11C-methionine positron emission tomography for target delineation of recurrent glioblastoma in re-irradiation planning

Aim

To define the optimal margin on MRI scans in the re-radiation planning of recurrent glioblastoma using methionine positron emission tomography (MET-PET).

Calcium-activated neutral protease and its endogenous inhibitor Activation at the cell membrane and biological function

The structures of calcium-activated neutral protease (CANP) and its endogenous inhibitor elucidated recently have revealed novel features with respect to their structure-function relationship and enzyme activity regulation. The protease is regarded as a proenzyme which can be activated at the cell membrane in the presence of Ca²⁺ and phospholipid, and presumably regulates the functions of proteins, especially membrane-associated proteins, by limited proteolysis. Protein kinase C is hydrolysed and activated by CANP at the cell membrane to a cofactor-independent form. These results are reviewed and the possible involvement of CANP in signal transduction is discussed.     

In vivo delivery of small interfering RNA targeting brain capillary endothelial cells

Brain capillary endothelial cells (BCECs) play an important role in blood-brain barrier (BBB) functions and pathophysiologic mechanisms in brain ischemia and inflammation. We try to suppress gene expression in BCECs by intravenous application of small interfering RNA (siRNA). After injection of large dose siRNA with hydrodynamic technique to mouse, suppression of endogenous protein and the BBB function of BCECs was investigated. The brain-to-blood transport function of organic anion transporter 3 (OAT3) that expressed in BCECs was evaluated by Brain Efflux Index method in mouse. The siRNA could be delivered to BCECs and efficiently inhibited endogenously expressed protein of BCECs. The suppression effect of siRNA to OAT3 is enough to reduce the brain-to-blood transport of OAT3 substrate, benzylpenicillin at BBB. The in vivo siRNA-silencing method with hydrodynamic technique may be useful for the study of BBB function and gene therapy targeting BCECs.     

A novel thermostable branching enzyme from an extremely thermophilic bacterial species, Rhodothermus obamensis

A branching enzyme (EC 2.4.1.18) gene was isolated from an extremely thermophilic bacterium, Rhodothermus obamensis. The predicted protein encodes a polypeptide of 621 amino acids with a predicted molecular mass of 72 kDa. The deduced amino acid sequence shares 42-50% similarity to known bacterial branching enzyme sequences. Similar to the Bacillus branching enzymes, the predicted protein has a shorter N-terminal amino acid extension than that of the Escherichia coli branching enzyme. The deduced amino acid sequence does not appear to contain a signal sequence, suggesting that it is an intracellular enzyme. The R. obamensis branching enzyme was successfully expressed both in E. coli and a filamentous fungus, Aspergillus oryzae. The enzyme showed optimum catalytic activity at pH 6.0-6.5 and 65 degrees C. The enzyme was stable after 30 min at 80 degrees C and retained 50% of activity at 80 degrees C after 16 h. Branching activity of the enzyme was higher toward amylose than toward amylopectin. This is the first thermostable branching enzyme isolated from an extreme thermophile.     

A novel action of collapsin: Collapsin-1 increases antero- and retrograde axoplasmic transport independently of growth cone collapse

Chick collapsin-1, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Collapsin-1 induces growth cone collapse via a pathway which may include CRMP-62 and heterotrimeric G proteins. CRMP-62 protein is related to UNC-33, a nematode neuronal protein required for appropriately directed axonal extension. Mutations in unc-33 affect neural microtubules, the basic cytoskeletal elements for axoplasmic transport. Using computer-assisted video-enhanced differential interference contrast microscopy, we now demonstrate that collapsin-1 potently promotes axoplasmic transport. Collapsin-1 doubles the number of antero- and retrograde-transported organelles but not their velocity. Collapsin-1 decreases the number of stationary organelles, suggesting that the fraction of time during which a particle is moving is increased. Collapsin-1-stimulated transport occurs by a mechanism distinct from that causing growth cone collapse. Pertussis toxin (PTX) but not its B oligomer blocks collapsin-induced growth cone collapse. The holotoxin does not affect collapsin-stimulated axoplasmic transport. Mastoparan and a myelin protein NI-35 induce PTX-sensitive growth cone collapse but do not stimulate axoplasmic transport. These results provide evidence that collapsin has a unique property to activate axonal vesicular transport systems. There are at least two distinct pathways through which collapsin exerts its actions in developing neurons. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 316–328, 1997     

The systematic position ofAsplenium cardiophyllum (Aspleniaceae)

Asplenium cardiophyllum is a morphologically unusual species with simple leaves and anastomosing venation, and is often placed in the segregate genusBoniniella. To determine its systematic position, character comparisons were made of vascular anatomy, raphides in leaf epidermis, chromosome number and perispore of this species and those ofAsplenium sect.Hymenasplenium. Asplenium cardiophyllum conforms with sect.Hymenasplenium in its dorsiventral dictyostele, the presence of raphides, a chromosome number of 2n=156 (x=39), and lophate peristore with spinulate projections on the lumina. We therefore propose to includeA. cardiophyllum in that section. Dedicated to the memory of the late Professor Kunio Mitui.     

Quantitative measurement of damage caused by 1064-nm wavelength optical trapping of Escherichia coli cells using on-chip single cell cultivation system

We quantitatively examined the possible damage to the growth and cell division ability of Escherichia coli caused by 1064-nm optical trapping. Using the synchronous behavior of two sister E. coli cells, the growth and interdivision times between those two cells, one of which was trapped by optical tweezers, the other was not irradiated, were compared using an on-chip single cell cultivation system. Cell growth stopped during the optical trapping period, even with the smallest irradiated power on the trapped cells. Moreover, the damage to the cell's growth and interdivision period was proportional to the total irradiated energy (work) on the cell, i.e., irradiation time multiplied by irradiation power. The division ability was more easily affected by a smaller energy, 0.36 J, which was 30% smaller than the energy that adversely affected growth, 0.54 J. The results indicate that the damage caused by optical trapping can be estimated from the total energy applied to cells, and furthermore, that the use of optical trapping for manipulating cells might cause damage to cell division and growth mechanisms, even at wavelengths under 1064 nm, if the total irradiation energy is excessive.     

The cyanobacterial PilT protein responsible for cell motility and transformation hydrolyzes ATP

The unicellular cyanobacterium, Synechocystis sp. PCC 6803 is motile. A homologue of the PilT protein family, required for twitching motility in Pseudomonas aeruginosa and social gliding motility in Myxococcus xanthus, was found to be necessarily associated with cyanobacterial motility. The pilT1 (slr0161) mutant shows a pleotropic phenotype, defects in individual cell motility, and an increased number of long surface pili. Furthermore, the mutant loses its ability of natural competency. These findings demonstrate that PilT1 is essential for both cell motility and competency. Since the pilT gene contains a consensus ATP-binding motif (Walker boxes), the PilT protein is suggested for supplying energy for cell motility. The product of pilT1, overproduced in Escherichia coli and purified by Ni-affinity chromatography, hydrolyzes ATP in vitro.