Diagnostic criteria for adult-onset leukoencephalopathy with axonal spheroids and pigmented glia due to CSF1R mutation.

BACKGROUND AND PURPOSE: To establish and validate diagnostic criteria for adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) due to colony-stimulating factor 1 receptor (CSF1R) mutation. METHODS: We developed diagnostic criteria for ALSP based on a recent analysis of the clinical characteristics of ALSP. These criteria provide 'probable' and 'possible' designations for patients who do not have a genetic diagnosis. To verify its sensitivity and specificity, we retrospectively applied our criteria to 83 ALSP cases who had CSF1R mutations (24 of these were analyzed at our institutions and the others were identified from the literature), 53 cases who had CSF1R mutation-negative leukoencephalopathies and 32 cases who had cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) with NOTCH3 mutations. RESULTS: Among the CSF1R mutation-positive cases, 50 cases (60%) were diagnosed as 'probable' and 32 (39%) were diagnosed as 'possible,' leading to a sensitivity of 99% if calculated as a ratio of the combined number of cases who fulfilled 'probable' or 'possible' to the total number of cases. With regard to specificity, 22 cases (42%) with mutation-negative leukoencephalopathies and 28 (88%) with CADASIL were correctly excluded using these criteria. CONCLUSIONS: These diagnostic criteria are very sensitive for diagnosing ALSP with sufficient specificity for differentiation from CADASIL and moderate specificity for other leukoencephalopathies. Our results suggest that these criteria are useful for the clinical diagnosis of ALSP.

Genetic basis for soma is present in undifferentiated volvocine green algae.

Somatic cellular differentiation plays a critical role in the transition from unicellular to multicellular life, but the evolution of its genetic basis remains poorly understood. By definition, somatic cells do not reproduce to pass on genes and so constitute an extreme form of altruistic behaviour. The volvocine green algae provide an excellent model system to study the evolution of multicellularity and somatic differentiation. In Volvox carteri, somatic cell differentiation is controlled by the regA gene, which is part of a tandem duplication of genes known as the reg cluster. Although previous work found the reg cluster in divergent Volvox species, its origin and distribution in the broader group of volvocine algae has not been known. Here, we show that the reg cluster is present in many species without somatic cells and determine that the genetic basis for soma arose before the phenotype at the origin of the family Volvocaceae approximately 200 million years ago. We hypothesize that the ancestral function was involved in regulating reproduction in response to stress and that this function was later co-opted to produce soma. Determining that the reg cluster was co-opted to control somatic cell development provides insight into how cellular differentiation, and with it greater levels of complexity and individuality, evolves.

Clinical and genetic characterization of adult-onset leukoencephalopathy with axonal spheroids and pigmented glia associated with CSF1R mutation.

BACKGROUND AND PURPOSE: The clinical characteristics of colony stimulating factor 1 receptor (CSF1R) related adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) have been only partially elucidated. METHODS: Clinical data from CSF1R mutation carriers who had been seen at our institutions or reported elsewhere were collected and analysed using a specific investigation sheet to standardize the data. RESULTS: In all, 122 cases from 90 families with CSF1R mutations were identified. The mean age of onset was 43 years (range 18-78 years), the mean age at death was 53 years (range 23-84 years) and the mean disease duration was 6.8 years (range 1-29 years). Women had a significantly younger age of onset than men (40 vs. 47 years, P = 0.0006, 95% confidence interval 3.158-11.177). There was an age-dependent penetrance that was significantly different between the sexes (P = 0.0013). Motor dysfunctions were the most frequent initial symptom in women whose diseases began in their 20s. Thinning of the corpus callosum, abnormal signalling in pyramidal tracts, diffusion-restricted lesions and calcifications in the white matter were characteristic imaging findings of ALSP. The calcifications were more frequently reported in our case series than in the literature (54% vs. 3%). Seventy-nine per cent of the mutations were located in the distal part of the tyrosine kinase domain of CSF1R (102 cases). There were no apparent phenotype-genotype correlations. CONCLUSIONS: The characteristics of ALSP were clarified. The phenotype of ALSP caused by CSF1R mutations is affected by sex.

Diagnostic Value of Brain Calcifications in Adult-Onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia.

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia is a rare neurodegenerative disease resulting from mutations in the colony stimulating factor 1 receptor gene. Accurate diagnosis can be difficult because the associated clinical and MR imaging findings are nonspecific. We present 9 cases with intracranial calcifications distributed in 2 brain regions: the frontal white matter adjacent to the anterior horns of the lateral ventricles and the parietal subcortical white matter. Thin-section (1-mm) CT scans are particularly helpful in detection due to the small size of the calcifications. These calcifications had a symmetric "stepping stone appearance" in the frontal pericallosal regions, which was clearly visible on reconstructed sagittal CT images. Intrafamilial variability was seen in 2 of the families, and calcifications were seen at birth in a single individual. These characteristic calcification patterns may assist in making a correct diagnosis and may contribute to understanding of the pathogenesis of leukoencephalopathy.

Calcineurin inhibitor, FK506, prevents reduction in the binding capacity of cyclic AMP-dependent protein kinase in ischemic gerbil brain.

We examined the effects of FK506, a specific inhibitor of calcineurin, on the binding capacity of cyclic AMP-dependent protein kinase (cAMP-DPK) in gerbils subjected to 2-h cerebral hemispheric ischemia. FK506 (0.1 mg/kg) was infused intravenously at 15 min prior to the induction of ischemia by common carotid artery occlusion. The binding capacity of cAMP-DPK was evaluated by autoradiographic analysis of the cAMP binding, and cerebral blood flow (CBF) was measured by the [14C] iodoantipyrine method. In the sham-operated gerbils. FK506 significantly increased mean arterial blood pressure and tended to decrease CBF, suggesting that FK506 may constrict systemic blood vessels as well as cerebral blood vessels. On the other hand, cAMP binding was not altered by FK506 in the sham-operated gerbils. In the ischemia group of gerbils, FK506 prevented any significant reduction of cAMP binding in the hippocampus CA1 and cerebral cortices on the ischemic side, whereas it exerted no significant influence on the cAMP binding of the nonischemic side. The values of CBF were comparable between the vehicle-treated gerbils and FK506-treated gerbils in the ischemic regions. Preservation of cAMP binding indicates that intracellular signal transduction via cAMP-DPK can be maintained by FK506 despite ischemia, suggesting that this agent may be beneficial for reducing ischemic tissue damage.

Flow threshold for reduction of cyclic AMP binding in the hippocampus CA1 and other brain regions during stroke development in gerbils.

The flow threshold for alterations of the in vitro [3H]cyclic AMP (cAMP) binding, an indicator of the total amount of particulate cAMP-dependent protein kinase, was evaluated in the gerbil brain after 30 min, 2 h, and 6 h of unilateral common carotid artery occlusion, respectively. The autoradiographic method developed in our laboratory enabled us to measure the [3H]cAMP binding and local CBF in each region of the same brain. The ischemic flow thresholds for reduction of the cAMP binding in the hippocampus CA1 were 18, 34, and 49 ml 100 g-1 min-1 after 30-min, 2-h, and 6-h ischemia, respectively. These values were higher than those in other regions such as the hippocampus CA, and temporal cerebral cortex in each duration of ischemia. These findings indicate that (a) the ischemic flow threshold for perturbation of the cAMP system may be higher in the hippocampus CA1 than in other brain regions, suggesting that the hippocampus CA1 could be especially vulnerable to acute ischemic stress; and (b) the level of the aforementioned threshold may increase progressively during the time course of ischemia in particular regions such as the hippocampus CA1 and CA3, suggesting that the duration of ischemia exerts a definite influence on the viability of the ischemic neuronal cells in these regions.

Molecular cloning of a 74-kDa regulatory subunit (B" or delta) of human protein phosphatase 2A.

Based on amino acid sequence data of a 74-kDa regulatory subunit (B" or delta) of a human heterotrimeric protein phosphatase 2A, a cDNA encoding the subunit was isolated from a human cerebral cortex library. The cDNA had an open reading frame encoding an M(r) 66,138 protein of 570 amino acids. Bacterial expression of the cDNA yielded a protein immunoreactive with antisera specific to the 74-kDa subunit. The predicted primary structure of the subunit had no similarity to already reported sequences of PP2A regulatory subunits including A, B, and PR72. Potential phosphorylation sites for protein kinases A and C, a bipartite motif of putative nuclear localization signal, and SH3 accessible proline-rich domain, and a unique PQ repeat were found in the sequence. The subunit mRNA of about 2.9 kb was ubiquitously expressed in rat tissues.

Alteration of inositol 1,4,5-trisphosphate receptor after six-hour hemispheric ischemia in the gerbil brain.

In order to evaluate the influence of cerebral ischemia on the inositol 1,4,5-trisphosphate receptor, the alterations of inositol 1,4,5-trisphosphate receptor binding sites and local cerebral blood flow were examined 6 h after occlusion of the right common carotid artery in the gerbil brain. The autoradiographic method developed in our laboratory enabled us to measure both parameters within the same brain. Animals attaining ischemic scores of more than 5, as assessed 1 h after occlusion, were utilized. The local cerebral blood flow was measured 6 h after occlusion by the [14C]iodoantipyrine method. The inositol 1,4,5-trisphosphate binding sites were evaluated in vitro using [3H]inositol 1,4,5-trisphosphate as a specific ligand. The local cerebral blood flow fell below 15 ml/100 g per min in most of the cerebral regions on the occluded side. In contrast, a significant reduction in inositol 1,4,5-trisphosphate binding sites was noted only in the hippocampus CA1 on the occluded side. Inositol 1,4,5-trisphosphate binding tended to decrease when the values of local cerebral blood flow were below 20 ml/100 g per min in this region. On the other hand, the inositol 1,4,5-trisphosphate receptor immunoreactivity in the brain examined with a monoclonal antibody against inositol 1,4,5-trisphosphate receptor protein did not reveal any differences between the ischemia and sham groups on both sides, suggesting that the inositol 1,4,5-trisphosphate receptors may not undergo significant morphological degradation. These findings indicate that the suppression of inositol 1,4,5-trisphosphate binding in the hippocampus CA1 may be attributable to a regionally specific perturbation of the inositol 1,4,5-trisphosphate metabolism in this region.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid reduction in ryanodine binding of hippocampus CA1 in cerebral ischemia.

Ryanodine receptors located on the sarcoplasmic or endoplasmic reticulum, play an important role in the regulation of the intracellular Ca2+ level via the mechanism of Ca(2+)-induced Ca2+ release (CICR). Perturbation of intracellular Ca2+ regulation has been considered to be one of the most important mechanisms underlying acute ischemic neuronal damage. The ryanodine binding, an indicator of intracellular channels of CICR, and local cerebral blood flow (LCBF) were therefore examined at 15 min post-ischemia in the gerbil brain. The autoradiographic method developed in our laboratory enabled us to determine both parameters within the same brain. Severe hemispheric cerebral ischemia was induced by occluding the right common carotid artery. LCBF was measured at the end of the experiment using [14C]iodoantipyrine method. The ryanodine binding was evaluated autoradiographically in vitro using [3H] ryanodine. A group of gerbils who underwent a sham procedure served as controls. LCBF was found to be significantly decreased in most cerebral regions on the occluded side. In contrast, a significant reduction in ryanodine binding was noted only in the hippocampus CA1 on the occluded side. Taken together, these findings indicate that the CICR in the hippocampus CA1 may be especially susceptible to acute ischemic stress, and be closely associated with the pathophysiological mechanisms of the selective vulnerability of this region.

Tissue and subcellular distributions, and characterization of rat brain protein phosphatase 2A containing a 72-kDa delta/B" subunit.

A 74-kDa delta/B" subunit was isolated by heparin-Sepharose column chromatography from human erythrocyte protein phosphatase 2A (PP2A) consisting of a 34-kDa catalytic subunit (alpha/C) and 63- and 74-kDa regulatory subunits (beta/A and delta/B") in a ratio of 1:1:1. The purified delta/B" was used as an immunogen in mice, to prepare specific antisera against delta/B". Immunoblot analyses with the antisera detected an immunoreactive 72-kDa protein in the cytosol from various rat tissues including erythrocytes, brain, lung, testis, adrenal gland, heart, spleen, kidney, and liver. The 72-kDa protein was highly abundant in brain and was distributed evenly in cerebral cortex, cerebellum, and brain stem. The 72-kDa protein was also detected in mitochondria and microsome fractions. An immunoreactive 68-kDa protein was detected mainly in nuclear and microsome fractions. The 72-kDa protein from rat brain cytosol copurified with phosphorylated H2B histone phosphatase activity during successive chromatographies on DEAE-Toyopearl, AH-Sepharose, Sephadex G-150, H1 histone-Toyopearl, TSK DEAE-5PW, protamine-Toyopearl, and TSK G3000SW columns. The purified enzyme migrated as a single protein band on nondenaturing PAGE and as three protein bands of 34, 63, and 72 kDa in a ratio of 1:1:1 on SDS-PAGE. The molecular weight of the enzyme was estimated to be 170,000 from the s20,W value of 7.2 +/- 0.3 S and the Stokes radius of 5.5 +/- 0.1 nm. The rat brain enzyme was classified as PP2A, based on the following properties; (1) an IC50 for okadaic acid of 10(-9) M; (2) its preferential dephosphorylation of the a subunit of phosphorylase kinase; (3) its insensitivity to protein inhibitor 2; and (4) its heterotrimeric subunit structure. The Km value and the molecular activity of the enzyme for phosphorylated H2B histone were 72.3 +/- 0.3 microM and 192 +/- 2 mol Pi released/min/mol enzyme, respectively, and were comparable to those of human erythrocyte PP2A (alpha1 beta1 delta1/ CAB"). The 72-kDa subunit in the purified rat brain PP2A was phosphorylated in vitro by cAMP-dependent protein kinase.

Importance of anticomplement immunofluorescence antibody titration for diagnosing varicella-zoster virus infection in Bell's palsy.

Anticomplement Immunofluorescence was used for antibody titration against varicella-zoster virus (VZV) in 43 patients with peripheral facial palsy. Nine of 31 patients (29%) with Bell's palsy and eight of 12 patients (75%) with Ramsey-Hunt syndrome had anticomplement immunofluorescence antibody titres of greater than or equal to 1/10. On the other hand, none of 14 patients with herpes simplex virus (HSV) infection and 51 healthy adults showed anticomplement immunofluorescence antibody titres of greater than or equal to 1/10. The anticomplement immunofluorescence antibody titre in two patients with Ramsey-Hunt syndrome increased later and decreased sooner than the indirect immunofluorescence antibody titre, becoming undetectable at 66 and 104 days, respectively, after onset of the disease. There was no cross reaction between anti-VZV and anti-HSV antibodies in the patients who showed a positive antibody rise for VZV. As the acute stage of VZV infection is obscure in the patients with peripheral facial palsy without herpes the screening of anticomplement immunofluorescence antibody to VZV at titres greater than or equal to 1/10 may be useful for the diagnosis of VZV infection in patients with peripheral facial palsy.

A newly designed flexible fiberoptic bronchoscope for use in intensive care units.

A newly designed flexible fibroptic bronchoscope has been manufactured for use in intensive care units (ICU). It has an inside channel of large caliber, diameter 2.5 mm, through which pulmonary secretions can be aspirated directly via the suction tube. Furthermore, prompt bedside use is possible since handle type batteries can easily be attached to the bronchofibroscope. This new instrument is now employed in our ICU for: 1. The diagnosis and treatment of atelectasis. 2. Suction of retained secretions. 3. Detection of tracheal obstruction. 4. Evaluation of endotracheal and tracheostomy tubes whilst in position. 5. Observation of tracheal and bronchial changes. 6. Help in endotracheal intubation. It was used most frequently for the diagnosis and treatment of atelectasis and suction of retained secretions.

Relationship between pupil size and acetylcholinesterase activity in patients exposed to sarin vapor.

OBJECTIVE: To elucidate the effect of sarin vapor on pupil size and erythrocyte acetylcholinesterase activity (AchE). DESIGN: Retrospective observational survey. SETTING: Emergency department of an urban teaching hospital. PATIENTS: 80 patients who were exposed to sarin in a terrorist attack in Tokyo subways. MEASUREMENTS AND RESULTS: Pupil size and AchE activity on the day of exposure were measured. Among the 80 patients, the pupils were miotic (< 3 mm) in 50 patients (62.5%), while AchE activity was below the normal range (< 1.2 U) in 34 patients (42.5%). AchE was significantly lower in the miotic group than in the group with normal pupils (1.0 +/- 0.5 U vs 1.5 +/- 0.3 U, p < 0.01). In the miotic group, AchE activity was lower than normal in 32 patients (64.0%) but was decreased in only 2 patients in the normal pupil group (6.7%) (p < 0.01). CONCLUSIONS: Miosis is a more sensitive index of exposure to sarin vapor than erythrocyte AchE. Systemic poisoning is apparently less likely to develop if the patient's pupil size is normal on arrival at the hospital.

Secondary exposure of medical staff to sarin vapor in the emergency room.

OBJECTIVE: To clarify the risk of secondary exposure of medical staff to sarin vapor in the emergency room, and to warn emergency room staffs of the hazard. DESIGN: Retrospective observational survey. SETTING: Emergency department of a university hospital in a metropolitan area of Japan. PARTICIPANTS: Fifteen doctors treating victims of a terrorist attack with sarin in the Tokyo subways on the day of the attack. MEASUREMENTS AND RESULTS: Of the 15 doctors who worked in the emergency room treating the victims, 13 became simultaneously aware of symptoms during the resuscitation of two victims who were exposed to sarin. Among 11 doctors (73%) who complained of dim vision, the pupils were severely miotic (<2 mm) in 8 (73%). Other symptoms included rhinorrhea in eight (53%), dyspnea or tightness of the chest in four (27%), and cough in two (13%). Atropine sulfate was given to six, and pralidoxime was given to one of these six doctors. To decontaminate the emergency room of sarin vapor, ventilation was facilitated and all belongings of the patients were sealed up. None of the doctors noticed worsening of their symptoms thereafter. CONCLUSIONS: Careful attention to the risks of secondary exposure to toxic gas in the emergency room and prompt decontamination if such exposure should occur are necessary in the case of large-scale disasters caused by sarin.

Inhibition of nitric oxide synthesis impairs autoregulation of local cerebral blood flow in the rat.

The effect of intravenous administration of NG-monomethyl-L-arginine (L-NMMA, 30 mg kg-1), a specific inhibitor of nitric oxide (NO) synthesis, on the autoregulation of local cerebral blood flow (LCBF) was examined in the rat using the [14C]iodoantipyrine autoradiographic method. LCBF was significantly lower in various superficial regions such as the cerebral cortices and cerebellar cortex and in several deep brain regions in animals with haemorrhagic hypotension induced after L-NMMA infusion (the L-NMMA + HEM group) compared with animals without haemorrhagic hypotension after L-NMMA infusion (the L-NMMA group). The present findings suggest that NO synthesis may play a crucial role in the autoregulation of LCBF in response to a reduction in blood pressure in the cerebral cortices, cerebellar cortex and several deep brain regions.

Acute ischemic vulnerability of PKA in the dendritic subfields of the hippocampus CA1.

Alterations of [3H]cyclic AMP (cAMP) binding, an indicator of the binding activity of particulate cyclic AMP-dependent protein kinase (PKA), were examined after 15 and 30 min of ischemia in the gerbil brain. Severe hemispheric cerebral ischemia was induced by occluding the right common carotid artery. Significant reductions in cAMP binding were noted only in the dendritic subfields of the hippocampus CA1 such as the strata oriens, radiatum and lacunosum-moleculare, on the ischemic side after 15 min of ischemia. After 30 min ischemia cAMP binding was significantly decreased not only in each dendritic subfield of the hippocampus CA1, but also in the layer of pyramidal cell bodies (stratum pyramidale) on the occluded side; other brain regions such as the hippocampus CA3, dentate gyrus and cerebral cortices revealed no significant changes in cAMP binding. These findings suggest that derangement of PKA may begin in the dendritic subfields of the hippocampus CA1 after as little as 15 min of severe ischemia, and proceed centrally to the neuronal cell bodies of the hippocampus CA1.

Immobilization stress induces alterations of second-messenger systems in the gerbil brain.

The effects of immobilization stress on the cerebral second messenger (adenylate cyclase and phosphoinositide) were investigated autoradiographically in mongolian gerbils. After 10 min (10-min stress group, n = 7), or after 6 h (6-h stress group, n = 7) of fixation on a flat board while supine, in vitro autoradiography was performed using [3H]forskolin (3H-FK) and [3H]phorbol-12,13-dibutyrate (3H-PDBu) as specific ligands to identify the distribution of adenylate cyclase and protein kinase C, respectively. In another group of 7 gerbils (control group), the same autoradiographic procedure was performed immediately after the animals were removed from the cage. In the 10-min stress group, FK binding was significantly decreased in the hypothalamus and amygdala, but significantly increased in the basal ganglia including the caudate-putamen and globus pallidus. FK binding in the 6-h stress group tended to increase throughout the brain, rising significantly in the basal ganglia. PDBu binding in either stress group did not change significantly compared to the control group in any region except the hippocampal CA3 region of the 6-h stress group. Under immobilization stress, the adenylate cyclase system may undergo time-dependent and regionally specific changes, while the phosphoinositide system remains relatively stable.

Binding capacity of FK506 binding protein after 2-hour hemispheric ischemia in gerbil brain.

The binding capacity of FK506 binding protein (FKBP) was examined after 2-h hemispheric ischemia in the gerbil brain in order to clarify the precise mechanism of the neuroprotective effects of FK506. Firstly, the FK506 binding was evaluated in vitro in the normal gerbil brain using 1 nM [3H]dihydro-FK506 as a specific ligand. FK506 binding sites were distributed in a rather homogeneous manner, although the greatest binding was noted in the hippocampus CA1. Secondly, Scatchard analysis demonstrated that the binding sites of FK506 could be composed of two components in each brain region. Thirdly, 18 Mongolian gerbils were divided into two groups: an ischemia group (n = 12) and a sham group (n = 6). The right common carotid artery was ligated to induce hemispheric ischemia for 2 h in the ischemia group. The local cerebral blood flow was measured at the end of the experiment by the [14C]iodoantipyrine method. The ligated animals with levels of local cerebral blood flow in the lateral nuclei of the thalamus of less than 50 ml/100 g/min were utilized as the ischemia group (n=6) for further data analysis. No significant differences in FK506 binding between the ischemia and sham groups were observed in any regions. The above data indicate that the binding capacity of FKBP tends to remain normal during 2-h ischemia, suggesting that FK506 may exert its neuroprotective effects through its binding to FKBP in the brain during the early phase of cerebral ischemia.

Development of the human pontine nuclei: a morphometric study.

The morphometric development of the pontine nuclei in the human fetus from 16 to 40 gestational weeks, in a 2-month-old infant and in a 63-year-old adult were examined employing a serial celloidin section method and computer assisted electronic planimeter. The results of our study can be summarized as follows: (1) the development of the human pontine nuclei accelerated in volume after 32 gestational weeks and continued after birth, (2) neuron numbers remained relatively constant after 27 gestational weeks. It was difficult to clearly distinguish neurons from glia before 27 gestational weeks. The total estimated neuronal numbers were not indicative of the gestational stages in infants 27 gestational weeks and older, (3) individual neurons appeared to continue to develop after 32 gestational weeks in accordance with size, distribution and circularity ratio, (4) many islet-shaped groups of large neurons appeared and were scattered throughout the pontine nuclei after 32 gestational weeks.

Gibbestatin B inhibits the GA-induced expression of alpha-amylase expression in cereal seeds.

The expression of alpha-amylase in aleurone layers of barley is known to be induced by gibberellin A3 (GA). In the present study, gibbestatin B (GNB) was isolated from Streptomyces sp. C-39 as an inhibitor of the GA-induced expression of alpha-amylase in barley and rice, with IC50 values of 125 and 70 microM, respectively. GNB suppressed accumulation of GA-induced barley high-pI type B and rice RAmylA alpha-amylase transcripts. However, GNB showed no inhibitory activity on GUS expression in transgenic tobacco harboring the auxin-inducible par B promoter:: GUS fusion gene. The transcription of an abscisic acid (ABA)-inducible gene, HVA1, was unaffected by GNB. In addition, GNB prevented aleurone cells from cell death induced by GA. In tobacco and Arabidopsis plants, GNB suppressed the germination and retarded the growth of seedlings without toxicity. The growth of gai, spy and abi mutants was also retarded by GNB. Normal plants treated with GA-biosynthesis inhibitors and GA-defective and GA-signaling mutants normally have dwarf dark green leaves. However, dwarfed healthy green leaves were observed in normal plants treated with GNB. GA-induced stem elongation of plants was also detected in the presence of GNB. These analyses indicate that GNB inhibits the GA-induced expression of alpha-amylase by regulating one of the steps involved in ABA signaling, but not by acting as a weak ABA analog.