S-100 Protein in Clonal Astroglioma Cells Is Released by Adrenocorticotropic Hormone and Corticotropin-Like Intermediate-Lobe Peptide

S-100 protein in clonal GA-1 and C6 rat glioma cell lines was released in serum-free medium supplemented with adrenocorticotropic hormone (ACTH). The induction of S-100 protein release by ACTH was dose-dependent, showing a half-maximal release at about 5 microM, and the S-100 protein concentration in the medium increased sharply within 3 min, but slightly during further incubation. The S-100 protein release was apparently accompanied by a decrease in the membrane-bound form of S-100 protein in the cell. The S-100 protein release was induced not by the ACTH1-24 fragment, which exhibits the known effects of ACTH, but by the ACTH18-39 fragment, which is designated as corticotropin-like intermediate-lobe peptide (CLIP). These results indicate that the C-terminal half of ACTH is responsible for the S-100 protein release. The enhancement of S-100 protein release by ACTH was also observed in normal rat glioblasts. The release induced by ACTH was apparently specific to S-100 protein, because little release of the cytoplasmic enzymes, creatine kinase, and enolase was observed under the same conditions. High concentrations (5 mM) of dibutyryl cyclic AMP or dibutyryl cyclic GMP were also found to induce S-100 protein release; however, catecholamines (epinephrine, norepinephrine, isoproterenol, and dopamine), acetylcholine, and glutamic acid did not enhance the release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical localization of plasma retinolbinding protein and prealbumin in human pancreatic islets

Summary This study was undertaken, employing the immunoenzyme method, to confirm the presence of retinol-binding protein in human pancreatic islets, and to compare its distribution with that of prealbumin, insulin, glucagon, somatostatin and pancreatic polypeptide. It was found that most islet cells contained retinol-binding protein, although centrally located cells showed stronger reactivity than those in the peripheral region. The distribution of each of the five polypeptides differed from that of retinolbinding protein, indicating that these peptides did not cross-react with anti-retinol-binding protein antibody. Islet cells which contained prealbumin, on the other hand, were mostly classified as A cells. Further studies are necessary to confirm whether the islet cells produce retinol-binding protein or only store it.     

Aminoacyl-tRNA exclusively in the 3'-isomeric form is bound to polypeptide chain elongation factor Tu

2' and 3'-O-(N-acetyl-L-phenylalanyl)adenosine (Ac-Phe-Ado) were chemically synthesized. These two isomers were clearly separated from each other by high-performance liquid chromatography (HPLC). From the two isomers of [3H]Phe-tRNA in equilibrium, Ac-[3H]Phe-Ado was prepared, without any change in the 2'/3'-isomer ratio, by acetylation of the phenylalanyl residue with acetic anhydride followed by digestion with pancreatic RNase A. By HPLC analysis of this preparation of Ac-[3H]Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H]Phe-tRNA was found to be 0.20:0.80. Further, [3H]Phe-tRNA was bound to Escherichia coli polypeptide chain elongation factor Tu (EF-Tu) with the ligand of GTP or guanosine 5'-[beta, gamma-imido]triphosphate (GMP-P(NH)P). The ternary complex was treated with phenol and acetic anhydride, and then digested with pancreatic RNase A. By HPLC analysis of Ac-[3H]Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H]Phe-tRNA was determined to be 0.07:0.93 in the complex with EF-Tu.GTP and 0.04:0.96 in the complex with EF-Tu.GMP-P(NH)P. These results clearly indicate that the 3'-isomer, rather than the 2'-isomer, of aminoacyl-tRNA is exclusively involved in the ternary complex.     

Exercise-induced splitting of the inorganic phosphate peak: investigation by time-resolved 31P-nuclear magnetic resonance spectroscopy

To investigate the splitting of the inorganic phosphate (Pi) peak during exercise and recovery, a time-resolved ³¹phosphorus nuclear magnetic resonance spectroscopy (³¹P-MRS) technique was used. Seven healthy young sedentary male subjects performed knee flexion exercise in the prone position inside a 2.1-T magnet, with the surface coil for ³¹P-MRS being placed on the biceps femoris muscle. After a 1-min warm-up without loading, the exercise intensity was increased by 0.41 W at 15-s intervals until exhaustion, followed by a 5-min recovery period. The ³¹P-MRS were recorded every 5 s during the rest-exercise-recovery sequence. Computer-aided contour analysis and pixel imaging of the Pi and phosphocreatine peaks were performed. Five of the seven subjects showed two distinct Pi peaks during exercise, suggesting two different pH distributions in exercising muscle (high pH and low pH region). In these five subjects, the high-pH increased rapidly just after the onset of exercise, while the low-pH peak increased gradually approximately 60 s after the onset of exercise. During recovery, the disappearance of the high-pH peak was more rapid than that of the low-pH peak. These findings suggest that our method ³¹P-MRS provides a simple approach for studying the kinetics of the Pi peak and intramuscular pH during exercise and recovery.     

31P-Nuclear magnetic resonance spectroscopy study of the time course of energy metabolism during exercise and recovery

This study evaluated the time courses of intracellular pH and the metabolism of phosphocreatine (PCr) and inorganic phosphate (P) at the onset of four exercise intensities and recoveries. Non-invasive evaluation of continuous changes in phosphorus metabolites has become possible using³¹P-nuclear magnetic resonance spectroscopy (³¹P-MRS). After measurements at rest, six healthy male subjects performed 4 min of femoral flexion exercise at intensities of 0 (loadless), 10, 20 and 30 kg · m · min^–1 in a 2.1 T superconducting magnet with a 67-cm bore. Measurements were continuously made during 5 min of recovery. During a series of rest-exercise-recovery procedures,³¹P-MRS were accumulated using 32 scans · spectrum^–1 requiring 12.8 s each. At the onset of exercise, PCr decreased exponentially with a time constant of 27–32 s regardless of the exercise intensity. The time constant PCr resynthesis during recovery was about 27–40 s. The PCr kinetics were independent of exercise intensity. There were similar Pi kinetics at the onset of all types of exercise, while those of Pi recovery became significantly longer at the higher exercise intensities (P < 0.05). Furthermore, the intracellular pH indicated temporary alkalosis just at the onset of exercise, probably due to absorption of hydrogen ions by PCr hydrolysis, and then decrease at a point about 40%–50% of the preexercise PCr. The pH recovery time was longer than that for the P_i or PCr kinetics. By using a more efficient resolution system it was possible to obtain the phosphorus kinetics during exercise and to follow PCr resynthesis within the first few minutes of recovery. From our results it was concluded that in general the time course of PCr and Pi metabolism were unaffected by the exercise intensity, both at the onset of exercise and during recovery, with the exception of P_i recovery.     

Changes in intracellular pH during repeated exercise

The rates of change in intracellular pH during repeated exercise sessions with rest periods was determined by 31 phosphorus-nuclear magnetic resonance spectroscopy (³¹P-MRS). Five long-distance runners and six healthy male subjects as controls performed a 2-min femoral flexion at 20 kg · m · min^–1 in a 2.1 T superconducting magnet with a 67-cm bore and repeated this exercise four times with 2-min rest periods intervening. In all cases during exercise the inorganic phosphate (P_i) peak split into two, the earlier increased rapidly (high-pH P_i) and the later (low-pH P_i) increased more slowly. The P_i peaks were separated by a fitting procedure using the least square mean method. The high-pH P_i area during exercise decreased as the number of repeated exercise periods increased, while the low-pH P_i area gradually increased. Although the total P_i area decreased exponentially during the recovery period, the high-pH P_i area decreased first and then the low-pH P_i area reduced gradually. The pH values were estimated from the chemical shift between the phosphocreatine peak and each split peak in the P_i. The high-pH in pooled data ranged from 6.6 to 7.0 during exercise and recovery, while the low pH decreased to 6.2 during exercise. As the number of exercise periods increased, each pH value gradually became less acidic, although there was a tendency to more acidity in the control subjects than in the long-distance runners. In conclusion, it was possible to obtain by non-invasive, continuous³¹P-MRS, a split pattern of P_i peaks during exercise and there were at least tow different intracellular pH values during exercise, suggesting that each P_i peak might be attributed to the types of muscle fibre recruited.     

Effects of photoperiod and aging on locomotor activity rhythms in the onion fly,Delia antiqua

At photoperiods longer than 8h per 24h, adults of the day-active onion fly Delia antiqua showed a major peak of locomotor activity in the late photophase and also bursts of activity induced by lights-on or lights-off. At shorter photoperiods the activity peaks fused. After transfer from long photoperiods to constant darkness (DD), the rhythm free-ran, but only the major peak persisted. This suggests that only the major peak is controlled by the circadian pacemaker. At long photoperiods, the daily phase of the major peak occurred progressively later with age. As a result, the activity at short photoperiods often shifted from photophase to scotophase in old flies. The free-running period (tau) also changed with age; tau was shorter than 24h until 14-20 days after eclosion and thereafter became longer, but a few individuals repeated changes in tau. The phase delay of locomotor activity with age in D. antiqua would be attributable to the increase in tau.     

The rate assay of alpha-toxin assembly in membrane

Abstract A rapid and easy method to determine the 'rate' of the assembly of α-toxin from Staphylococus aureus in erythrocyte membrane was described. Upon addition of a small amount of α-toxins into erythrocyte suspension, absorbance at 700 nm decreased linearly after a short period of lag time. From the linear portion of the record the rate of the assembly of α-toxin was calculated. An optimum temperature and an optimum pH for the assembly of the toxin on erythrocyte membranes were found to be 25–30°C and pH 5.     

Guest-induced conformational change of β-cyclodextrin capped with an environmentally sensitive chromophore

A capped cyclodextrin, 6-deoxy-6-(p-hydroxy-m-nitrophenacylthio)-β-cyclodextrin, was prepared in order to detect any conformational change of the host upon the guest binding. The association constant between the cyclodextrin and 1-adamantanecarboxylate, cyclohexanecarboxylate, p-methylbenzoate, 3,3-dimethylbutyrate, or 2,2-dimethylpropionate was enhanced 20, 5.3, 3.7, 2.3, or 2.0 times, respectively, by chromophore capping. The changes in the electronic, NMR, and circular dichroism spectra as well as pK_a of this cyclodextrin upon binding of the guest strongly indicate a conformational change around the chromophoric moiety of the cyclodextrin.