Synthesis of N-4909 analogs. Part I. A stimulant of apolipoprotein E secretion in human hepatoma G2 cells.

Analogs of N-4909 (1), which had a stimulating activity for apolipoprotein E (apo E) secretion in Human hepatoma Hep G2 cells, were prepared and their activities examined. Cyclic analogs which had different kinds of amino acids or different number of amino acids from N-4909 (1) showed less effect on apo E secretion from Hep G2 cells. The length of acyl chain was found to be an important factor for the activity. Shorter chain reduced the activity. Linear analogs were also prepared. One of their analogs, N-5849 (17), which had six amino acids was found to have strong activity.

Deletion of Ala144-Lys145 in Thermus thermophilus inorganic pyrophosphatase suppresses thermal aggregation.

The regions contributing to the thermostability of inorganic pyrophosphatase (PPase, EC 3.6.1.1) from Thermus thermophilus (Tth) were deduced in our previous study by random chimeragenesis, one of them being estimated to be Ala144-Lys145 [Satoh, T., Takahashi, Y., Oshida, N., Shimizu, A., Shinoda, H., Watanabe, M., and Samejima, T. (1999) Biochemistry 38, 1531-1536]. Therefore, we investigated the contributions of these two residues in Tth by preparing a deletion mutant (del.144-145 mutant) of Tth PPase. We examined its thermostability in terms of the CD and fluorescence spectra, and the thermal change in the enzymatic activity. The thermostability of the enzymatic activity of the del.144-145 mutant was similar to that of the wild type Tth PPase, whereas this mutant was more stable against heating. Furthermore, we compared the thermal aggregation of the wild type with that of the del.144-145 mutant. We found that the thermal aggregation of the mutant was reduced relative to that of the wild type. Moreover, the molecular weight of the mutant after heating at 90 degrees C was higher than that of the unheated one, whereas the wild type aggregated under the same conditions. Therefore, we can conclude that although the Ala144-Lys145 residues in Tth PPase may partly cause thermal aggregation, the deletion of these residues may stabilize the Tth PPase molecule structurally against heating and suppress thermal aggregation.

A chimeric inorganic pyrophosphatase derived from Escherichia coli and Thermus thermophilus has an increased thermostability.

Factors contributing to the thermostability of inorganic pyrophosphatase (PPase) were investigated by examining chimeric PPases from Escherichia coli and Thermus thermophilus (Tth). Two chimeric PPase genes, T1-135E (residues 1-135 from the N terminus are comprised of Tth PPase and residues 136-173 are derived from the C terminus of E. coli PPase) and T1-149E [residues 1-149 from the N terminus are from Tth PPase and the rest (150-175) are from E. coli PPase], were constructed by random chimeragenesis. After the genes were overexpressed in the E. coli BL21(DE3) strain and the expression products were purified, we compared the characteristics of these chimeric PPases with those of the parental PPases. We found that the two chimeras had higher activity than either parent PPase at the optimum temperature. We also examined thermal stability in terms of CD spectra, fluorescence spectra, and thermal changes in enzyme activity. The results revealed that the thermal stability of T1-149E is similar to that of Tth PPase, but T1-135E is much more stable. This suggests that the four residues that are different between T1-135E and T1-149E may be critical for thermostability between the two chimeras. By comparing the three-dimensional structures of Tth and E. coli PPases, we deduced that the following two factors may contribute to differences in thermostability. (1) Two residues (Thr138 and Ala141 in the Tth PPase and His140 and Asp143 in the E. coli PPase) in the vicinity of the trimer-trimer interface were different. (2) The Ala144-Lys145 loop in the Tth PPase was deleted in the E. coli PPase and also in the T1-135E chimera. Therefore, we conclude that T1-135E was thermostabilized by these two factors, and also, the Tth PPase moiety may contribute to the structural integrity of the chimeric enzymes.

[Clinical effects of sodium ozagrel and urokinase in patients with acute cerebral infarction in the territory of the internal carotid artery].

In this study, the clinical effects were compared between a thromboxane synthetase inhibitor (sodium ozagrel) and a thrombolytic agent (urokinase) in patients with acute cerebral infarction. The subjects consisted of 598 patients admitted on the day of the onset of the cerebral infarction in the territory of the internal carotid artery who showed a low density area on CT images within 5 days. Of these patients, 300 were treated with sodium ozagrel and classified as Group Oz, while the remaining 298 were treated with urokinase and classified as Group Ur. The results were as follows: 1. In group Oz, complete recovery of motor impairment was seen in 209 (69.7%) patients. Complete recovery within 3 weeks after onset was seen in 186 (62.0%) patients. In group Ur, complete recovery of motor impairment was seen in 175 (58.7%) patients. Complete recovery within 3 weeks after onset was seen in 120 (40.3%) patients. Therefore, a higher incidence of complete recovery of the motor impairment was noted in group Oz [p < 0.001: chi 2 test]. Similarly, complete recovery within 3 weeks after onset was more frequent in group Oz [p < 0.001: chi 2 test]. 2. In group Oz, complete recovery was made contribution statistically by Anosognosia (Ag) and unilateral neglect (UN) on admission [multivariate analysis: p < 0.01]. In group Ur, complete recovery was made contribution statistically by Ag (p < 0.01), UN (p < 0.01) and aphasia (p < 0.05). 3. Progressive stroke was observed in 29 (9.5%) patients in the group Oz and in 71 (23.0%) patients in group Ur. There was a higher incidence of progressive stroke in group Ur [p < 0.001: chi 2 test] 4. All patients with progressive stroke had initial evidence of deterioration of neurological deficits within 6 days after the onset in group Oz, and within 5 days after the onset in group Ur. The maximal period from the beginning to the end of the deterioration of neurological deficit was 7 days. 5. In group Oz, progressive stroke was only seen in 29 (29.9%) of the patients who were admitted with motor disturbances and unilateral neglect. In group Ur, progressive stroke was seen in 8 (4.3%) of the 187 patients with motor disturbances without higher cortical dysfunction, in 17 (47.2%) of the 36 patients with motor disturbances and higher cortical dysfunction without unilateral neglect and was seen in 46 (61.3%) of the patients with motor disturbances and unilateral neglect. 6. Hemorrhagic infarction was observed in 14 (4.6%) patients in group Oz and in 31 (10.0%) patients in group Ur. There was a higher incidence of hemorrhagic infarction in group Ur [p < 0.001: chi 2 test]. 7. In group Oz, there was a higher incidence of hemorrhagic infarction among patients with atrial fibrillation (Af) on the ECG [p < 0.001: chi 2 test]. Similarly, in group Ur, hemorrhagic infarction was more frequent among patients with atrial fibrillation (Af) on the ECG [p < 0.001: chi 2 test]. Therefore, sodium ozagrel was clinically more efficient and safer than urokinase in patients with acute cerebral infarction.

[Motor function outcome and changes in motor impairment level of the upper limbs and fingers in patients with acute carotid-system cerebral infarction].

Early diagnosis of motor function status and changes in motor impairment level of the upper limbs and fingers in patients with acute cerebral infarction is important in establishing a treatment plan. This study investigated a method of predicting motor function outcome and changes in motor impairment based on the characteristics of symptoms on admission and severity according to the CT classification. The subjects were 309 patients with carotid-system cerebral infarction admitted on the day of onset of symptoms and who exhibited a low density area in the territory of the middle cerebral artery on CT images within 5 day of the onset of symptoms. The motor function level was evaluated according to Brunnstrom's stage. The findings were as follows: 1) The motor function level of the upper limbs and fingers improved to stage 3 or more in patients without unilateral neglect. 2) Complete recovery of motor impairment of the upper limbs and fingers was observed in 163 patients (54.7%). Motor impairment of the upper limbs recovered within 3 weeks after the onset in 121 patients, and from 22 days to 3 months after the onset in the remaining 42 patients. Similarly, motor impairment of the fingers recovered within 3 weeks after the onset in 113 patients, and between 22 days and 3 months after the onset in the remaining 50 patients. 3) Stage 4 or higher motor impairment grade on admission was seen in 107 (88.4%) of the patients with recovery of impaired motor function in the upper limbs within 3 weeks after the onset. Similarly, stage 4 or higher was seen in 101 (89.4%) patients with recovery of impaired motor function of the fingers within 3 weeks after the onset. 4) Of the 44 patients with recovery of impaired motor function of the upper limbs between 22 days and 3 months after the onset, 30 improved to stage 4 by 2 weeks after the onset and to stage 6 by one month. The remaining 12 patients improved to stage 5 by one month after the onset and to stage 6 by 3 months. Of the 50 patients with recovery of impaired motor function of the fingers between 22 days and 3 months after the onset, 44 improved to stage 4 by 2 weeks after the onset and to stage 6 by one month. The remaining 6 patients improved to stage 5 by one month after the onset and to stage 6 by 3 months. 5) Progressive stroke was observed in 60 patients (20.1%). All patients with progressive stroke showed unilateral neglect on admission. 6) Completed stroke, excludise of progressive stroke, was seen in 75 patients (24.3%). In the patients with completed stroke, there was clearly no improvement in the motor impairment of the limbs and fingers over 3 months after onset. 7) It was difficult to predict motor function level at the time of discharge based on the evaluation of stage on admission and the location of the low density area on CT. 8) Our CT classification was closely correlated with the motor function outcome of the upper limbs and fingers. Therefore, this classification and assessment of whether unilateral neglect is present on admission may be useful in predicting motor function outcome and changes in motor impairment of the upper limbs and fingers early after the onset of acute cerebral infarction.

[Progressive stroke during the acute state of cerebral infarction in the territory of the internal carotid artery].

Diagnosis of deterioration of neurological deficits in the early stage after hospital admission immediately after the onset of cerebral infarction is important in establishing a treatment plan. This study investigated the clinical characteristics of progressive stroke on admission. The subjects were 309 patients admitted on the day of the onset of symptoms and showed a low density area on CT images within 5 days. There were 202 with cerebral infarction in the territory of the deep perforate arteries, 77 with cerebral infarction in the territory of the cortical branch of the middle cerebral artery (MCA), and the remaining 36 had cerebral infarction in the territory of MCA. The results were as follows: 1) Progressive stroke was observed in 71 patients (23.0%): 60 with completed stroke and 11 with reversible ischemic neurological deficits (RIND). 2) The patients with progressive stroke were clearly older than those with non progressive stroke (p < 0.05: Cochran Cox's test). 3) Progressive stroke was seen more frequently in patients with cerebral infarction of the cortical branch of the MCA and patients with occlusion of the internal carotid artery and MCA trunk than patients with cerebral infarction of the deep perforate arteries (p < 0.001: chi 2 test). 4) All patients with progressive stroke had initial evidence of deterioration of neurological deficits within 5 days after the onset, with 39 patients showing deterioration on day 2 and 13 patients showing deterioration on day 3. 5) Deterioration of neurological deficits usually stopped the day after the start of progression. The maximum period from the beginning to the end of the deterioration of neurological deficits was 7 days. 6) Progressive stroke was not seen in 11 patients who were admitted with higher cortical dysfunction and without sensory or motor disturbances, and was seen in only 8 (4%) of the 187 patients with sensory or motor disturbances without higher cortical dysfunction. 7) Progressive stroke was seen in 63 (57%) patients with sensory or motor disturbances and higher cortical dysfunction. 8) Progressive stroke was seen frequently in patients with atrial fibrillation (Af) on the ECG [P < 0.001). 9) Fifty-six of 71 patients with progressive stroke showed persistent severe motor impairment in the upper limbs. Therefore, to diagnose progressive stroke, it is useful to assess the higher cortical dysfunction and examine for Af on admission.