Dynamic Organization of Microtubules in Guard Cells of Viciafaba L. with Diurnal Cycle

Stomatal movement is regulated by changes in the volume of guardcells, thought to be mainly controlled by an osmo-regulatorysystem. In the present study, we examined the additional involvementof cytoskeletal events in the regulation of stomatal movement.Microtubules (MTs) in guard cells of Viciafaba L., grown undersunlight, were observed during the day and night by immunofluorescencemicroscopy. Cortical MTs began to be organized in a radial arrayat dawn and increased in numbers in the morning following theincrease in the stomatal aperture size. Thereafter, MTs becamelocalized near the nucleus and began to be destroyed from theevening to midnight, following the decrease in stomatal aperturesize. These diurnal changes in MT organization were observedeven two days after transfer from natural light condition tototal darkness, and were accompanied by corresponding changesin stomatal aperture. The increase in stomatal aperture sizein the early morning was inhibited by 50 µM propyzaraide,which destroys cortical MTs in guard cells, whereas the decreasein aperture size in the evening was suppressed by 10 µMtax-ol, which stabilizes cortical MTs. These results suggestthat radially-organized cortical MTs of guard cells may controldiurnal stomatal movement. (Received September 3, 1997; Accepted November 5, 1997)     

Cytotaxonomic study of genusHymenasplenium (Aspleniaceae) in Xishuangbanna, Southwestern China

The gametic chromosome numbers of sevenHymenasplenium (Aspleniaceae) species from Xishuangbanna, Yunnan Prov., China, were investigated. All the examined individuals ofH. obscurum, H. cheilosorum andH. latipinnum were sexual diploids with n=39 chromosomes. Intraspecific cytological variation was found inH. excisum, which has a sexual diploid (n=39) and a tetraploid (n=78). Only a triploid apogamous cytotype (n=ca.117) was found inH. laterepens. Hymenasplenium apogamum showed the most complicated intraspecific variation and included a sexual diploid (n=39), a sexual tetraploid (n=78) and an apogamous triploid (n=ca.117). This work reports for the first time the sexual diploids ofH. cheilosorum andH. apogamum, which are only apogamous elsewhere in east Asia, Himalayas and Indochina. These results may indicate that this area is one of the diversity centers ofHymenasplenium. Most of the above species have chromosome numbers based on x=39. In contrast,H. costarisorum contains a sexual diploid (n=36) and a sexual tetraploid (n=72), indicating that its basic number is x=36.     

Altered function of factor I caused by amyloid beta: implication for pathogenesis of age-related macular degeneration from Drusen

The results of recent studies have implicated local inflammation and complement activation as the processes involved in the pathogenesis of age-related macular degeneration (AMD). We have demonstrated that amyloid beta (Abeta), which is deposited in drusen, causes an imbalance in the angiogenesis-related factors in retinal pigment epithelial cells. We have also shown that neprilysin gene-disrupted mice accumulate Abeta, and develop several features of AMD. The purpose of this study was to investigate the mechanisms involved in the development of AMD that are triggered by Abeta. Our results showed that Abeta binds to complement factor I which inhibits the ability of factor I to cleave C3b to inactivated iC3b. Factor H and factor I are soluble complement-activation inhibitors, and preincubation of factor I with Abeta in the presence of factor H abolished the ability of Abeta to cleave C3b, and also abolished the ability of factor I to cleave FGR-AMC. In contrast, Abeta did not affect the function of factor H even after binding. The production of iC3b was significantly decreased when C3b and factor H were incubated with the eyes from neprilysin gene-disrupted mice as compared with when C3b and factor H were incubated with eyes from age-matched wild-type mice. These results suggest that Abeta activates the complement system within drusen by blocking the function of factor I leading to a low-grade, chronic inflammation in subretinal tissues. These findings link four factors that have been suggested to be associated with AMD: inflammation, complement activation, Abeta deposition, and drusen.     

Polysaccharide Lyase: Molecular Cloning, Sequencing, and Overexpression of the Xanthan Lyase Gene of Bacillus sp. Strain GL1

When grown on xanthan as a carbon source, the bacterium Bacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520–2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus (identity score, 37.7%). Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4°C, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) from Bacillus sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of Bacillus sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.     

Molecular cloning and characterization of neural activity-related RING finger protein (NARF): a new member of the RBCC family is a candidate for the partner of myosin V

Activity-dependent synaptic plasticity has been thought to be a cellular basis of memory and learning. The late phase of long-term potentiation (L-LTP), distinct from the early phase, lasts for up to 6 h and requires de novo synthesis of mRNA and protein. Many LTP-related genes are enhanced in the hippocampus during pentyrenetetrazol (PTZ)- and kainate (KA)-mediated neural activation. In this study, mice were administered intraperitoneal injections of PTZ 10 times, once every 48 h, and showed an increase in seizure indexes. Genes related to plasticity were efficiently induced in the mouse hippocampus. We used a PCR-based cDNA subtraction method to isolate genes that are expressed in the hippocampus of repeatedly PTZ-treated mice. One of these genes, neural activity-related RING finger protein (NARF), encodes a new protein containing a RING finger, B-box zinc finger, coiled-coil (RBCC domain) and beta-propeller (NHL) domain, and is predominantly expressed in the brain, especially in the hippocampus. In addition, KA up-regulated the expression of NARF mRNA in the hippocampus. This increase correlated with the activity of the NMDA receptor. By analysis using GFP-fused NARF, the protein was found to localize in the cytoplasm. Enhanced green fluorescent protein-fused NARF was also localized in the neurites and growth cones in neuronal differentiated P19 cells. The C-terminal beta-propeller domain of NARF interacts with myosin V, which is one of the most abundant myosin isoforms in neurons. The NARF protein increases in hippocampal and cerebellar neurons after PTZ-induced seizure. These observations indicated that NARF expression is enhanced by seizure-related neural activities, and NARF may contribute to the alteration of neural cellular mechanisms along with myosin V.     

Determination of nitrate in blood by gas chromatography and gas chromatography–mass spectrometry

We devised a sensitive and simple method for determining nitrate in whole blood, using an extractive alkylation technique. Nitrate in whole blood was reduced to nitrite by hydrazine sulfate in the presence of Cu²⁺ and Zn²⁺ as catalysts, and alkylated with pentafluorobenzyl bromide using tetradecyldimethylbenzylammonium chloride as the phase-transfer catalyst. The obtained derivative was analyzed qualitatively by gas chromatography–mass spectrometry and quantitatively by gas chromatography with electron-capture detection. The detection limit of nitrate in whole blood was 0.01 mM. The calibration curve was linear over the concentration range from 0.02 to 1.0 mM for nitrate in whole blood. The accuracy and precision of the method were evaluated and the relative standard deviations were found to be within 10%. Using this method, the blood nitrate levels of two victims who committed suicide by inhaling automobile exhaust gas were determined.     

Replacing the cyclohexene-linker of FR181157 leading to novel IP receptor agonists: orally active prostacyclin mimetics. Part 6

The synthesis and biological activity of novel derivatives of our previously reported IP receptor agonist FR181157 is described. SAR studies to replace the cyclohexene-linker of FR181157 led to the discovery of compound 1i (FR207845) as a potent non-prostanoid PGI2 mimetic with good oral bioavailability.     

Improved isotopic deuterium labeling at the diastereotopic methyl group of leucine: a synthetic route to (4S)- and (4R)-[5-2H1]leucine

Two isotopomers of deuterium-labeled leucine in the diastereotopic methyl group were synthesized from inexpensively available (R)- and (S)-citronellol in a more convenient way than by previous methods.