cAMP-GEFII is a direct target of cAMP in regulated exocytosis.

Although cAMP is well known to regulate exocytosis in many secretory cells, its direct target in the exocytotic machinery is not known. Here we show that cAMP-GEFII, a cAMP sensor, binds to Rim (Rab3-interacting molecule, Rab3 being a small G protein) and to a new isoform, Rim2, both of which are putative regulators of fusion of vesicles to the plasma membrane. We also show that cAMP-GEFII, through its interaction with Rim2, mediates cAMP-induced, Ca2+-dependent secretion that is not blocked by an inhibitor of cAMP-dependent protein kinase (PKA). Accordingly, cAMP-GEFII is a direct target of cAMP in regulated exocytosis and is responsible for cAMP-dependent, PKA-independent exocytosis.

Activating transcription factor-2 is a positive regulator in CaM kinase IV-induced human insulin gene expression.

Insulin plays a crucial role in the regulation of glucose-homeostasis, and its synthesis is regulated by several stimuli. The transcription of the human insulin gene, enhanced by an elevated intracellular concentration of calcium ions, was completely blocked by Ca2+/calmodulin-dependent protein kinase inhibitor. The activity of the transcription factor activating transcription factor-2 (ATF-2), which binds to the cAMP responsive elements of the human insulin gene, was enhanced by Ca2+/calmodulin-dependent protein kinase IV (CaMKIV). Mutagenesis studies showed that Thr69, Thr71, and Thr73 of ATF-2 are all required for activation by CaMKIV. CaMKIV-induced ATF-2 transcriptional activity was not altered by activation of cJun NH2-terminal protein kinase (JNK) or p38 mitogen-activated protein (MAP) kinase. Furthermore, when transfected into rat primary cultured islets, ATF-2 enhanced glucose-induced insulin promoter activity, whereas cAMP response element-binding protein (CREB) repressed it. These results suggest a mechanism in which ATF-2 regulates insulin gene expression in pancreatic beta-cells, with the transcriptional activity of ATF-2 being increased by an elevated concentration of calcium ions.

Evaluation of preload reserve during isometric exercise testing in patients with old myocardial infarction: Doppler echocardiographic study.

To estimate the preload reserve in response to an increase in afterload in patients with old myocardial infarction, the relation between the Doppler echocardiographic inflow velocity pattern and left ventricular end-diastolic pressure was investigated during isometric handgrip exercise testing. The study population consisted of 16 normal subjects and 40 patients with old myocardial infarction. The 40 patients were subdivided into two groups according to left ventricular end-diastolic pressure at rest: group I (22 patients), less than 18 mm Hg; group II (18 patients), 18 mm Hg or more. At rest, the ratio of peak velocity in atrial contraction phase to peak velocity in early diastolic filling phase (A/E) was significantly higher in the patients with old myocardial infarction than in normal subjects; values in the two subgroups of myocardial infarction did not differ significantly. The A/E ratio and left ventricular end-diastolic pressure increased significantly during exercise in group I. Conversely, the change in left ventricular end-diastolic pressure during exercise in group II was significantly greater than that in group I, and was associated with a decrease in the A/E ratio. Thus, an atrial compensatory mechanism operated effectively in response to the increase in afterload in patients with a normal left ventricular filling pressure, whereas this compensatory mechanism deteriorated in patients with elevated left ventricular filling pressure due to a limited preload reserve.

Biochemical characteristics of cardiac myosin: the pH dependence of Ca-ATPase activity, and that of the absorption spectrum of 2,4,6-trinitrophenyl groups attached to myosin.

For both cardiac and skeletal myosin, the Ca-ATPase activity of myosin at acidic pH was shown to be different from that at alkaline pH, in the susceptibility to heat-inactivation, the effects of organic solvents, and the effect of trinitrophenylation of the myosin. It is therefore suggested that there are two different types of Ca-ATPase of both cardiac and skeletal myosin. Differences in the Ca-ATPase activity were also found between cardiac and skeletal myosins. (a) The Ca-ATPase activity of cardiac myosin was more susceptible to heat-inactivation at alkaline pH than at acidic pH. In contrast, the activity of skeletal myosin was more susceptible to heat-inactivation at acidic pH than at alkaline pH. (b) Dioxane weakly stimulated the activity of cardiac myosin at acidic pH, but strongly activated that of skeletal myosin at acidic pH. Acetone very strongly inhibited the activity of cardiac myosin at alkaline pH, but not so strongly that of skeletal myosin at alkaline pH. (c) Trinitrophenylation of the myosin resulted in loss of the activity optimum at acidic pH with skeletal myosin but not with cardiac myosin. As reported by Srivastava et al. (J. Biochem. 86, 725-731, 1979), 1 mol of lysine residue per mol of cardiac myosin quickly reacted with 2,4,6-trinitrobenzene sulfonate (TNBS) either in the absence or presence of inorganic pyrophosphate (PPi). However, trinitrophenyl (TNP) groups bound to cardiac myosin in the presence of PPi were significantly different, in the pH dependence of the absorption spectrum, from those bound (to cardiac myosin) in the absence of PPi.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of mitiglinide (KAD-1229), a new anti-diabetic drug, on ATP-sensitive K+ channels and insulin secretion: comparison with the sulfonylureas and nateglinide.

Mitiglinide (KAD-1229), a new anti-diabetic drug, is thought to stimulate insulin secretion by closing the ATP-sensitive K+ (K(ATP)) channels in pancreatic beta-cells. However, its selectivity for the various K(ATP) channels is not known. In this study, we examined the effects of mitiglinide on various cloned K(ATP) channels (Kir6.2/SUR1, Kir6.2/SUR2A, and Kir6.2/SUR2B) reconstituted in COS-1 cells, and compared them to another meglitinide-related compound, nateglinide. Patch-clamp analysis using inside-out recording configuration showed that mitiglinide inhibits the Kir6.2/SUR1 channel currents in a dose-dependent manner (IC50 value, 100 nM) but does not significantly inhibit either Kir6.2/SUR2A or Kir6.2/SUR2B channel currents even at high doses (more than 10 microM). Nateglinide inhibits Kir6.2/SUR1 and Kir6.2/SUR2B channels at 100 nM, and inhibits Kir6.2/SUR2A channels at high concentrations (1 microM). Binding experiments on mitiglinide, nateglinide, and repaglinide to SUR1 expressed in COS-1 cells revealed that they inhibit the binding of [3H]glibenclamide to SUR1 (IC50 values: mitiglinide, 280 nM; nateglinide, 8 microM; repaglinide, 1.6 microM), suggesting that they all share a glibenclamide binding site. The insulin responses to glucose, mitiglinide, tolbutamide, and glibenclamide in MIN6 cells after chronic mitiglinide, nateglinide, or repaglinide treatment were comparable to those after chronic tolbutamide and glibenclamide treatment. These results indicate that, similar to the sulfonylureas, mitiglinide is highly specific to the Kir6.2/SUR1 complex, i.e., the pancreatic beta-cell K(ATP) channel, and suggest that mitiglinide may be a clinically useful anti-diabetic drug.

Troglitazone but not pioglitazone affects ATP-sensitive K(+) channel activity.

We compared the effects of the two thiazolidinedione derivatives, troglitazone and pioglitazone, on ATP-sensitive K(+) (K(ATP)) channel activities. Pancreatic beta-cell type and cardiac type K(ATP) channels were reconstituted in COS-1 cells (SV 40-transformed African green monkey kidney (AGMK) cells) by heterologously expressing sulfonylurea receptor 1 (SUR1) plus Kir6.2 and sulfonylurea receptor 2A (SUR2A) plus Kir6.2, respectively. Troglitazone inhibited [86Rb(+)] efflux in both K(ATP) channel types in the presence of metabolic inhibitors, which was confirmed by electrophysiological techniques. The [86Rb(+)] efflux increased by the channel openers diazoxide and pinacidil was abolished by troglitazone. In contrast, pioglitazone did not affect these channel activities in either type K(ATP) channel. These results suggest that troglitazone modulates the various cellular functions including insulin secretion by inhibiting the K(ATP) channels, while pioglitazone has no effect on K(ATP) channel activity.

Urinary N-acetyl-beta-glucosaminidase and guanidinoacetic acid levels in epileptic patients treated with anti-epileptic drugs.

We investigated potential renal functional impairment induced by chronic use of anti-epileptic drugs (AEDs) in 79 epileptic children. They were divided into five groups: valproic acid (VPA) monotherapy where the serum concentration (SC) of VPA was no less than 60 micrograms/ml (VPA [SC > or = 60]) (15 cases), VPA monotherapy where the SC VPA was less than 60 micrograms/ml (VPA [SC < 60]) (29 cases), phenobarbital monotherapy (PB) (7 cases), carbamazepine monotherapy (CBZ) (16 cases), and polytherapy containing VPA (12 cases). Urinalysis (proteinuria and hematuria) and serum creatinine were normal except for two cases of proteinuria and two cases of hematuria. The level of urinary excretion of N-acetyl-beta-glucosaminidase (u-NAG) was high in 29% of all patients, and 47% of VPA (SC > or = 60), 38% of CBZ, 25% of polytherapy, and 24% of VPA (SC < 60) groups. There was a significant positive correlation between serum concentration of VPA and u-NAG/urinary creatinine (u-Cr). The level of guanidinoacetic acid (u-GAA) excreted in the urine was normal except in one patient. U-NAG/u-Cr may be a more sensitive marker than u-GAA/u-Cr for renal functional impairment in AED therapy.

Two siblings with partial trisomy 1(q42.3-ter).

Two brothers, aged 6 and 4 years, with an unbalanced chromosome translocation (partial trisomy 1), and their mother, a balanced carrier of the translocation, t(1;3)(q42.3;p26.3), were described. Both patients show minor anomalies; a large head with a prominent forehead, low-set and soaring ears, a high-arched palate, a shallow nasal bridge, hypertelorism, and slender hands and feet. The manifestations in our cases were very mild compared to in the previously reported cases of partial trisomy 1. And our patients exhibit psychomotor retardation and ventricular dilatation on brain CT. We speculated that the amount of extra material reflects the phenotype. Our cases and previous reports indicate that the minimum clinical features of partial trisomy 1 are poor psychomotor development, a prominent forehead, and slender hands and feet. And many cases have macrocephaly with ventricular dilatation or hydrocephalus. So these features may be a key for the diagnosis of very mild partial trisomy 1.

Auditory brainstem responses in congenital heart disease.

To evaluate the effect of chronic hypoxemia on brainstem maturation, auditory brainstem responses were examined in 70 children (32 with and 38 without cyanosis) who had congenital heart disease. Ninety-one age-matched normal children served as controls. At 1-3 months of age, the I-V interpeak latencies of cyanotic infants (mean +/- S.D.; 5.17 +/- 0.17 ms) were more prolonged than were those of controls (4.95 +/- 0.11 ms) and those without cyanosis (4.84 +/- 0.22 ms; P < .05; P < .01). At 4-11 months of age, the I-V interpeak latencies of cyanotic infants (4.85 +/- 0.13 ms) were more prolonged than were those of controls (4.67 +/- 0.19 ms) and those not experiencing cyanosis (4.5 +/- 0.17 ms; P < .05; P < .01). In the cyanotic children, there was a significant negative correlation between the I-V interpeak latency and oxygen partial pressure (P < .01) or oxygen saturation (P < .05). Three of the 70 patients (4.3%) with congenital heart disease had absent auditory brainstem response. These data indicate that chronic hypoxemia may be one of the factors in retarded brainstem maturation.

Acute cerebellar ataxia with abnormal MRI lesions after varicella vaccination.

A 2-year-old boy, with the primary difficulties of nausea and vomiting, developed a staggering gait and dysarthria 10 days after varicella vaccination. Magnetic resonance imaging demonstrated multiple areas of high signal intensity in the white matter of the cerebellum, predominantly in the parieto-occipital white matter and both globus pallidi. He did not present any signs of myelitis or encephalitis and thus his cerebellar dysfunction was diagnosed as acute cerebellar ataxia, which is, generally speaking, not an etiologic entity but a clinical syndrome. Magnetic resonance imaging may reveal a variety of abnormalities of the central nervous system in acute cerebellar ataxia.

P300 event-related potentials in epileptic children.

To evaluate the cognitive function of epileptic children, we examined P300 in 50 patients, 32 with idiopathic generalized epilepsy (IGE) and 18 with temporal lobe epilepsy (TLE), and 39 normal children. There were significant negative correlations between age and P300 latencies at Pz and Cz in normal controls. For data analysis, we used the age-corrected latency, which was calculated as the interval between the actual and predicted P300 latencies. The predicted latency was calculated with the regression equation as the relationship between P300 latency and age in normal controls. The age-corrected P300 latencies recorded from Pz and Cz were significantly longer in patients with IGE (41.5 +/- 13.1, 42.0 +/- 12.5) than in control subjects (0 +/- 7.5, 0 +/- 7.9). There were no significant differences in age-corrected P300 latencies between patients with TLE (21.2 +/- 17.6, 31.5 +/- 17.0) and controls, or IGE and TLE. Recently, it was considered that the mesencephalic reticular formation and thalamus may play major roles in the genesis of generalized epilepsy, so we speculate that dysfunction of these systems may contribute to the prolongation of P300 in children with idiopathic generalized epilepsy.

Digoxin-like immunoreactive substance in urine of patients with mucocutaneous lymph node syndrome (MCLS).

Levels of digoxin-like immunoreactive substance (DLIS) and dehydroepiandrosterone sulfate (DHEA) in urine from patients with mucocutaneous lymph node syndrome (MCLS) were measured by radioimmunoassay. Because DLIS of stored samples was often below the level of detection by conventional immunoassay, the authors used 80% ammonium sulfate and extraction with phosphate buffer and then 80% hot ethanol. To study the origin of raised levels of DLIS in urine, the synthesis of DLIS by cultured human umbilical vein endothelial cells (HUVEC) was tested in vitro. The correlation between DLIS and DHEA levels was not significant. Mean levels of urinary DLIS corrected for creatinine in the patients with MCLS were significantly higher than in both normal and diseased controls. The culture medium of HUVEC was found to contain DLIS activity. These results show that MCLS should be added to the clinical states associated with an increased urinary concentration of DLIS and that the endothelial cells are one source of DLIS in man.

[Nemaline myopathy with type 2 fiber predominance; a case report].

A 4-year-old boy was admitted to our hospital because of dyspnea and muscle-weakness. His developmental milestones were delayed. His face was long with opened mouth. He spoke with nasal voice. He had proximally dominant muscle weakness and his deep tendon reflexes were absent. Laboratory examination revealed normal serum creatine kinase level and a myopathic EMG pattern. Blood gas analysis revealed metabolic acidosis with PH 7.35, PCO2 55.4 mmHg, PO2 62.4 mmHg, BE 3.0, probably from the metabolic acidosis by respiratory muscle weakness. In the biopsied left biceps brachii muscle, there were scattered fibers with nemaline bodies and abnormal fiber type distribution; type 1, 2 A, 2 B and 2 C fibers comprised 7%, 70%, 21% and 2% respectively. Small type 1 fibers and type 1 fiber predominance are the characteristic and common histochemical findings in nemaline myopathy. Accordingly, type 2 fiber predominance in the present patient is a unique, rare phenomenon. The finding might result from a preferential loss of type 1 motoneurons or muscle fiber type transformation from type 1 to type 2 fibers due to a certain abnormal neuronal influence on developing muscles.

[MRI findings of moyamoya disease in children].

Magnetic resonance imaging (MRI) findings were evaluated in two cases with moyamoya disease. A twelve-year-old girl and a five-year-old boy were admitted to our hospital with the complaints of transient ischemic attack. They were diagnosed as having moyamoya disease by cerebral angiogram. MRI clearly presented cerebral infarction by T2-weighted imaging and multiple small round or tortuous hypo_signal areas around the basal ganglia by proton density weighted imaging. These findings were consistent with moyamoya vessels shown by cerebral angiogram. Moyamoya vessels were visualized more clearly on proton density weighted imaging than on T1-weighted imaging. MRI is less invasive than cerebral angiogram and repeated safely. It might play an important role in a follow-up study of morphological changes on moyamoya vessels.

[A female child with Down syndrome complicated by spinal cord compression by atlanto-axial dislocation].

A 3-year-old girl with Down syndrome was admitted to our hospital, associated with hypotonia and dyspnea. She presented frog-posture and shallow breathing. The blood gas analysis revealed hypercapnic acidosis with pH 7.371, PO2 74.6 mmHg, PCO2 52.6 mmHg, and BE 3.5. The cervical X-ray films with flexion and extension of head showed anterior dislocation of the atlanto-axial articulation. Magnetic resonance imaging clearly showed a severe cord compression between C1 and C2. In children, spinal cord compression induced by dislocation of the atlanto-axial articulation is very rare. However, we should take into consideration of this insidious risk associated often with Down syndrome.

[Two cases of brain infarction associated with hypertriglyceridemia].

We reported two cases of brain infarction. They were cousins. Case 1 was a 12-year-old girl, who complained of aphasia, dyscalculia, right-left disorientation and right homonymous hemianopsia. CT showed low density areas in left superior and middle temporal gyri. Case 2 was a 15-year-old boy, who had left hemiplegia and hypesthesia to pain, temperature and touch on the left side of the body. CT showed low density areas from the genu of the internal capsule to the corona radiata, and from the posterior portion of putamen to the posterior limb of the internal capsule on the right side. Both cases had hypertriglyceridemia which might be associated with the etiology of infarction.