Novel Method for Detection of Butanolides in Streptomyces coelicolor Culture Broth, Using a His-Tagged Receptor (ScbR) and Mass Spectrometry

γ-Butyrolactone derivative molecules in Streptomyces play a crucial role in cell density control, secondary metabolism, and cell differentiation. As their synthesis level in the cell is very low compared to those of similar N-acyl homoserine lactone molecules from gram-negative bacteria, it is very hard to analyze them even with several hundredfold concentration of the culture broth. We have developed a very quick and easy detection method using an affinity capture technique with His-tagged receptor proteins and electrospray tandem mass spectrometry. Using Streptomyces coelicolor as a model system, SCB1 was detected from only 100 ml of the culture broth after solvent extraction. This method can be further applied to detection and quantitative analysis of butanolides and inhibitor screening of the receptor molecules.     

A Putative Calcium-Permeable Cyclic Nucleotide-Gated Channel, CNGC18, Regulates Polarized Pollen Tube Growth

A tip-focused Ca^2＋ gradient is tightly coupled to polarized pollen tube growth, and tip-localized influxes of extracellular Ca^2＋ are required for this process. However the molecular identity and regulation of the potential Ca^2＋ channels remains elusive. The present study has implicated CNGC18 （cyclic nucleotide-gated channel 18） in polarized pollen tube growth, because its overexpression induced wider and shorter pollen tubes. Moreover, CNGC18 overexpression induced depolarization of pollen tube growth was suppressed by lower extracellular calcium （[Ca^2＋]ex）. CNGC18-yellow fluorescence protein （YFP） was preferentially localized to the apparent post-Golgi vesicles and the plasma membrane （PM） in the apex of pollen tubes. The PM localization was affected by tip-localized ROP1 signaling. Expression of wild type ROP1 or an active form of ROP1 enhanced CNGC18-YFP localization to the apical region of the PM, whereas expression of RopGAP1 （a ROP1 deactivator） blocked the PM localization. These results support a role for PM-Iocalized CNGC18 in the regulation of polarized pollen tube growth through its potential function in the modulation of calcium influxes.     

Assignment of a human serum glycoprotein SP-40,40 gene (CLI) to chromosome 8.

SP-40,40 is a serum glycoprotein consisting of two different subunits (alpha and beta) assembled into a dimer by disulfide bonds. Northern blot hybridization, using total RNA from several cell lines, showed that SP-40,40 is expressed in glioblastoma and testicular tumor cells, as well as hepatoma cells. Spot blot hybridization of flow-sorted human chromosomes, using a SP-40,40 cDNA fragment as a probe, localized the gene for SP-40,40 to human chromosome 8. This gene has been given the designation CLI, for complement lysis inhibitor, by the Human Gene Nomenclature Committee.     

Dissociable Perceptual Effects of Visual Adaptation

Neurons in the visual cortex are responsive to the presentation of oriented and curved line segments, which are thought to act as primitives for the visual processing of shapes and objects. Prolonged adaptation to such stimuli gives rise to two related perceptual effects: a slow change in the appearance of the adapting stimulus (perceptual drift), and the distortion of subsequently presented test stimuli (adaptational aftereffects). Here we used a psychophysical nulling technique to dissociate and quantify these two classical observations in order to examine their underlying mechanisms and their relationship to one another. In agreement with previous work, we found that during adaptation horizontal and vertical straight lines serve as attractors for perceived orientation and curvature. However, the rate of perceptual drift for different stimuli was not predictive of the corresponding aftereffect magnitudes, indicating that the two perceptual effects are governed by distinct neural processes. Finally, the rate of perceptual drift for curved line segments did not depend on the spatial scale of the stimulus, suggesting that its mechanisms lie outside strictly retinotopic processing stages. These findings provide new evidence that the visual system relies on statistically salient intrinsic reference stimuli for the processing of visual patterns, and point to perceptual drift as an experimental window for studying the mechanisms of visual perception.     

Responses of Mentha suspension-cultured cells to 2,4-dichlorophenoxyacetic acid and accumulation of esterified phenolic acids in their cell walls.

Two distinct types of cell growth of suspension-cultured Mentha were formed when the cells maintained in the medium containing 1000 micrograms l-1 2,4-D were subcultured into different 2,4-D concentrations. Few cell elongation of Mentha (average cell length: 34-40 microns) was observed after division in the medium containing 1-200 micrograms l-1 2,4-D; and significant cell elongation (average cell length: 95-130 microns) was observed after cell division in the medium containing 500-2000 micrograms l-1 2,4-D. A close correlation between culture medium and water content in the cells indicated that 2,4-D promoted cell elongation by water uptake. Amounts of phenolic acid in cell walls were much higher in unelongated cell walls than in elongated ones during the cultivation, and there was a close correlation between the amounts and the level of PAL activity in elongated and unelongated cells. However, there was no significant difference in cell wall components and its neutral sugar composition between elongated and unelongated cells.     

Change of hepatitis B virions (Dane particles) phosphorylation pattern by human hepatoma cell particulate fraction

Protein kinase activity has been found in hepatitis B virions (Dane particles) purified from the plasma of hepatitis B surface antigen carriers [Albin, C., and Robinson, W.S. (1980) J. Virol. 34, 297-302]. Dane particles were purified from the pooled, HBeAg-positive plasma. When this preparation was incubated with [gamma 32P]ATP in the presence of 10mM MnCl2 and 0.5% NP-40 for 15 seconds at 30 degrees C, several phosphorylated polypeptides of 20,000, 42,000, 48,000, 50,000 and 56,000 daltons were detected in sodium dodecyl sulfate-polyacrylamide gels. When the Dane particles were incubated with [gamma 32P]ATP, 10 mM MnCl2, and 0.5% NP-40 in the presence of human hepatoma cell (J-5) particulate fraction at 30 degrees C, 15 seconds, the 42,000, 48,000 and 50,000 daltons phosphorylated polypeptides were not found. When human peripheral blood lymphocytes particulate fraction was incubated with Dane particles under the same conditions, no change of Dane particle phosphorylated polypeptides was detected. Previous publications [Albin, C., and Robinson, W.S. (1980) J. Virol. 34, 297-302; Gerlich, W.H. et al. (1982) J. Virol. 42, 761-766] showed that when hepatitis B core particles purified from hepatoma tissues contained protein kinase activity, only phosphorylated polypeptide was 20,000 daltons. Our data suggested that when Dane particles were put in an environment of hepatoma cells (or tissues), the protein kinase could only phosphorylate selected polypeptides in these particles.     

NMR study of isoleucine transfer RNA from Thermus thermophilus

An NMR and nuclear Overhauser effect (NOE) analysis of Thermus thermophilus tRNAIle1a is presented. This species contains modifications including s2T54 and s4U8 [Horie, N., Hara-Yokoyama, M., Yokoyama, S., Watanabe, K., Kuchino, Y., Nishimura, S., & Miyazawa, T. (1985) Biochemistry 24, 5711-5715]. All the expected secondary and reverse Hoogsteen AU pairs were identified, with one possible exception. The general geometry of the T psi C loop is the same as the Escherichia coli species, and there is NOE evidence for an A9-UA12 triple. Preliminary measurements of solvent exchange rates of internally hydrogen-bonded bases suggest that this tRNA is more stable than previously studied E. coli and yeast tRNAs.     

Genetics and Mechanism of Resistance to Meloidogyne arenaria in Peanut Germplasm

Segregation of resistance to Meloidogyne arenaria in six BC₅F₂ peanut breeding populations was examined in greenhouse tests. Chi-square analysis indicated that segregation of resistance was consistent with resistance being conditioned by a single gene in three breeding populations (TP259-3, TP262-3, and TP271-2), whereas two resistance genes may be present in the breeding populations TP259-2, TP263-2, and TP268-3. Nematode development in clonally propagated lines of resistant individuals of TP262-3 and TP263-2 was compared to that of the susceptible cultivar Florunner. Juvenile nematodes readily penetrated roots of all peanut genotypes, but rate of development was slower (P = 0.05) in the resistant genotypes than in Florunner. Host cell necrosis indicative of a hypersensitive response was not consistently observed in resistant genotypes of either population. Three RFLP loci linked to resistance at distances of 4.2 to 11.0 centiMorgans were identified. Resistant and susceptible alleles for RFLP loci R2430E and R2545E were quite distinct and are useful for identifying individuals homozygous for resistance in segregating populations.