicants still require development.
Objective
In this study, we identified ...the characteristic cellular responses and target genes affected by three nephrotoxic drugs (cisplatin, amphotericin B, and geneticin) using human oligonucleotide microarray and applied these genes to nephrotoxicity assessment in human kidney proximal tubular cells (HK-2).
Results
Through analysis of the gene expression profiles, it was identified 21 up- and 58 down-regulated genes changed by all three nephrotoxicants. Especially, the nephrotoxicants induced an increase in expression of a family of growth arrest and DNA-damage (GADD)-inducible genes (GADD34, GADD45A, GADD45G, and GADD153). In addition, these nephrotoxicants induced phosphorylation of p38 MAPK, which acts upstream of the GADD-related apoptosis pathway. The influence of other types of nephrotoxicants on expression of GADD family genes was also examined.
Conclusion
Our findings support that cisplatin, amphotericin B, and geneticin mediate induction of the GADD family of genes via the p38 MAPK pathway, resulting in the induction of apoptosis in HK-2 cells. We suggest that the apoptosis-related genes GADD45A and GADD153 may be reasonable candidate biomarkers for the screening of nephrotoxicity of drugs.
Serotonin antagonist reuptake inhibitor (SARI) drugs that block both 5-HT2 receptors and the serotonin transporters have been developed. The human 5-HT2A/2C receptor has been implicated in several ...neurological conditions, and potent selective 5-HT2A/2C ligands may have therapeutic potential for treatment of CNS diseases such as depression. An imidazole moiety usually provides good pharmacokinetic properties as a drug substance, and thus considerable efforts have been devoted to develop imidazole derivatives into drug candidates. The imidazole series of compounds was evaluated against 5-HT2A/2C and serotonin reuptake inhibition. A few of the compounds in the series showed promising IC50 values and antidepressant-like effect in in vivo forced swimming test (FST). On the basis of these results, further lead optimization studies resulted in identifying promising compounds potentially for therapeutic use.
The molecular level details of the displacement of surface adsorbed fibrinogen from silica substrates were studied by atomic force microscopy, immunochemical assays, fluorescence microscopy, and ...vibrational sum frequency spectroscopy. The results showed that human plasma fibrinogen (HPF) can be readily displaced from the interface by other plasma proteins near neutral pH because the positively charged αC domains on HPF sit between the rest of the macromolecule and the underlying surface. The αC domains make weak electrostatic contact with the substrate, which is manifest by a high degree of alignment of Lys and Arg residues. Upon cycling through acidic pH, however, the αC domains are irreversibly removed from this position and the rest of the macromolecule is free to engage in stronger hydrogen bonding, van der Waals, and hydrophobic interactions with the surface. This results in a 170-fold decrease in the rate at which HPF can be displaced from the interface by other proteins in human plasma.
Matrix metalloproteinases (MMPs) are endopeptidases that take significant roles in extracellular matrix degradation and therefore linked with several complications such as metastasis of cancer ...progression, oxidative stress, and hepatic fibrosis.
Ishige okamurae
, a brown alga, has been reported to possess various bioactivities including but not limited to antiviral, antimicrobial, and anti-inflammatory. In this study, the potential of
I. okamurae
against cancer cell invasion was evaluated through MMP inhibitory effect in HT1080 fibrosarcoma cells in vitro.
I. okamurae
crude extract was fractionated with organic solvents, namely water (H
2
O),
n-
butanol (
n-
BuOH), 85% aqueous methanol (85% aq. MeOH), and
n-
hexane (
n-
Hex). The non
-
toxicity of fractions was confirmed by MTT assay. All fractions inhibited the enzymatic activities of MMP-2 and MMP-9 according to gelatin zymography assay. Cell migration was also inhibited by 85% aq. MeOH fraction, significantly. In addition, both gene and protein expressions of MMP-2 and MMP-9, and tissue inhibitor of MMPs (TIMPs) were evaluated by RT-PCR and Western blotting, respectively. Fractions suppressed the mRNA and protein levels of MMP-2 and MMP-9 while elevating the TIMP-1 and TIMP-2, H
2
O fraction being the least effective while 85% aq. MeOH fraction the most. Collectively, 85% aq. MeOH fraction from brown algae could be a potential inhibitor of MMPs, suggestively considering presence of poly-unsaturated fatty acids in high amounts.
Diacylglycerol (DAG) is an intermediate lipid involved in the synthesis of phospholipids and triglycerides. As signaling regulators, DAG activate novel protein kinase C leading to decreased response ...to insulin in skeletal muscle. Alteration of DAG contents correlates with development of metabolic dysregulation in obese and diabetic conditions. Recent advances in lipidomics using mass spectrometry allow expanded measurements of various lipid species. This study describes a rapid measurement of DAG species using the triple quadrupole mass spectrometry using atmospheric pressure chemical ionization in a positive ion mode. DAG in the cells and muscle tissues were separated depending on differences in chain lengths and degree of unsaturation. The limit of detection and quantification for DAG was 0.2 to 17 pmol for this method. When C2C12 cells were treated with palmitate or oleate, we found a 12-fold and 2-fold DAG increase respectively compared to the no-treatment control. In the muscles of obese db/db mice, DAG levels were elevated by 6-fold compared to those of wild-type skeletal muscles. The present analytical method provides a rapid and sensitive quantification of DAG molecular species from various biological samples and can be used to correlate the degree of metabolic dysregulation with lipotoxic metabolites.
Transcriptional repression of pathogen defense-related genes is essential for plant growth and development. Several proteins are known to be involved in the transcriptional regulation of plant ...defense responses. However, mechanisms by which expression of defense-related genes are regulated by repressor proteins are poorly characterized. Here, we describe the in planta function of CBNAC, a calmodulin-regulated NAC transcriptional repressor in Arabidopsis. A T-DNA insertional mutant (cbnac1) displayed enhanced resistance to a virulent strain of the bacterial pathogen Pseudomonas syringae DC3000 (PstDC3000), whereas resistance was reduced in transgenic CBNAC overexpression lines. The observed changes in disease resistance were correlated with alterations in pathogenesis-related protein 1 (PR1) gene expression. CBNAC bound directly to the PR1 promoter. SNI1 (suppressor of nonexpressor of PR genes1, inducible 1) was identified as a CBNAC-binding protein. Basal resistance to PstDC3000 and derepression of PR1 expression was greater in the cbnac1 sni1 double mutant than in either cbnac1 or sni1 mutants. SNI1 enhanced binding of CBNAC to its cognate PR1 promoter element. CBNAC and SNI1 are hypothesized to work as repressor proteins in the cooperative suppression of plant basal defense.
Osteoporosis, a prevalent bone disease in an aging population, is considered to be closely related to osteoblastogenesis and adipogenesis. As a part of an ongoing trend to develop natural substances ...that attenuate osteoporotic conditions, edible brown algae E. cava and its bioactive constituents were tested for their effects on adipogenic differentiation in 3T3-L1 fibroblasts and osteoblast differentiation in MC3T3-E1 pre-osteoblasts. Following an activity-based isolation, three phlorotannin derivatives, triphlorethol-A (1), eckol (2) and dieckol (3), were isolated. Anti-adipogenesis effect of phlorotannins at the concentration of 20 µM was observed by reduced lipid accumulation and the suppressed expression of adipogenic differentiation markers. In addition, isolated phlorotannins successfully enhanced the osteoblast differentiation as indicated by increased alkaline phosphatase activity along with raised levels of osteoblastogenesis indicators and intracellular calcification at the concentration of 20 µM. In conclusion, E. cava is suggested as a source for functional food ingredients, especially phlorotannin derivatives that can be utilized for extenuating osteoporosis and obesity.
Limonium tetragonum has been well-known for its antioxidative properties as a halophyte. This study investigated the antimetastasis effect of solvent-partitioned L. tetragonum extracts (LTEs) and ...isolated compounds on HT1080 mouse melanoma cell model with a focus on matrix metalloproteinase (MMP) activity and TIMP and MAPK pathways. Upregulation and stimulation of MMPs result in elevated degradation of extracellular matrix which is part of several complications such as metastasis, cirrhosis, and arthritis. The anti-MMP capacity of LTEs was confirmed by their MMP-inhibitory effects, regulation of MMP and TIMP expression, and suppression of MAPK pathway. Among all tested LTEs, 85% aq. MeOH and n-BuOH were found to be most active fractions which later yielded two known flavonoid glycosides, myricetin 3-galactoside and quercetin 3-o-beta-galactopyranoside. Anti-MMP potential of the compounds was confirmed by their ability to regulate MMP expression through inhibited MAPK pathway activation. These results suggested that L. tetragonum might serve as a potential source of bioactive substances with effective anti-MMP properties.
To investigate the feasibility and preliminary effects of a simultaneous stage-matched exercise and diet (SSED) intervention in breast cancer survivors.
Randomized, controlled trial.
Oncology ...outpatient treatment clinics at the National Cancer Center in South Korea.
45 women with breast cancer who completed their cancer therapy.
Participants were assigned to the SSED intervention group (n = 23) or a control group (n = 22). Participants in the SSED group received a 12-week individualized intervention promoting prescribed exercise and a balanced diet through stage-matched telephone counseling and a workbook.
Program feasibility, behavioral outcomes (stage of motivational readiness for exercise and diet, physical activity, and diet quality), and quality-of-life (QOL) outcomes (functioning and global QOL, fatigue, anxiety, and depression).
Participant evaluations of the SSED intervention indicated that it was feasible and acceptable. All women felt that the overall intervention contents were appropriate, and 95% believed that the intervention helped to promote healthy behaviors. Objective data also supported the SSED intervention's feasibility (i.e., 91% completed the trial and 100% of intervention calls were received). When compared to control, the SSED intervention group showed significantly greater improvement in motivational readiness for exercise and diet, emotional functioning, fatigue, and depression.
Preliminary results suggest that the SSED intervention delivered via telephone counseling and workbook is feasible and beneficial for positive behavioral and QOL outcomes.
Nurse-led lifestyle interventions may improve QOL for cancer survivors.
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