Horizontal visual search in a large field by patients with unilateral spatial neglect.

In this study, we investigated the horizontal visual search ability and pattern of horizontal visual search in a large space performed by patients with unilateral spatial neglect (USN). Subjects included nine patients with right hemisphere damage caused by cerebrovascular disease showing left USN, nine patients with right hemisphere damage but no USN, and six healthy individuals with no history of brain damage who were age-matched to the groups with brain right hemisphere damage. The number of visual search tasks accomplished was recorded in the first experiment. Neck rotation angle was continuously measured during the task and quantitative data of the measurements were collected. There was a strong correlation between the number of visual search tasks accomplished and the total Behavioral Inattention Test Conventional Subtest (BITC) score in subjects with right hemisphere damage. In both USN and control groups, the head position during the visual search task showed a balanced bell-shaped distribution from the central point on the field to the left and right sides. Our results indicate that compensatory strategies, including cervical rotation, may improve visual search capability and achieve balance on the neglected side.

Effect of a centrally active angiotensin converting enzyme inhibitor, perindopril, on cognitive performance in chronic cerebral hypo-perfusion rats.

We have previously demonstrated that perindopril, an angiotensin-converting enzyme (ACE) inhibitor, ameliorated the cognitive deficits in Alzheimer's disease model animals, independently of its anti-hypertensive effect. In this study, we again investigated the effects of perindopril on cognitive function in a vascular dementia model animal, comparing it with other ACE inhibitors. We also determined ACE activity in the brain and extracellular acetylcholine (ACh) concentration in the perirhinal cortex in order to elucidate the mechanism(s) responsible for the effects of these ACE inhibitors on cognitive function. Perindopril was suggested to be more centrally active than imidapril and enalapril, in consideration of the relative distribution of their active metabolites in the brain. This property was at least partially attributed to the lipophilicity of the compound. While the 3 day treatment with perindopril, imidapril or enalapril lowered blood pressure to the same level in spontaneous hypertensive rats, only perindopril reversed the decline in the recognition index in chronic cerebral hypo-perfusion rats, regarded as an animal model of vascular dementia, during an object recognition task. Using the same dosing regimen, perindopril inhibited the brain ACE activities of rats more than imidapril or enalapril. Moreover, a single treatment with perindopril enhanced the extracellular level of ACh in the perirhinal cortex of normal rats. Therefore, we confirmed that only centrally active ACE inhibitors, such as perindopril, can inhibit the ACE in the brain, augmenting cholinergic neurotransmission and thereby ameliorating cognitive impairment in the animal model of vascular dementia.

ATP-induced osteoclast function: the formation of sealing-zone like structure and the secretion of lytic granules via microtubule-deacetylation under the control of Syk.

Osteoclasts are bone-resorbing cells which play an exclusive role in bone remodeling, but the molecular mechanisms of osteolysis, how osteoclasts are activated and how the lytic granules are finally released towards the bone matrix are poorly understood. Here we show that an energy molecule ATP induces osteolysis via P2X(7)-nucleotide receptor and that deacetylation of alpha-tubulin is essential for the whole process of osteolysis under the control of a tyrosine kinase Syk. By developing a traceable and reproducible in vitro analyzing system for osteoclast function, we found that ATP-signaling gives rise to two events simultaneously (i) cytoskeletal reorganization for the formation of sealing zones, ring-like adhesion structures which delimit the contact surface, and (ii) the delivery and secretion of lytic granules towards the delimited site on the matrix. We further found that deacetylation of alpha-tubulin is a critical reaction for osteoclast function. Pharmacological inhibition of alpha-tubulin deacetylation resulted in (i) failure of the sealing-zone like structure formation and (ii) ceased secretion of lytic granules. Additionally, kinetics of deacetylation was found to be regulated by Syk. These data suggest a novel P2X(7) microtubular regulation pathway related to Syk for a therapeutic target in osteolytic diseases.

Characterization of a flavin reductase from a thermophilic dibenzothiophene-desulfurizing bacterium, Bacillus subtilis WU-S2B.

Bacillus subtilis WU-S2B is a thermophilic dibenzothiophene (DBT)-desulfurizing bacterium and produces a flavin reductase (Frb) that couples with DBT and DBT sulfone monooxygenases. The recombinant Frb was purified from Escherichia coli cells expressing the frb gene and was characterized. The purified Frb exhibited high stability over wide temperature and pH ranges of 20-55 degrees C and 2-12, respectively. Frb contained FMN and exhibited both flavin reductase and nitroreductase activities.

[Paroxysmal bilateral ballism induced by hypoglycemia].

Ballism is a kind of involuntary movement presenting as irregular but stereotyped jerking and writhing movements of the limbs with proximal dominance. The most common type is hemiballism, and bilateral ballism is extremely rare. In contrast to hemiballism with preferential involvement of the contralateral subthalamic nucleus, bilateral ballism results from diffuse lesions on both sides of the basal ganglia. Here, we present a patient in whom bilateral ballism occurred as a result of hypoglycemia. A 75-year-old man with long-standing insulin-dependent diabetes mellitus had suffered recurrent episodes of paroxysmal and transient involuntary movements of all 4 limbs over a 6-week period. When he visited our hospital first, he was alert and well-oriented. Neurological examination revealed no deficits except right hemianopsia. Violent involuntary movements suddenly manifested 10 days later during MRI scans. Involuntary movements occurred in all 4 limbs, more prominently on the left side, lasted over 15 min in total, and ceased spontaneously. These movements were considered as bilateral ballism. The patient was awake, but rather confused. Serum glucose level during this attack was 25 mg/dl. Intravenous glucose was administered, and he became aware. Brain CT and MRI showed no evilence of ischemic or other basal ganglia pathology and no significant vascular lesions were detected by MR angiograhy. EEG revealed no epileptic discharges. He was admitted to our hospital and insulin dosage was adjusted. No further episodes occurred during a 6-month follow up. Our patient experienced an episode of bilateral ballism associated with documented hypoglycemia. In addition, good control of serum glucose with an appropriate insulin treatment has abolished the involuntary movement episodes described above. Conversely, the basal ganglia is known to be vulnerable to hypoglycemia, due to large metabolic demand and poor vascularization. Recurrent episodes of bilateral ballism in this case may thus have been caused by transient dysfunction of the basal ganglia due to hypoglycemia. We proposed a hypothesis to explain why the involuntary movements disappeared spontaneously.

Gene cloning and characterization of Mycobacterium phlei flavin reductase involved in dibenzothiophene desulfurization.

Mycobacterium phlei WU-F1 possesses the ability to convert dibenzothiophene (DBT) to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range from 20 degrees C to 50 degrees C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and a flavin reductase is essential in combination with these flavin-dependent monooxygenases. The flavin reductase gene (frm) of M. phlei WU-F1, which encodes a protein of 162 amino acid residues with a molecular weight of 17,177, was cloned and the deduced amino acid sequence shares approximately 30% identity with those of several flavin reductases in two protein-component monooxygenases. It was confirmed that the coexpression of frm with the DBT-desulfurization genes (bdsABC) from M. phlei WU-F1 was critical for high DBT-desulfurizing ability over a wide temperature range from 20 degrees C to 55 degrees C. The frm gene was overexpressed in Escherichia coli cells, and the enzyme (Frm) was purified to homogeneity from the recombinant cells. The purified Frm was found to be a 34-kDa homodimeric protein with a monomeric molecular mass of 17 kDa. Frm exhibited high flavin reductase activity over a wide temperature range, and in particular, the turnover rate for FMN reduction with NADH as the electron donor reached 564 s(-1) at 50 degrees C, which is one of the highest activities among all of the flavin reductases previously reported. Intriguingly, Frm also exhibited a high ferric reductase activity.

Cloning of a gene encoding flavin reductase coupling with dibenzothiophene monooxygenase through coexpression screening using indigo production as selective indication.

The thermophilic dibenzothiophene (DBT)-desulfurizing bacterium, Bacillus subtilis WU-S2B, possesses the ability to convert DBT to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range up to 50 degrees C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and flavin reductase is essential in combination with these flavin-dependent monooxygenases. The recombinant Escherichia coli cells expressing the DBT monooxygenase gene (bdsC) from B. subtilis WU-S2B also oxidize indole to blue pigment indigo in the presence of a heterologous flavin reductase. Thus, to clone a gene encoding flavin reductase from B. subtilis WU-S2B, indigo production by coexpression of the gene with bdsC in E. coli was used as a selection. Using this method, the corresponding gene (frb) was obtained from a recombinant strain forming a blue colony due to indigo production on a nutrient agar plate, and it was confirmed that this gene product Frb exhibited flavin reductase activity. The deduced amino acid sequence of frb consists of 174 amino acid residues and shares 61% identity with that of nitroreductase (YwrO) of Bacillus amyloliquefaciens. In addition, coexpression of frb with the DBT-desulfurization genes (bdsABC) from B. subtilis WU-S2B was critical for high DBT-desulfurizing ability over a wide temperature range of 20-55 degrees C. This coexpression screening using indigo production as selective indication may be widely applicable for cloning novel genes encoding either component of flavin reductase or flavin-dependent monooxygenase which efficiently couples with the other component in two-component monooxygenases.

Potent and selective inhibitors of platelet-derived growth factor receptor phosphorylation. 3. Replacement of quinazoline moiety and improvement of metabolic polymorphism of 4-[4-(N-substituted (thio)carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives.

We have previously reported that a series of 4-[4-(N-substituted (thio)carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives were potent and selective inhibitors of platelet-derived growth factor receptor (PDGFR) phosphorylation and demonstrated several biological effects such as suppression of neointima formation following balloon injury in rat carotid artery by oral administration. Here, we investigated structure-activity relationships of the 6,7-dimethoxyquinazolinyl moiety. In regard to 6,7-dimethoxy groups, ethoxy analogues showed potent activity (IC(50) of 16b is 0.04 microM; IC(50) of 17a is 0.01 microM) and further extension of the alkyl group reduced activity. Interestingly, methoxyethoxy (IC(50) of 16j is 0.02 microM; IC(50) of 17h is 0.01 microM) and ethoxyethoxy (IC(50) of 17j is 0.02 micro M) analogues showed the most potent activity, suggesting that the inserted oxygen atom significantly interacts with beta-PDGFR. Among tricyclic quinazoline derivatives, the 2-oxoimidazo[4,5-e]quinazoline derivative 21a showed potent activity (IC(50) = 0.10 microM). Regarding replacements of quinazoline by other heterocyclic rings, pyrazolo[3,4-d]pyrimidine (39a, IC(50) = 0.17 microM) and quinoline (IC(50) of 40a is 0.18 microM; IC(50) of 40b is 0.09 microM) derivatives showed potent activity. Isoquinoline and some pyridopyrimidine derivatives were completely inactive; therefore, 1-aza has an important role. Also 7-aza and 8-aza substitution on the parent quinazoline ring has a detrimental effect on the interaction with beta-PDGFR. We also demonstrated that the substituents on the quinazoline ring possess major consequences for metabolic polymorphism. Although there existed extensive metabolizers and poor metabolizers in Sprague-Dawley rats administrated 6,7-dimethoxyquinazoline derivatives (1b and 1c), 6-(2-methoxy)ethoxy-7-methoxyquinazoline analogue 16k showed no metabolic polymorphism.

Role for Fes/Fps tyrosine kinase in microtubule nucleation through is Fes/CIP4 homology domain.

We have previously demonstrated that Fes/Fps (Fes) tyrosine kinase is involved in Semaphorin3A-mediated signaling. Here we report a role for Fes tyrosine kinase in microtubule dynamics. A fibrous formation of Fes was observed in a kinase-dependent manner, which associated with microtubules and functionally correlated with microtubule bundling. Microtubule regeneration assays revealed that Fes aggregates colocalized with gamma-tubulin at microtubule nucleation sites in a Fes/CIP4 homology (FCH) domain-dependent manner and that expression of FCH domain-deleted Fes mutants blocked normal centrosome formation. In support of these observations, mouse embryonic fibroblasts derived from Fes-deficient mice displayed an aberrant structure of nucleation and centrosome with unbundling and disoriented filaments of microtubules. Our findings suggest that Fes plays a critical role in microtubule dynamics including microtubule nucleation and bundling through its FCH domain.

Isolation and expression of a novel mitochondrial septin that interacts with CRMP/CRAM in the developing neurones.

BACKGROUND: Collapsin response mediator proteins (CRMPs) and CRAM belong to the unc-33 gene family which is implicated in axon guidance and outgrowth during neural development. However, their exact roles remain largely unknown. To understand the molecular basis of CRMP/CRAM function, we have undertaken to identify CRMP/CRAM interacting proteins. RESULTS: We have identified a novel mitochondrial septin (M-septin) as one of the CRMP/CRAM interacting proteins from the developing rat brain. M-septin is a major, alternatively spliced variant of the H5 gene in developing mouse brain and its expression is up-regulated during the neuronal differentiation of embryonal carcinoma P19 cells. In COS-7 cells, M-septin is specifically localized to mitochondria whereas H5 is diffusely distributed to the perinuclear cytoplasm and plasma membranes. In contrast to H5, M-septin induces the mitochondrial translocation of CRAM but not CRMP2. Finally, M-septin is found to be transiently translocated to mitochondria before the induction of the neurites and then dissociates from the mitochondria after neurite extension in P19 cells. CONCLUSIONS: Our results suggest that M-septin has a role which is distinct from H5, and together with CRMP/CRAM, may play an important role in the neuronal differentiation and axon guidance through the control of mitochondrial function.

Involvement of Fes/Fps tyrosine kinase in semaphorin3A signaling.

Collapsin response mediator proteins (CRMPs)/TOAD64/Ulips/DRPs and CRAM have emerged as strong candidates for a role in semaphorin signaling. In this study we identified Fes/Fps (Fes) tyrosine kinase in the CRMP-CRAM complex and investigated whether Fes was involved in semaphorin3A (Sema3A) signaling. In COS-7 cells, the interaction between Fes and plexinA1 (PlexA1) and the tyrosine phosphorylation of PlexA1 by Fes were observed; however, these events were significantly attenuated by co-expression of neuropilin-1 (NP-1). Even with NP-1 co-expression, Sema3A was able to enhance the association of Fes with PlexA1 and Fes-mediated tyrosine phosphorylation of PlexA1, CRAM and CRMP2. Co-expression of Fes with PlexA1 exhibited COS-7 cell contraction activity, indicating that Fes can convert inactive PlexA1 to its active form, whereas combination of Fes/NP-1/PlexA1 or Fes kinase-negative mutants/PlexA1 did not alter cell morphology. Finally, Sema3A-induced growth cone collapse of dorsal root ganglion neurons was suppressed by expression of Fes kinase-negative mutants. Taken together, our findings suggest that Fes links Sema3A signals to CRMP-CRAM, and that NP-1 negatively regulates PlexA1 activation by Fes in resting condition.