Antibacterial properties of a new isoflavonoid from Erythrina poeppigiana against methicillin-resistant Staphylococcus aureus.

A new isoflavonoid, together with four known isoflavonoids, was isolated from the roots of Erythrina poeppigiana. The chemical structure was determined by extensive spectroscopic studies, and then its antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) was investigated. The new isoflavonoid was identified as 3,9-dihyroxy-10-gamma,gamma-dimethylallyl-6a,11a-dehydropterocarpan (compound 1). Compound 1 inhibited bacterial growth most potently of the five isolates, and had a minimum inhibitory concentration (MIC) of 125 microg/ml against thirteen MRSA strains. Inhibitory activity was based on bactericidal action and viable cell number reduced by approximately 1/10,000 after 4 h incubation with compound 1. Despite intense bactericidal action against MRSA, compound 1 never resulted in leakage of 260 nm-absorbing substances from bacterial cells. Compound 1 (12.5 microg/ml) completely inhibited incorporation of radio-labeled thymidine, uridine and leucine into MRSA cells. Although glucose incorporation was also markedly inhibited by the compound, the amount of glucose incorporated by bacterial cells increased gradually with incubation time. These findings suggest that compound 1 exhibits anti-MRSA activity by interfering with incorporation of metabolites and nutrients into bacterial cells or by affecting the nucleic acids of MRSA cells. Furthermore, this new compound could be a potent phytotherapeutic agent for treating MRSA infections.     

Experimental study of seita-fitting

The purpose of this study was to verify the difference between carrying a load on the sacrum (LOS) and on the lumbar vertebrae (LOL) in oxygen uptake, muscle activities, heart rate, cadence, and subjective response. Nine males (26.7 +/- 3.1 years old), each carrying a 7.5 kg carrier frame and a 40 kg load, walked on a treadmill at a speed of 50 m/min. EMGs were recorded from the trapezius, rectus abdominis, erector spinae, vastus lateralis, rectus femoris, vastus medialis, biceps femoris long head, tibial anterior, soleus, medial head of gastrocnemius, and the lateral head of gastrocnemius. For each subject the integrated EMG (IEMG) was normalized by dividing the IEMG in the LOL and LOS by the IEMG in a no-load condition (NL) for each investigated muscle. The following was significantly higher in LOL than in LOS: oxygen uptake; IEMG of the tibial anterior, soleus, and medial head of gastrocnemius; cadence; and rated perceived exertion. However, IEMG of the erector spinae was significantly lower in LOL than in LOS. These results suggest that seita-fitting in LOS causes a decrease of leg muscle activities, which causes oxygen uptake to decrease beyond the increase of the erector spinae activity.     

Lipid-Protein Interplay in Dimerization of Juxtamembrane Domains of Epidermal Growth Factor Receptor

Transmembrane (TM) helix and juxtamembrane (JM) domains (TM-JM) bridge the extracellular and intracellular domains of single-pass membrane proteins, including epidermal growth factor receptor (EGFR). TM-JM dimerization plays a crucial role in regulation of EGFR kinase activity at the cytoplasmic side. Although the interaction of JM with membrane lipids is thought to be important to turn on EGF signaling, and phosphorylation of Thr654 on JM leads to desensitization, the underlying kinetic mechanisms remain unclear. In particular, how Thr654 phosphorylation regulates EGFR activity is largely unknown. Here, combining single-pair FRET imaging and nanodisc techniques, we showed that phosphatidylinositol 4,5-bis phosphate (PIP₂) facilitated JM dimerization effectively. We also found that Thr654 phosphorylation dissociated JM dimers in the membranes containing acidic lipids, suggesting that Thr654 phosphorylation electrostatically prevented the interaction with basic residues in JM and acidic lipids. Based on the single-molecule experiment, we clarified the kinetic pathways of the monomer (inactive state)-to-dimer (active state) transition of JM domains and alteration in the pathways depending on the membrane lipid species and Thr654 phosphorylation.     

Transient Acceleration of Epidermal Growth Factor Receptor Dynamics Produces Higher-Order Signaling Clusters

Cell signaling depends on spatiotemporally regulated molecular interactions. Although the movements of signaling proteins have been analyzed with various technologies, how spatial dynamics influence the molecular interactions that transduce signals is unclear. Here, we developed a single-molecule method to analyze the spatiotemporal coupling between motility, clustering, and signaling. The analysis was performed with the epidermal growth factor receptor (EGFR), which triggers signaling through its dimerization and phosphorylation after association with EGF. Our results show that the few EGFRs isolated in membrane subdomains were released by an EGF-dependent increase in their diffusion area, facilitating molecular associations and producing immobile clusters. Using a two-color single-molecule analysis, we found that the EGF-induced state transition alters the properties of the immobile clusters, allowing them to interact for extended periods with the cytoplasmic protein, GRB2. Our study reveals a novel correlation between this molecular interaction and its mesoscale dynamics, providing the initial signaling node.     

Purification and characterization of the intracellular β-glucosidase from Aureobasidium sp ATCC 20524

β-Glucosidase hydrolyzing cellobiose was extracted from Aureobasidium sp ATCC 20524 and purified to homogeneity. The molecular mass was estimated to be about 331 kDa. The enzyme contained 26.5% (w/w) carbohydrate. The optimum pH and temperature for the enzyme reaction were pH 4 and 80°C, respectively. The enzyme was stable at a wide range of pH, 2.2–9.8, after 3 h and at 75°C for 15 min. The kinetic parameters were determined. The enzyme was relatively stable against typical organic enzyme inhibitors. The enzyme also hydrolyzed gentiobiose, p-nitrophenyl-β-glucoside and salicin. Received 05 November 1998/ Accepted in revised form 14 February 1999     

A cambialistic SOD in a strictly aerobic hyperthermophilic archaeon, Aeropyrum pernix.

The superoxide dismutase (SOD) gene of Aeropyrum pernix, a strictly aerobic hyperthermophilic archaeon, was cloned and expressed in Escherichia coli, and its gene product was characterized. The molecular mass of the protein, based on the deduced amino acid sequence, was 24.6 kDa. The sequence showed overall similarity to the sequences of known Mn- and Fe-SODs. The metal binding residues conserved in Mn- and Fe-SODs were also found in A. pernix SOD. When the SOD gene was expressed in E. coli cells, the product formed a homodimer, and contained both Mn and Fe. Metal reconstitution experiments showed that A. pernix SOD is cambialistic, i.e. active with either Fe or Mn. The specific activities were 906 U/mg with Mn and 175 U/mg with Fe. No loss of activity of Mn-reconstituted SOD was observed at 105 degrees C even after 5 h incubation. Sodium azide, an inhibitor of SODs, did not inhibit the Mn-reconstituted SOD from A. pernix even at concentrations up to 400 mM. This SOD from an aerobic hyperthermophilic archaeon, Aeropyrum pernix, was extremely thermostable and active with either Mn or Fe. With Mn as a metal cofactor, it was more thermostable, and less sensitive to sodium azide and sodium fluoride than with Fe.     

A Highlights from MBoC Selection: p52Shc regulates the sustainability of ERK activation in a RAF-independent manner

p52SHC (SHC) and GRB2 are adaptor proteins involved in the RAS/MAPK (ERK) pathway mediating signals from cell-surface receptors to various cytoplasmic proteins. To further examine their roles in signal transduction, we studied the translocation of fluorescently labeled SHC and GRB2 to the cell surface, caused by the activation of ERBB receptors by heregulin (HRG). We simultaneously evaluated activated ERK translocation to the nucleus. Unexpectedly, the translocation dynamics of SHC were sustained when those of GRB2 were transient. The sustained localization of SHC positively correlated with the sustained nuclear localization of ERK, which became more transient after SHC knockdown. SHC-mediated PI3K activation was required to maintain the sustainability of the ERK translocation regulating MEK but not RAF. In cells overexpressing ERBB1, SHC translocation became transient, and the HRG-induced cell fate shifted from a differentiation to a proliferation bias. Our results indicate that SHC and GRB2 functions are not redundant but that SHC plays the critical role in the temporal regulation of ERK activation.     

Multiple mechanisms for accumulation of myosin II filaments at the equator during cytokinesis

Total internal reflection fluorescence microscopy revealed how individual bipolar myosin II filaments accumulate at the equatorial region in dividing Dictyostelium cells. Direct observation of individual filaments in live cells provided us with much convincing information. Myosin II filaments accumulated at the equatorial region by at least two independent mechanisms: (i) cortical flow, which is driven by myosin II motor activities and (ii) de novo association to the equatorial cortex. These two mechanisms were mutually redundant. At the same time, myosin II filaments underwent rapid turnover, repeating their association and dissociation with the actin cortex. Examination of the lifetime of mutant myosin filaments in the cortex revealed that the turnover mainly depended on heavy chain phosphorylation and that myosin motor activity accelerated the turnover. Double mutant myosin II deficient in both motor and phosphorylation still accumulated at the equatorial region, although they displayed no cortical flow and considerably slow turnover. Under this condition, the filaments stayed for a significantly longer time at the equatorial region than at the polar regions, indicating that there are still other mechanisms for myosin II accumulation such as binding partners or stabilizing activity of filaments in the equatorial cortex.