Highly sensitive SERS analysis of the cyclic Arg–Gly–Asp peptide ligands of cells using nanogap antennas

The cyclic RGD (cRGD) peptide ligands of cells have become widely used for treating several cancers. We report a highly sensitive analysis of c(RGDfC) using surface enhanced Raman spectroscopy (SERS) using single dimer nanogap antennas in aqueous environment. Good agreement between characteristic peaks of the SERS and the Raman spectra of bulk c(RGDfC) with its peptide's constituents were observed. The exhibited blinking of the SERS spectra and synchronization of intensity fluctuations, suggest that the SERS spectra acquired from single dimer nanogap antennas was dominated by the spectrum of single to a few molecules. SERS spectra of c(RGDfC) could be used to detect at the nanoscale, the cells' transmembrane proteins binding to its ligand.

SERS of cyclic RGD on nanogap antenna.     

Hedgehog creates a gradient of DPP activity in Drosophila wing imaginal discs

Hedgehog (HH) and Decapentaplegic (DPP) direct anteroposterior patterning in the developing Drosophila wing by functioning as short- and long-range morphogens, respectively. Here, we show that the activity of DPP is graded and is directly regulated by a novel HH-dependent mechanism. DPP activity was monitored by visualizing the activated form of Mothers against dpp (MAD), a cytoplasmic transducer of DPP signaling. We found that activated MAD levels are highest near the source of DPP but are unexpectedly low in the cells that express dpp. HH induces dpp in these cells; it also attenuates their response to DPP by downregulating expression of the DPP receptor thick veins (tkv). We suggest that regulation of tkv by HH is a key part of the mechanism that controls the level and distribution of DPP.     

Osteoblastic activity and estrogenic response in the regenerating scale of goldfish, a good model of osteogenesis

Osteogenesis in the teleost was morphologically observed using regenerating scales of goldfish. Histological observations indicated that osteoblasts around the regenerating scales on days 7 to 10 were greater in size and number than those at other stages. Therefore, further experiments were carried out to examine the activity of osteoblasts in the regenerating period. To quantify their osteoblastic activities, scales on the left side of the body were taken, and the regenerating scales were then used to measure the activities of alkaline phosphatase (ALP), a marker of osteoblasts, on days 7, 10, and 15. The ontogenic scales on the right side of the body were also collected and used to measure ALP activity on the same days. Osteoblasts at all stages of regenerating scales were more active than those in the remaining ontogenic scales. The regenerating scales on day 10 had the highest activity. Furthermore, we found that estrogen receptor (ER) mRNA was expressed in the regenerating scales because estrogen participates in osteoblastic growth and differentiation in mammals. Therefore, using a scale culture system reported previously, the estrogenic response was examined in the ontogenic and regenerating scales on day 10. The reactivity was much higher in regenerating scales, although estrogen treatment significantly activated the osteoblastic activities in both scales. We are the first to demonstrate that ER is expressed in regenerating scales and that estrogen participates in osteogenesis as it does in mammalian bone. Our findings strongly suggest that regenerating scales can be used as a model of osteogenesis in vertebrates.     

Soybean Seed Extracts Preferentially Express Genomic Loci of Bradyrhizobium japonicum in the Initial Interaction with Soybean, Glycine max (L.) Merr

Initial interaction between rhizobia and legumes actually starts via encounters of both partners in the rhizosphere. In this study, the global expression profiles of Bradyrhizobium japonicum USDA 110 in response to soybean (Glycine max) seed extracts (SSE) and genistein, a major soybean-released isoflavone for nod genes induction of B. japonicum, were compared. SSE induced many genomic loci as compared with genistein (5.0 µM), nevertheless SSE-supplemented medium contained 4.7 µM genistein. SSE markedly induced four predominant genomic regions within a large symbiosis island (681 kb), which include tts genes (type III secretion system) and various nod genes. In addition, SSE-treated cells expressed many genomic loci containing genes for polygalacturonase (cell-wall degradation), exopolysaccharide synthesis, 1-aminocyclopropane-1-carboxylate deaminase, ribosome proteins family and energy metabolism even outside symbiosis island. On the other hand, genistein-treated cells exclusively showed one expression cluster including common nod gene operon within symbiosis island and six expression loci including multidrug resistance, which were shared with SSE-treated cells. Twelve putatively regulated genes were indeed validated by quantitative RT-PCR. Several SSE-induced genomic loci likely participate in the initial interaction with legumes. Thus, these results can provide a basic knowledge for screening novel genes relevant to the B. japonicum- soybean symbiosis.Key words: soybean seed extracts, Bradyrhizobium japonicum, expression clusters, genistein, symbiosis     

Manzamenones L–N,new dimeric fatty-acid derivatives from an Okinawan marine sponge Plakortis sp.

Three new polyketides, manzamenones L–N (1–3), have been isolated from an Okinawan marine sponge of the genus Plakortis. The structures of 1–3 were elucidated on the basis of spectroscopic data. Manzamenones L–N (1–3) were new dimeric fatty-acid derivatives consisting of a tetrahydroindenone with three carboxy groups and two hexadecanyl chains. Manzamenones M (2) and N (3) showed antimicrobial activity against several bacteria and fungi.     

Electrotonic Coupling between Adjacent Internodal Cells of Chara braunii: Transmission of Action Potentials beyond the Node

Many nodal cells are interposed between two internodal cellsof Chara braunii. When an action potential conducted in an internodereached the node, no electrical activation in the nodal cellscould be found, although an area of the membrane bordering thenodal cells in this internode was partially activated (end-membraneaction potential). When the action potential approached thenode along the stimulated internode, an electrotonic potentialchange (depolarization) was produced in the other internode.This depolarization was greatly depressed by the end-membraneaction potential of the stimulated internode, so that hardlyany transmission took place. The ratio of the potential changein the surface membrane of the adjoining ("postsynaptic") internode(cell b) to that of the stimulated one (cell a), the couplingratio, e_b/e_a, can be estimated from a simple equivalent circuitof the nodal region composed of two surface-membrane resistances(R_a, R_b) and intercellular resistance (R_n). If R_n remains thesame, a higher ratio should be produced with a larger R_b, butthe ratio does not depend on any change in R_a, which could beproved experimentally. Transmission of the action potentialbeyond the node was frequent when the coupling ratio was increasedand when the threshold that elicits the action potential waslowered by immersing the node in a K or Na salt solution. ¹ Present address: Department of Physiology, Tohoku UniversitySchool of Dentistry, Sendai 980, Japan. (Received December 1, 1980; Accepted January 23, 1981)     

A large scale analysis of cDNA in Arabidopsis thaliana: generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries.

For comprehensive analysis of genes expressed in the model dicotyledonous plant, Arabidopsis thaliana, expressed sequence tags (ESTs) were accumulated. Normalized and size-selected cDNA libraries were constructed from aboveground organs, flower buds, roots, green siliques and liquid-cultured seedlings, respectively, and a total of 14,026 5'-end ESTs and 39,207 3'-end ESTs were obtained. The 3'-end ESTs could be clustered into 12,028 non-redundant groups. Similarity search of the non-redundant ESTs against the public non-redundant protein database indicated that 4816 groups show similarity to genes of known function, 1864 to hypothetical genes, and the remaining 5348 are novel sequences. Gene coverage by the non-redundant ESTs was analyzed using the annotated genomic sequences of approximately 10 Mb on chromosomes 3 and 5. A total of 923 regions were hit by at least one EST, among which only 499 regions were hit by the ESTs deposited in the public database. The result indicates that the EST source generated in this project complements the EST data in the public database and facilitates new gene discovery.     

Ephaptic Transmission and Conduction Velocity of an Action Potential in Chara Internodal Cells Placed in Parallel and in Contact with One Another

When two separated Chara internodal cells were kept in contactover a length of 14 mm or more in moist air, an action potentialof one cell could be transmitted to the other cell in about40% of cases (ephaptic transmission). The action potential ofthe former cell was sometimes eliminated transiently when anew action potential was elicited in the latter cell. The newaction potential reactivated the former cell. The conductionvelocity of the action potential was reduced from 0.30?0.11cm/s (mean?SD) to 0.15?0.05 cm/s by ephaptic transmission ofthe action potential. Substitution of an artificial pond waterfor moist air or contact over a shorter length (4 mm insteadof 14 mm or more) between the two cells reduced the couplingratio, the ratio of the change in membrane potential of onecell to that of the other cell, and no transmission was observed.After ephaptic transmission, the action potentials of the twocells were conducted at almost the same velocity along the cells.The velocity was increased to 0.68?0.06 cm/s by simultaneousconduction of the action potentials in the two adjacent cells.When the simultaneously conducted action potentials reachedthe node of one cell, at the point at which another cell madecontact, they were able very frequently to trigger a new actionpotential in the adjoining cell. (Received September 28, 1989; Accepted April 5, 1990)     

Therapeutic effect of risedronate on cancellous and cortical bone in ovariectomized osteopenic rats: a comparison with the effects of alfacalcidol.

The purpose of the present study was to compare the therapeutic effects of risedronate (RIS) and alfacalcidol (ALF) on cancellous and cortical bone in ovariectomized osteopenic rats. Forty-two female Sprague-Dawley rats, 7 months of age, were randomized by the stratified weight method into six groups: the sham-operated control (Sham) group, and five ovariectomized groups: treated with vehicle, RIS (0.1, 1.0, or 2.5 mg/kg, p.o., daily), and ALF (0.5 microg/kg, p.o., daily). Treatment was started 6 weeks after surgery and continued for 6 weeks. Evaluation at 12 weeks after surgery revealed that ovariectomy (OVX) decreased the cancellous bone volume/total tissue volume (BV/TV) of the proximal tibial metaphysis as a result of an increase of the bone formation rate/bone surface (BFR/BS), BFR/BV, and eroded surface (ES/BS), while having no effect on the cortical area (Ct Ar) of the tibial diaphysis. OVX also decreased the maximum load of the femoral distal metaphysis, while having no effect on any mechanical property parameters of the femoral diaphysis. RIS (at all the doses) increased the BV/TV relative to the value in the OVX-Vehicle group, but the value was not restored to that observed in the Sham group. The effects of RIS (1.0 mg/kg and 2.5 mg/kg) were similar, and greater than those of RIS (0.1 mg/kg). ALF also increased the BV/TV relative to the OVX-Vehicle group, but the value was not restored to that observed in the Sham group, similar to the results of RIS (1.0 mg/kg and 2.5 mg/kg) treatment. The alterations of the structural parameters induced by RIS (at the doses) were attributable to suppression of the increase of ES/BS, BFR/BS, and BFR/BV. The alterations of the structural parameters induced by ALF were attributable to suppression of the increase of ES/BS and attenuation of the increase of BFR/BV, while the BFR/BS was maintained. ALF also increased the Ct Ar to beyond the value observed in the Sham group. RIS (at all the doses) had no effect on the mechanical properties of the femoral distal metaphysis, whereas ALF prevented the loss of the maximum load of the femoral distal metaphysis. Thus, the results of the present study show differential effects of RIS and ALF on cancellous and cortical bone in ovariectomized osteopenic rats.     

Effect of oxygen supply on berberine production in cell suspension cultures and immobilized cells ofThalictrum minus

The ample supply of O₂ proved to be of great importance for berberine production in cell suspension culture ofThalictrum minus, as the specific O₂ consumption rate of berberine-producing cells was twice as high as that of non-producing cells. Furthermore, berberine yield increased with increases in the volumetric O₂ transfer coefficient (K_La). Estimation of the optimum conditions of oxygen supply in suspension cultures and immobilized cells according to a known theoretical model assuming O₂ uptake by cells to be a zero-order reaction was in good agreement with the experimental data. The O₂ supply to immobilized cells could be improved by reducing the cell density and radius of the bead.