Orientation of Microtubules during Regeneration of Cell Wall in Selected Giant Marine Algae

The microtubules in highly synchronized aplanospores of twogiant marine algae, Boergesenia forbesii and Valonia ventricosa,were examined by immunofluorescence microscopy throughout theregeneration of the cell wall. Microtubule orientation was alwaysrandom up to 20 h after wounding, although the orientation ofcellulose microfibrils changed from random to parallel withinthat time period. When the rhizoid cells were in the stage ofelongation at 7 to 10 days after wounding, highly ordered microtubuleswere always observed along the longitudinal cell axis exceptat the very tip of the cells where random ones were found. Incontrast, the microfibrils in the innermost lamellae of newlysynthesized cell walls showed three different orientations,that is, transverse, longitudinal and oblique to the longitudinalcell axis. These observations suggest that microtubules maycontrol cell shape, but not the orientation of microfibrils.The mechanism of cell wall construction in these algae is discussedin relation to the self-assembly mechanism thought to operatein the construction of helicoidal cell walls. ³ Present address: Polymer Research Laboratory, Mitsui ToatsuChemicals, Inc., Yokohama, Kanagawa 244, Japan. (Received November 18, 1987; Accepted April 11, 1988)     

Amino Acid Sequence of Triculamin

The amino acid sequence of triculamin is established from the amino acid composition of the peptides, which have been obtained by partial acid hydrolysis. It is a branched peptides, and a possible structure of the branched parts is proposed.     

H89 sensitive kinase regulates the translocation of Sar1 onto the ER membrane through phosphorylation of ER-coupled β-tubulin

ER-to-Golgi protein transport is carried out by transport vesicles which are formed at the ER-exit sites with recruitment of cytoplasmic coat proteins. Vesicle formation is initiated by assembly of the small G protein (Sar1) onto the ER membrane. Sar1 assembly onto the ER membrane is suppressed by protein kinase inhibitor H89, suggesting participation of H89-sensitive kinase in this process. The present study identified an effector of H89-sensitive kinase by LC-MS PMF analysis combined with 1D- and 2D-PAGE autoradiography, and examined the changes on the effector and Sar1 translocation induced by H89. H89 significantly suppressed the phosphorylation of 55 kDa protein with dosage dependency, and phosphorylation of 55 kDa, pI 5.5 protein spot in 2-D-autoradiography was drastically diminished by H89. LC-MS PMF analysis showed that the protein spot was β-tubulin. H89 significantly suppressed Sar1 translocation onto the ER. These findings indicate that β-tubulin is one of downstream effectors of H89-sensitive kinase, and that suppression of ER-coupled β-tubulin phosphorylation decreases Sar1 translocation onto the ER, suggesting that phosphorylation of β-tubulin regulates Sar1 translocation.     

Breaking the proximal Fe(II)-N(His) bond in heme proteins through local structural tension: lessons from the heme b proteins nitrophorin 4, nitrophorin 7, and related site-directed mutant proteins

The factors leading to the breakage of the proximal iron-histidine bond in the ferroheme protein soluble guanylate cyclase (sGC) are still a matter of debate. This event is a key mechanism in the sensing of NO that leads to the production of the second-messenger molecule cGMP. Surprisingly, in the heme protein nitrophorin 7 (NP7), we noticed by UV-vis absorbance spectroscopy and resonance Raman spectroscopy that heme reduction leads to a loss of the proximal histidine coordination, which is not observed for the other isoproteins (NP1-4). Structural considerations led to the generation and spectroscopic investigation of site-directed mutants NP7(E27V), NP7(E27Q), NP4(D70A), and NP2(V24E). Spectroscopic investigation of these proteins shows that the spatial arrangement of residues Glu27, Phe43, and His60 in the proximal heme pocket of NP7 is the reason for the weakened Fe(II)-His60 bond through steric demand. Spectroscopic investigation of the sample of NP7 reconstituted with 2,4-dimethyldeuterohemin ("symmetric heme") demonstrated that the heme vinyl substituents are also responsible. Whereas the breaking of the iron-histidine bond is rarely seen among unliganded ferroheme proteins, the breakage of the Fe(II)-His bond upon binding of NO to the sixth coordination site is sometimes observed because of the negative trans effect of NO. However, it is still rare among the heme proteins, which is in contrast to the case for trans liganded nitrosyl model hemes. Thus, the question of which factors determine the Fe(II)-His bond labilization in proteins arises. Surprisingly, mutant NP2(V24E) turned out to be particularly similar in behavior to sGC; i.e., the Fe(II)-His bond is sensitive to breakage upon NO binding, whereas the unliganded form binds the proximal His at neutral pH. To the best of our knowledge, NP2(V24E) is the first example in which the ability to use the His-on ? His-off switch was engineered into a heme protein by site-directed mutagenesis other than the proximal His itself. Steric tension is, therefore, introduced as a potential structural determinant for proximal Fe(II)-His bond breakage in heme proteins.     

Disease-Association Analysis of an Inflammation-Related Feedback Loop

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Highlights? The IL-6 amplifier is associated with various human diseases and disorders ? Genome-wide screens can isolate IL-6-amplifier-related genes ? These genes include those associated with human diseases ? Identified genes, e.g., the epiregulin-ErbB1 axis, may be therapeutic targets     

Xanthine Oxidase Mediates Axonal and Myelin Loss in a Murine Model of Multiple Sclerosis

Objectives

Oxidative stress plays an important role in the pathogenesis of multiple sclerosis (MS). Though reactive oxygen species (ROS) are produced by various mechanisms, xanthine oxidase (XO) is a major enzyme generating ROS in the context of inflammation. The objectives of this study were to investigate the involvement of XO in the pathogenesis of MS and to develop a potent new therapy for MS based on the inhibition of ROS.

Methods

XO were assessed in a model of MS: experimental autoimmune encephalomyelitis (EAE). The contribution of XO-generated ROS to the pathogenesis of EAE was assessed by treating EAE mice with a novel XO inhibitor, febuxostat. The efficacy of febuxostat was also examined in in vitro studies.

Results

We showed for the first time that the expression and the activity of XO were increased dramatically within the central nervous system of EAE mice as compared to naïve mice. Furthermore, prophylactic administration of febuxostat, a XO inhibitor, markedly reduced the clinical signs of EAE. Both in vivo and in vitro studies showed infiltrating macrophages and microglia as the major sources of excess XO production, and febuxostat significantly suppressed ROS generation from these cells. Inflammatory cellular infiltration and glial activation in the spinal cord of EAE mice were inhibited by the treatment with febuxostat. Importantly, therapeutic efficacy was observed not only in mice with relapsing-remitting EAE but also in mice with secondary progressive EAE by preventing axonal loss and demyelination.

Conclusion

These results highlight the implication of XO in EAE pathogenesis and suggest XO as a target for MS treatment and febuxostat as a promising therapeutic option for MS neuropathology.     

Effects of Hydrocarbons on the Morphology of Candida tropicalis pK 233

Candida tropicalis pK 233 exhibited marked morphological changes depending upon carbon sources for growth. Although the yeast showed a typical yeast-like development when grown on glucose, the cells grown on hydrocarbon or ethanol were composed of a mixture of filamentous-form (F-cells) and yeast-form cells (Y-cells). The carbon chain lengths of n-alkanes tested as growth substrates had a significant influence on the ratio of F-cells to Y-cells. Electronmicroscopic observation revealed that a hypha was divided by septa into several cells.

Separation of Y-cells and F-cells was achieved by using a suitable filter cloth. F-cells gave a high Qo₂ value compared with Y-cells when hydrocarbon was used as oxidation substrate, even though there was little difference between the respiratory activities of these two cells measured with glucose.     

Syntheses of New Quarternary Ammonium Iodides as Plant Growth Retardants and Their Biological Activities

Twelve new quarternary ammonium compounds were synthesized and their plant growth retarding activities were examined. Among the candidates, N,N,N-trimethyl-l-methyl-3-(3′,3′5′-trimethylcyclohexyl)- and N,N,N-trimethyl-l-methyl-3-(3′,3′,5′,5′-tetramethylcyclohexyl)-2-propenylammonium iodides were the most effective to suppress the growth of rice and cucumber seedlings, and their activities were far stronger than those of any growth retardants hitherto known.     

Induction of Catalase Activity in a Methanol-utilizing Yeast,Kloeckera sp. No. 2201

The methanol-grown cells of Kloeckera sp. No. 2201 exhibit a markedly high catalase activity as compared with the glucose-grown and ethanol-grown cells. In this connection, specific organelles (“microbodies”) appear only in the methanol-grown cells. When the yeast cells harvested from a methanol medium (cells whose catalase activity had been enhanced to an appreciable extent: “partially induced cells”) were transferred into media containing glucose, ethanol or methanol as the sole carbon and energy source, further increase of catalase activity was mediated only by methanol. This induction of catalase activity was partially inhibited by cycloheximide at its high concentration, but chloramphenicol did not show any effect. Glucose inhibited strongly the induction by methanol, while galactose gave no effect. Electron microscopical observation revealed that the development of microbodies in the cells growing on methanol was hardly affected by cycloheximide. Disappearance of microbodies was observed electron microscopically after the methanol-grown cells (partially induced cells) were transferred to a methanol-glucose medium and cultivated for 8 hr. 3′,5′-Cyclic AMP or dibutyryl-3′,5′-cyclic AMP could not eliminate the inhibitory effect of glucose on the catalase induction. Addition of caffeine or theophylline did not promote the action of the cyclic nucleotides. 3-Amino-1,2,4-triazole inhibited only 40% of the hydrogen peroxide-decomposing activity in the cell homogenate of methanol-grown cells even at its concentration of as high as 10 mm, while sodium azide inhibited the enzyme activity completely at the concentration of 1 mm.