ERK induces p35, a neuron-specific activator of Cdk5, through induction of Egr1.

The classical mitogen-activated protein kinase (MAPK; also known as extracellular-signal-regulated kinase), ERK cascade has been shown to have a crucial role in cell proliferation and differentiation. In PC12 cells, sustained activation of ERK induced by nerve-growth factor (NGF) is essential for neuronal differentiation. However, downstream targets of ERK that are essential for neuronal differentiation have not been defined. Here we show that NGF induces strong, sustained expression of p35, the neuron-specific activator of cyclin-dependent kinase 5 (Cdk5), through activation of the ERK pathway. The induced kinase activity of Cdk5 is required for NGF-induced neurite outgrowth. Our results indicate that sustained activation of ERK is necessary and sufficient for strong induction of p35. Furthermore, the transcription factor Egr1, is induced by NGF through the ERK pathway and mediates induction of p35 by ERK. Our results thus define an essential signalling pathway, downstream of ERK/MAPK, that leads to neuronal differentiation.

Purification and properties of a membrane-bound phospholipase B from baker's yeast (Saccharomyces cerevisiae).

Phospholipase B bound tightly to the membrane fraction of baker's yeast (Saccharomyces cerevisiae) was purified approximately 226-fold by extraction with sodium deoxycholate (DOC), acetone precipitation, ammonium sulfate fractionation, and column chromatographies on Phenyl-Sepharose CL-4B, DEAE-Sepharose CL-6B, and Sepharose 4B. Only one major activity peak with an apparent molecular weight of 330,000 was detected by gel filtration on Sepharose 4B. However, two glycoprotein bands, one major and one minor, were evident on SDS-polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol. Isoelectric focusing also revealed two activities, pI 3.4 (major) and pI 3.0 (minor). The purified enzyme had phospholipase B activity (in the presence of DOC) and lysophospholipase activity in a ratio of 1:4.8. Both activities had an optimum pH of 3.5-4.0. Phospholipase B activity was appreciably stimulated only by DOC among bile acids, but lysophospholipase activity was markedly inhibited by them. Both activities were not stimulated by Ca2+ and inhibited by SDS, Triton X-100, Fe3+ and Al3+. The purified enzyme hydrolyzed the acyl ester bonds of phosphatidylcholine sequentially, first the 2-acyl and then the 1-acyl groups. It preferentially hydrolyzed 1-acyl-sn-glycero-3-phosphocholine and, to a lesser degree, 2-acyl-sn-glycero-3-phosphocholine.

Differential control of cellular gene expression by diffusible and non-diffusible EGF.

Cell gene expression is affected by both the kind and mode of growth factor stimulation (diffusive vs. non-diffusive). Epidermal growth factor (EGF) was pattern-immobilized on a polystyrene plate. Although the growth of the rat phaeochromocytoma cell line PC12 is stimulated by diffusible EGF, and differentiation is stimulated by diffusible nerve growth factor (NGF), immobilized (non-diffusible) EGF stimulated PC12 differentiation. The immobilized EGF caused a long-lasting stimulation of the intracellular signal protein mitogen-associated protein MAP kinase (MAPK, also known as ERK) and p38 (a subfamily of the MAPK superfamily) in cells, as did diffusible NGF. The switching between growth stimulation and differentiation is considered to be due to the duration of the stimulus. The function of the biosignal conjugate was regulated using conjugation methodology.

Mental stress test is an effective inducer of vasospastic angina pectoris: comparison with cold pressor, hyperventilation and master two-step exercise test.

BACKGROUND: Cold pressor, hyperventilation and exercise stress tests were usually used for inducing an angina attack in patients with vasospastic angina pectoris. We induced vasospastic angina attack using the mental calculation stress test, and compared the results with those using other stress tests. SUBJECTS AND METHODS: Subjects were 29 patients with vasospastic angina pectoris. Their ages were 60.8+/-8.4 years. Coronary vasospasm was induced by an acetylcholine infusion test during coronary angiography. The mental stress test was performed as follows; after memorizing six digits numbers, they repeated these numbers in reverse for 5 min, and performed serial subtraction of 17 from 1000 for 5 min. Blood pressure, heart rate and ECG were recorded every 1-5 min during the mental stress test. The serum concentrations of epinephrine and norepinephrine were measured before and during the mental stress test. We compared these results with those obtained using cold pressor, hyperventilation and the Master two-step exercise stress test. RESULTS: (1) Eight of the 29 patients (28%) showed ischemic ST-T change, which was caused by the mental stress test. (2) The increase in norepinephrine was greater in patients with an ST-T change than without an ST-T change (0.11+/-0.06 vs. 0.04+/-0.04 ng/ml, P<0.01). (3) The incidence of the ST-T change caused by the mental stress test (28%) was similar to the cold pressor test (27%) and greater than that caused by the hyperventilation test (13%). The incidence of ST-T change caused by the Master two-step test was 55%. CONCLUSIONS: The mental stress test is an effective inducer of vasospastic angina attack, and attack may be induced by neurohumoral vasoconstrictive reflex and/or increased left ventricular afterload.

[Isolation of verotoxin-producing Escherichia coli from pediatric patients suffering from enteritis with bloody stool and intussusception].

We tried to isolate verotoxin-producing Escherichia coli (VTEC) on sorbitol-MacConkey (SMAC) agar and in part by the polymerase chain reaction (PCR) method from sporadic enteritis patients with bloody stools and intussusception patients who came to three pediatric clinics in the Fukuoka area from October 1990 to September 1994. VTEC O157:H7 strains were isolated from 6 (10.5%) of 57 patients with bloody stools, Campylobacter spp. 15 (26.3%), Salmonella spp. 14 (24.6%) and Yersinia enterocolitica 2. We were not able to detect VT genes by PCR from 11 of 20 patients from whose stools no causative bacteria were isolated. Massive fresh bloody stools following frequent watery diarrhea were typical of the VTEC enteritis patients. Only 1 patient had fever and 2 had leukocytosis, but the C-reactive protein in all of them was below 1+. The VTEC strains were isolated during the summer season, 1 in June, 2 in July, and 3 in September. Since in the area O157:H7 appeared to be the most prevalent VTEC serotype, SMAC is very useful for screening VTEC in bloody stools. VTEC seems to be a rare pathogen of intussusception because the organisms were detected from none of the 30 patients.

[A study for serodiagnosis of verotoxin-producing Escherichia coli (enterohemorrhagic E. coli) O157 by the bacterial agglutination technique].

We evaluated the usefulness of bacterial agglutination antibodies for serodiagnosis of verotoxin-producing Escherichia coli (enterohemorrhagic E. coli) O157 infections. We examined 50 serum samples from 50 control children (whiout diarrhea 31, with diarrhea 19), 24 samples from 8 diarrhea cases due to O157:H7, 37 samples from 14 cases of hemolytic uremic syndrome (HUS) for antibodies to heat-killed E. coli E32511 (O157:H.-) strain using the bacterial agglutination technique. Of the control sera all but one (x80) showed 20 > or = in the antibody. All the diarrhea patients due to O157:H7 showed a significant rise (x160-x5120) of the titers in the sera at 5-7 days on illness, after that the titers fell rapidly. Significant antibody rise (x160-x5120) was detected in twelve out of 14 HUS patients at the early stage of the illness which fell in the convalescent phase. The assay appeared to be a useful serodiagnostic technique because of its easiness and simplicity as well as because of its high sensitivity and specificity.

[A case of encephalitis due to Mycoplasma pneumoniae: detection of specific DNA from cerebrospinal fluid and elevation of interleukin-6].

A 9-year-old female was admitted to our hospital due to a generalized seizure and consciousness disturbance. The patient had a fever and rash four days before admission, but she had no respiratory symptoms. The seizure and consciousness disturbance was prolonged and intractable. We diagnosed the patient as having encephalitis because of the increase in the cell count in the cerebrospinal fluid (CSF) and a diffuse slow EEG wave. The computed tomography of the head was normal. The causative agent was identified as Mycoplasma pneumoniae because of the increase of antibodies, and the detection of a specific DNA with a polymerase chain reaction. The interleukin (IL)-6 level of CSF was high (384 pg/ml). In spite of intensive treatment she had severe neurological sequelae. The invasion of Mycoplasma pneumoniae to the central nervous system appeared to have a role in the development of encephalitis in the patient. We speculated that there is a possible relationship between the IL-6 levels of CSF and clinical severity of encephalitis.

[Epidemiology of sporadic pediatric enteritis patients due to verotoxin-producing Escherichia coli].

We tried to isolate verotoxin-producing Escherichia coli (VTEC) from 323 sporadic pediatric enteritis patients who came to three clinics in the Fukuoka area. We used the sorbitol-MacConkey medium for the isolation of VTEC O157:H7. For non-O157 VTEC strains we used the V1/PECS method. VT/PECS method was applied. VTEC strains were isolated from three patients (0.9%). None of the patients were seriously, ill or developed the hemolytic uremic syndrome. The three patients were all seen in the summer season, July and August. O157:H7 strains were isolated from two patients, and O145:NM from one. This study showed that sporadic enteritis cases due to VTEC exist in the Fukuoka area. In the future a rapid and easy method for the detection of VT or VTEC should be developed and commercialized to proceed with epidemiological studies of VTEC infections throughout Japan.

[Analysis of verotoxin-producing Escherichia coli (O157:H7) strains isolated in the Fukuoka area in 1994 by pulsed-field gel electrophoresis].

Nine verotoxin-producing Escherichia coli O157:H7 strains were isolated from 9 pediatric patients with sporadic enteritis between July and September 1994 at four clinics in the Fukuoka area. The patients included two families with two cases each. These strains were analyzed by pulsed-field gel electrophoresis for Xba I-digested DNA fragments. The restriction patterns were identical between each two strains within the two family outbreaks, but different among the seven strains of the distinct seven sporadic cases. It is strongly suggested that the seven sporadic cases were infected through distinct sources, and that the two family cases were due to a common source of infection or person to person infection.

Measurement of the back fat layer in beagles for estimation of obesity using two-dimensional ultrasonography.

The purpose of this study was to select a suitable location for measuring the subcutaneous fat layer in beagles as an indicator of excess body fat deposition. A location suitable for such a measurement should meet five conditions--it should be easy to: (1) apply the probe, (2) detect the anatomical character of the measurement location, and (3) obtain a reproducible ultrasonogram at all times. In addition, the fat layer should be (4) thick enough for measuring, and (5) located in an area that varies markedly with the degree of obesity. Ultrasonograms made in the transverse planes on the top of the spinous process of the sixth lumbar vertebra, the seventh lumbar vertebra and the first sacral vertebra were found to be suitable for measurement. The depth and area of the back fat layer at these locations were closely related to the degree of obesity.

[Effect of colforsin daropate hydrochloride after cardiopulmonary bypass].

We evaluated the effects of colforsin daropate hydrochloride (CDH) after cardiopulmonary bypass (CPB) in comparison with milrinone. CDH or milrinone were given during and after CPB combined with cathecholamines so as to maintain mean arterial pressure (mABP > 60 mmHg) and cardiac index (CI > 3.0 l.min-1.m-2). Hemodynamics measurement was done immediately after CPB (A 1), one hour after CPB (A 2) and after the chest closure (A 3). In CDH group, mABP was significantly higher compared with milrinone group. Also, CDH group showed a significant reduction in the cases of combined use of dobutamine (at A 2, A 3) and norepinephrine (at A 1, A 2) compared with milrinone group. In conclusion, colforsin daropate hydrochloride exerts more inotropic effect and could reduce the necessity of combined use of cathecholamine compared with milrinone.

[Assessment of anesthesia satisfaction using direct interviews at post-anesthesia clinic].

The authors studied 5,034 consecutive patients undergoing elective surgery. Preoperative, intraoperative, and postoperative variables were gathered and patient satisfaction was assessed using direct interviews at pre- and post-anesthesia clinic. Fifty-nine percent of the 4,717 responders showed satisfaction and 4% showed dissatisfaction with anesthesia. The most undesirable perioperative outcome was vomiting/nausea. Other undesirable outcomes include discomfort of urine catheter, sore throat, memory of extubation, postoperative pain and so on. Anesthesiologist can improve the quality of anesthesia by preoperative explanation and preventative management for undesirable perioperative outcomes.

Effect of telmisartan on cholesterol levels in patients with hypertension - Saga Telmisartan Aggressive Research (STAR).

Saga Telmisartan Aggressive Research (STAR) is a single-arm, prospective multi-center trial to evaluate the effectiveness of treatment with telmisartan in patients with hypertension. A total of 197 patients with a systolic blood pressure of > or =140 or a diastolic blood pressure of > or =90 mmHg were enrolled in this study, and were prescribed 20 to 80 mg/day of telmisartan for 6 months. In all patients, both systolic and diastolic blood pressures decreased (159+/-20 to 135+/-12 mmHg, p<0.0001, 87+/-12 to 75+/-8 mmHg, p<0.0001, respectively). In addition, total cholesterol (TC) levels decreased from 200+/-40 to 188+/-33 mg/dl (p<0.05). In patients with TC > or =220 mg/dl, the change was more striking (249+/-33 to 204+/-31 mg/dl, p<0.0001). Even in patients receiving statins, TC levels still were decreased (216+/-51 to 190+/-31 mg/dl, p<0.02). In addition, TC levels were also decreased even in patients receiving telmisartan in exchange for other ARBs with TC > or =220 mg/d. Triglyceride (TG) levels were decreased (270+/-199 to 175+/-74 mg/dl, p<0.005) in patients with TG levels > or =150 mg/dl. Fasting blood glucose (FBG) was decreased (158+/-68 to 138+/-60 mg/dl, p<0.05) in patients with FBG > or =110 mg/dl. These results suggest that telmisartan may have favorable effects on lipid and glucose metabolism, in addition to lowering blood pressure. The profound effect of telmisartan to lower cholesterol suggests a potential use in hypertensive patients with dyslipidemia.

Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells.

Nerve growth factor (NGF) induces sustained activation of classical MAP kinase (MAPK, also known as ERK) and neuronal differentiation in PC12 cells, whereas epidermal growth factor (EGF) induces transient activation of ERK/MAPK and stimulates proliferation of the cells. Although previous studies showed that sustained activation of ERK/MAPK is important for neuronal differentiation of the cells, a recent report revealed that inhibition of the sustained phase of ERK/MAPK activation alone does not block neurite outgrowth caused by NGF. These results suggest requirement for an additional signaling pathway(s) triggered by NGF in neuronal differentiation. Here we show that NGF induces sustained activation of p38, a subfamily member of the MAPK superfamily, and that inhibition of the p38 pathway blocks neurite outgrowth in PC12 cells. Surprisingly, expression of constitutively active MAPK/ERK kinase (MAPKK, also known as MEK) results in p38 activation as well as ERK/MAPK activation, and a p38 inhibitor blocks neurite outgrowth caused by the constitutively active MAPKK/MEK. Moreover, constitutive activation of p38 is able to induce neurite outgrowth when combined with EGF treatment. These results reveal an essential role of p38 in neuronal differentiation in PC12 cells.

Prevention of exercise-induced bronchospasm in asthmatic children. Effect of aerosol and oral procaterol hydrochloride.

This double-blind crossover study compared the therapeutic value of bronchodilator procaterol hydrochloride at 5 micrograms per puff by aerosol inhalation using a metered-dose inhaler and at 25 micrograms by oral administration using tablets with respective inert placebos in preventing exercise-induced bronchospasm (EIB) in 10 children with bronchial asthma. The two active dosage formulations produced a significant EIB inhibitory effect compared with the placebos. In a comparison of the two active preparations, the aerosol was more efficacious than the tablet in improving all respiratory parameters examined, and statistical significance was obtained in FEV1, MMEF, PEFR, V50, and V25 5 minutes after exercise. Further, the aerosol was beneficial in all subjects, while the tablet was beneficial in seven of 10.

Morphological differences in flagella in Campylobacter fetus subsp. intestinalis and C. fetus subsp. jejuni.

Intact flagella were isolated from human pathogenic strains of Campylobacter, C. fetus subsp. intestinalis and C. fetus subsp. jejuni, by the method of DePamphilis and Adler and examined by electron microscopy. The isolated flagella were composed of a filament, a hook, a basal body, and a large disk associated with the end of the hook region covering the basal body. The width of the hook was approximately 28 nm, somewhat greater than that of the filament (20 nm in diameter). The hook region of C. fetus subsp. intestinalis was curved, but it was straight in C. fetus subsp. jejuni. The structure of the basal body of the two subspecies was similar to that reported for other gram-negative bacteria. The large disk detached from the flagella showed concentrically arranged circular structures. This structure was more clearly observed in the disk of C. fetus subsp. jejuni than in C. fetus subsp. intestinalis. Observations of thin-sectioned profiles at the attachment site of the flagellum revealed that the large disk is located on the inner side of the outer membrane. The role of the large disk in bacterial movement is not clear, but it is assumed that it acts as an organ to protect the flagellar insertion site from vigorous rotation of the polar end inflicted during bacterial movement.