Low density lipoprotein and apoprotein B induce increases in inositol trisphosphate and cytosolic free Ca2+ via pertussis toxin-sensitive GTP-binding protein in vascular smooth muscle cells

Low density lipoprotein (LDL), a major cholesterol-carrying lipoprotein in the plasma, binds to its receptor through apoprotein B (Apo-B). The addition of LDL and Apo-B induced rapid (5 s), but transient increase in the inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) level with K0.5 values of 1.1 and 0.07 microgram/ml, accompanied by increases of cytosolic free Ca2+ concentration [( Ca2+]i), in vascular smooth muscle cells (VSMC). The increases by LDL and Apo-B were both reduced by pretreatment of the VSMC with pertussis toxin. The early change in Ins-1,4,5-P3 involving a GTP-binding protein may function as an initial signal for the action of LDL in VSMC.     

Cascading effects of larval Crucian carp introduction on phytoplankton and microbial communities in a paddy field: top-down and bottom-up controls

Effects of fish predation propagate through aquatic food webs, where the classical grazing food chain and microbial loop are interwoven by trophic interactions. The overall impact on aquatic food webs is further complicated because fish may also exert bottom-up controls through nutrient regeneration. Yet, we still have limited information about cascading effects among fish, zooplankton, phytoplankton, and microbes. In this study, we performed a mesocosm experiment to evaluate effects of fish introduction on plankton communities. Six plots were set in factorial combination with fish introduction and rice straw plowing in a paddy field, and the experiment was continued for 4 weeks. Introduction of fish significantly increased chlorophyll a concentrations in smaller size fractions (<15 μm) and abundances of filamentous bacteria (>5 μm in length) and heterotrophic nanoflagellates in 3–15 μm fraction. Microbes in 0.8–3 μm fraction showed increasing but not significant trends in response to fish introduction. These results indicate cascading effects of fish predation operating via two pathways, one through grazing food chain and the other through microbial food web. Phytoplankton community compositions shifted in similar fashion in all plots until 1 week after fish introduction, and then diverged between plots with and without fish thereafter. Bottom-up effects of fish introduction were suggested by increases of total chlorophyll a and inedible phytoplankton species in response to fish introduction. This study provides an example of how fish predation regulates biomass and structure of phytoplankton and microbial communities.     

Stress fibers in the mesenteric mesothelial cells of the large intestine of the bullfrog,Rana catesbeiana

Summary Actin-containing cytoplasmic fibers were visualized in the mesenteric mesothelial cells of the large intestine of bullfrog tadpoles by rhodamine-phalloidin staining of en face preparations of mesothelial cells. These fibers were concurrently stained by immunofluorescence using antibodies to myosin or -actinin. Electron microscopy showed the presence of bundles of microfilaments in the basal cytoplasm of the cells. Such fibers in the mesothelial cells may be comparable to the stress fibers present in cultured cells. The mesothelial cells initially formed axially oriented stress fibers when they changed from a rhombic to a slender spindle-like shape. On the other hand, stress fibers disappeared as cells transformed from elongated to polygonal shapes during the period of metamorphic climax. Expression of stress fibers in these cells appears to be related to the degree of tension loaded on the mesentery, which may be generated by mesenteric winding. These stress fibers in the mesothelial cells may serve to regulate cellular transformation. They may also help to maintain cellular integrity by strengthening the cellular attachment to subepithelial tissue against tensile stress exerted on the mesentery.     

Purification and properties of a novel thermostable galacto-oligosaccharide-producing beta-galactosidase from Sterigmatomyces elviae CBS8119.

A thermostable beta-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was solubilized from a cell wall preparation of Sterigmatomyces elviae CBS8119. The enzyme was purified to homogeneity by means of chromatography on DEAE-Toyopearl, Butyl-Toyopearl, Chromatofocusing, and p-aminobenzyl 1-thio-beta-D-galactopyranoside agarose columns. The molecular weight of the purified enzyme was estimated to be about 170,000 by gel filtration with a Highload-Superdex 200pg column and 86,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its isoelectric point, determined by polyacrylamide gel electrofocusing, was 4.1. The optimal temperature for enzyme activity was 85 degrees C. It was stable at temperatures up to 80 degrees C for 1 h. The optimal pH range for the enzyme was 4.5 to 5.0, it was stable at pH 2.5 to 7.0, and its activity was inhibited by Hg2+. The Km values for o-nitrophenyl-beta-D-galactopyranoside and lactose were 9.5 and 2.4 mM, respectively, and the maximum velocities for these substrates were 96 and 240 mumol/min per mg of protein, respectively. In addition, this enzyme possessed a high level of transgalactosylation activity. Galacto-oligosaccharides, including tri- and tetrasaccharides, were produced with a yield, by weight, of 39% from 200-mg/ml lactose.     

Molecular Dissection of the Rho-associated Protein Kinase (p160ROCK)-regulated Neurite Remodeling in Neuroblastoma N1E-115 Cells

A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho–ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells.     

Targeting Aurora B to the Equatorial Cortex by MKlp2 Is Required for Cytokinesis

Although Aurora B is important in cleavage furrow ingression and completion during cytokinesis, the mechanism by which kinase activity is targeted to the cleavage furrow and the molecule(s) responsible for this process have remained elusive. Here, we demonstrate that an essential mitotic kinesin MKlp2 requires myosin-II for its localization to the equatorial cortex, and this event is required to recruit Aurora B to the equatorial cortex in mammalian cells. This recruitment event is also required to promote the highly focused accumulation of active RhoA at the equatorial cortex and stable ingression of the cleavage furrow in bipolar cytokinesis. Specifically, in drug-induced monopolar cytokinesis, targeting Aurora B to the cell cortex by MKlp2 is essential for cell polarization and furrow formation. Once the furrow has formed, MKlp2 further recruits Aurora B to the growing furrow. This process together with continuous Aurora B kinase activity at the growing furrow is essential for stable furrow propagation and completion. In contrast, a MKlp2 mutant defective in binding myosin-II does not recruit Aurora B to the cell cortex and does not promote furrow formation during monopolar cytokinesis. This mutant is also defective in maintaining the ingressing furrow during bipolar cytokinesis. Together, these findings reveal that targeting Aurora B to the cell cortex (or the equatorial cortex) by MKlp2 is essential for the maintenance of the ingressing furrow for successful cytokinesis.     

Suppression of stop codon UGA in acrB can contribute to antibiotic resistance in Klebsiella pneumoniae ATCC10031

We previously reported that Klebsiella pneumoniae MGH78578 exhibited higher resistance against various antimicrobials than K. pneumoniae ATCC10031. In this study, we showed that the plasmid, pKPN5, in K. pneumoniae MGH78578 played an important role in resistance against aminoglycosides, ampicillin, tetracycline, and chloramphenicol, while genome-derived β-lactamases and drug efflux pumps appeared to be more important in resistance to cloxacillin. acrAB, encoding a potent multidrug efflux pump, was cloned from K. pneumoniae MGH78578 and ATCC10031, to investigate reasons for the high drug resistance of K. pneumoniae MGH78578, and the results revealed that AcrAB from K. pneumoniae ATCC10031 conferred weaker drug resistance than AcrAB from K. pneumoniae MGH78578. DNA sequencing revealed that acrB from K. pneumoniae ATCC10031 carried the nonsense mutation, UGA, which was not found in acrB from K. pneumoniae MGH78578. However, acrB from K. pneumoniae ATCC10031 conferred slightly elevated resistant levels to several antimicrobials. The intact length of AcrB was detected in K. pneumoniae ATCC10031 by Western blot analysis, even though its quantity was small. Therefore, the stop codon UGA in acrB was thought to be overcome to some extent in this strain. We artificially introduced the nonsense mutation, UGA to the cat gene on pACYC184, and the plasmid also elevated the MIC of chloramphenicol in K. pneumoniae ATCC10031. These results suggest that a mechanism to overcome the nonsense mutation in acrB sustained resistance against a few β-lactams, dyes, and cholic acid in K. pneumoniae ATCC10031.     

Increase of (Ca2++Mg2+)-ATPase activity of renal basolateral membrane by parathyroid hormone via cyclic AMP-dependent membrane phosphorylation

Studies were made on the mechanism of the effect of parathyroid hormone (PTH) on the activity of (Ca2++Mg2+)-ATPase, a membrane bound Ca2+-extrusion pump enzyme from the basolateral membranes (BLM) of canine kidney (Km for free Ca2+ = 1.3 X 10(-7) M, Vmax = 200 nmol Pi/mg/min). At 1 X 10(-7) M free Ca2+, both PTH (10(-7)-10(-6) M) and cAMP (10(-6)-10(-4) M) stimulated (Ca2++Mg2+)-ATPase activity dose-dependent and their stimulatory effects were inhibited completely by 5 microM H-8, an inhibitor of cAMP-dependent protein kinase. PTH (10(-7) M) also caused 40% increase in 32P incorporation into the BLM and 5 microM H-8 inhibited this increase too. PTH (10(-7) M) was found to stimulate phosphorylation of a protein of Mr 9000 by cAMP dependent protein kinase and 5 microM H-8 was found to block this stimulation also. From these results, it is proposed that PTH stimulates (Ca2++Mg2+)-ATPase activity by enhancing its affinity for free Ca2+ via cAMP-dependent phosphorylation of a BLM protein of Mr 9000.