Recording site dependence of the neuronal spiking statistics

Spiking characteristics of neurons in the middle temporal (MT) area and the medial superior temporal (MST) area in the visual cortex of a monkey are compared with the ones in the principal sulcus (PS) area in the prefrontal cortex. The comparison is based on the basis of three inter-spike interval statistical measures: the coefficient of variation (CV), the skewness coefficient (SK) and the correlation coefficient of consecutive intervals (COR). Even for the spike sequences recorded from the same neuron, three coefficients computed from 100 intervals do not always exhibit similar values, but distribute rather widely. The distribution of three coefficients obtained from a single neuron in the MST area does not largely deviate from the distribution obtained from multiple neurons in MT and MST areas. Those distributions, however, largely deviate from the distribution obtained from neurons in the PS area. In this way, the distribution of those statistical coefficients reflects the nature of the recording site.     

Interactions among PIN-FORMED and P-glycoprotein auxin transporters in Arabidopsis

Directional transport of the phytohormone auxin is established primarily at the point of cellular efflux and is required for the establishment and maintenance of plant polarity. Studies in whole plants and heterologous systems indicate that PIN-FORMED (PIN) and P-glycoprotein (PGP) transport proteins mediate the cellular efflux of natural and synthetic auxins. However, aromatic anion transport resulting from PGP and PIN expression in nonplant systems was also found to lack the high level of substrate specificity seen in planta. Furthermore, previous reports that PGP19 stabilizes PIN1 on the plasma membrane suggested that PIN-PGP interactions might regulate polar auxin efflux. Here, we show that PGP1 and PGP19 colocalized with PIN1 in the shoot apex in Arabidopsis thaliana and with PIN1 and PIN2 in root tissues. Specific PGP-PIN interactions were seen in yeast two-hybrid and coimmunoprecipitation assays. PIN-PGP interactions appeared to enhance transport activity and, to a greater extent, substrate/inhibitor specificities when coexpressed in heterologous systems. By contrast, no interactions between PGPs and the AUXIN1 influx carrier were observed. Phenotypes of pin and pgp mutants suggest discrete functional roles in auxin transport, but pin pgp mutants exhibited phenotypes that are both additive and synergistic. These results suggest that PINs and PGPs characterize coordinated, independent auxin transport mechanisms but also function interactively in a tissue-specific manner.     

Type-specific regulation of adenylyl cyclase. Selective pharmacological stimulation and inhibition of adenylyl cyclase isoforms

Crystallographic studies have elucidated the binding mechanism of forskolin and P-site inhibitors to adenylyl cyclase. Accordingly, computer-assisted drug design has enabled us to identify isoform-selective regulators of adenylyl cyclase. After examining more than 200 newly synthesized derivatives of forskolin, we found that the modification at the positions of C6 and C7, in general, enhances isoform selectivity. The 6-(3-dimethylaminopropionyl) modification led to an enhanced selectivity for type V, whereas 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] and 6-(4-acrylbutyryl) modification led to an enhanced selectivity for type II. In contrast, 2'-deoxyadenosine 3'-monophosphate, a classical and 3'-phosphate-substituted P-site inhibitor, demonstrated a 27-fold selectivity for inhibiting type V relative to type II, whereas 9-(tetrahydro-2-furyl) adenine, a ribose-substituted P-site ligand, showed a markedly increased, 130-fold selectivity for inhibiting type V. Consequently, on the basis of the pharmacophore analysis of 9-(tetrahydro-2-furyl) adenine and adenylyl cyclase, a novel non-nucleoside inhibitor, 2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80), was identified after virtual screening of more than 850,000 compounds. NKY80 demonstrated a 210-fold selectivity for inhibiting type V relative to type II. More importantly, the combination of a type III-selective forskolin derivative and 9-(tetrahydro-2-furyl) adenine or NKY80 demonstrated a further enhanced selectivity for type III stimulation over other isoforms. Our data suggest the feasibility of adenylyl cyclase isoform-targeted regulation of cyclic AMP signaling by pharmacological reagents, either alone or in combination.     

Multiple signalling pathways mediate fungal elicitor-induced beta-thujaplicin biosynthesis in Cupressus lusitanica cell cultures

The biosynthesis of a phytoalexin, beta-thujaplicin, in Cupressus lusitanica cell cultures can be stimulated by a yeast elicitor, H(2)O(2), or methyl jasmonate. Lipoxygenase activity was also stimulated by these treatments, suggesting that the oxidative burst and jasmonate pathway may mediate the elicitor-induced accumulation of beta-thujaplicin. The elicitor signalling pathway involved in beta-thujaplicin induction was further investigated using pharmacological and biochemical approaches. Treatment of the cells with calcium ionophore A23187 alone stimulated the production of beta-thujaplicin. A23187 also enhanced the elicitor-induced production of beta-thujaplicin. EGTA, LaCl(3), and verapamil pretreatments partially blocked A23187- or yeast elicitor-induced accumulation of beta-thujaplicin. These results suggest that Ca(2+) influx is required for elicitor-induced production of beta-thujaplicin. Treatment of cell cultures with mastoparan, melittin or cholera toxin alone or in combination with the elicitor stimulated the production of beta-thujaplicin or enhanced the elicitor-induced production of beta-thujaplicin. The G-protein inhibitor suramin inhibited the elicitor-induced production of beta-thujaplicin, suggesting that receptor-coupled G-proteins are likely to be involved in the elicitor-induced biosynthesis of beta-thujaplicin. Indeed, both GTP-binding activity and GTPase activity of the plasma membrane were stimulated by elicitor, and suramin and cholera toxin affected G-protein activities. In addition, all inhibitors of G-proteins and Ca(2+) flux suppressed elicitor-induced increases in lipoxygenase activity whereas activators of G-proteins and the Ca(2+) signalling pathway increased lipoxygenase activity. These observations suggest that Ca(2+) and G-proteins may mediate elicitor signals to the jasmonate pathway, and the jasmonate signalling pathway may then lead to the production of beta-thujaplicin.     

Species separation in <Emphasis Type="Italic">Curvularia</Emphasis> “geniculata” group inferred from <Emphasis Type="Italic">Brn1</Emphasis> gene sequences

 Brn1, a reductase gene involved in the melanin biosynthetic pathway, was adopted for species delimitation among members in the “geniculata” group of Curvularia species and proved to be useful for this purpose. Phylogenetic trees of these fungal members were constructed from nucleotide sequences of this region. The so-called geniculata group of Curvularia was separated into several clusters. The conidial morphology of the members in each cluster is closely similar but clearly different among discrete clusters. The phylogenetic groups almost concurred with the morphological grouping. Thus, the synonymous treatment of Curvularia affinis, C. fallax, and C. senegalensis to C. geniculata in a previous study was supported. The isolates with warping hilum conidia were clearly different from C. geniculata and separated into two clusters. C. geniculata ATCC 6671 made an independent cluster situated near these clusters. The protuberant hilum species were located separately in the phylogenetic trees. For sound taxonomic treatment of these isolates, we should accumulate more information and retain our species determination for them. Received: September 26, 2002 / Accepted: March 12, 2003     

Tight binding of influenza virus hemagglutinin to its receptor interferes with fusion pore dilation

Deletion of oligosaccharide side chains near the receptor binding site of influenza virus A/USSR/90/77 (H1N1) hemagglutinin (HA) enhanced the binding of HA to erythrocyte receptors, as was also observed with A/FPV/Rostock/34 (H7N1). Correlated with the enhancement of binding activity, the cell fusion activity of HA was reduced. A mutant HA in which three oligosaccharide side chains were deleted showed the highest level of binding and the lowest level of fusion among the HAs tested. The cell fusion activity of the oligosaccharide deletion mutant of HA, however, was drastically elevated when the binding activity was reduced by deletion of four amino acids adjacent to the receptor binding site. Thus, a reciprocal relationship was observed between the receptor binding and the cell fusion activities of H1/USSR HA. No difference was observed, however, in lipid mixing activity, so-called hemifusion, between wild-type (WT) and oligosaccharide deletion mutant HAs. Soluble dye transfer testing showed that even the HA with the lowest cell fusion activity was able to form fusion pores through which a small molecule such as calcein could pass. However, electron microscopic studies revealed that a large molecule such as hemoglobin hardly passed through the fusion pores formed by the mutant HA, whereas hemoglobin did efficiently pass through those formed by the WT HA. These results suggested that interference in the process of dilation of fusion pores occurs when the binding of HA to the receptor is too tight. Since the viral nucleocapsid is far larger than hemoglobin, appropriate receptor binding affinity is important for virus entry.     

Relationship between the lifestyle of a Siboglinid (Pogonophoran) polychaete, Oligobrachia mashikoi, and the total sulfide and nitrogen levels in its habitat

A gutless polychaete of the family Siboglinidae, Oligobrachia mashikoi, known in the past as a beard worm of the group Pogonophora, inhabits Tsukumo Bay of the Noto Peninsula in the Sea of Japan. Photographs were taken of this polychaete projecting about one third of the length of its tentacles outside of its tube. The tube protruded several mm from the sea bottom. These are the first field photographs of beard worms. The trophosome of this beard worm harbors sulfur-oxidizing bacteria. In fact, the muddy sediment where this worm inhabits smells slightly of hydrogen sulfide. Total sulfide levels, which can be an indicator of the generation of hydrogen sulfide gas, were measured at 10 locations in the bay. Furthermore, at the location which this species inhabits, the total sulfide levels in the vertical direction were determined. In addition, the total nitrogen levels, which can indicate the quantity of organic substances, were measured. The sediment inhabited by this worm was determined to have total sulfide levels of 0.24-0.39 mg/g dry mud, measured in the form of acid-volatile sulfide-sulfur. The total nitrogen levels were 1.0-1.5 microg/mg dry mud. These values suggest that the bottom of Tsukumo Bay has not been deteriorated by eutrophication. The levels were, however, highest in the surface layer of the sediment. These results suggest that hydrogen sulfide is generated in the surface of the sediment by sulfate-reducing bacteria, and that O. mashikoi appears to able to live in an environment that contains a slight amount of sulfide.     

The Arabidopsis OBERON1 and OBERON2 genes encode plant homeodomain finger proteins and are required for apical meristem maintenance

Maintenance of the stem cell population located at the apical meristems is essential for repetitive organ initiation during the development of higher plants. Here, we have characterized the roles of OBERON1 (OBE1) and its paralog OBERON2 (OBE2), which encode plant homeodomain finger proteins, in the maintenance and/or establishment of the meristems in Arabidopsis. Although the obe1 and obe2 single mutants were indistinguishable from wild-type plants, the obe1 obe2 double mutant displayed premature termination of the shoot meristem, suggesting that OBE1 and OBE2 function redundantly. Further analyses revealed that OBE1 and OBE2 allow the plant cells to acquire meristematic activity via the WUSCHEL-CLAVATA pathway, which is required for the maintenance of the stem cell population, and they function parallel to the SHOOT MERISTEMLESS gene, which is required for preventing cell differentiation in the shoot meristem. In addition, obe1 obe2 mutants failed to establish the root apical meristem, lacking both the initial cells and the quiescent center. In situ hybridization revealed that expression of PLETHORA and SCARECROW, which are required for stem cell specification and maintenance in the root meristem, was lost from obe1 obe2 mutant embryos. Taken together, these data suggest that the OBE1 and OBE2 genes are functionally redundant and crucial for the maintenance and/or establishment of both the shoot and root meristems.