A Nerve Growth Factor-Sensitive S6 Kinase in Cell-Free Extracts from PC 12 Cells

Soluble extracts from nerve growth factor (NGF)-stimulated PC12 cells prepared by alkaline lysis show a two- to 10-fold greater ability to phosphorylate the 40S ribosomal protein S6 than do extracts from control cells. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half-maximal incorporation of 32P from [32P]ATP into S6 occurred after 4-7 min of NGF treatment. The partially purified NGF-sensitive S6 kinase has a molecular weight of 45,000. It is not inhibited by NaCl, chlorpromazine, or the specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase, nor is it activated by addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF-treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP-dependent protein kinase or protein kinase C nor the result of tryptic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cAMP or 5'-N-ethylcarboxamideadenosine also increases the subsequent cell-free phosphorylation of S6. This observation suggests that cAMP-dependent protein kinase may be involved in the phosphorylation of S6 kinase.     

Clinical Use of a New Mitral Disc Valve

A disc valve of new design was used successfully for the replacement of the mitral valve in patients with rheumatic mitral valve disease. This valve would appear to have the following advantages over the mitral ball valve prosthesis:• Lower left atrial pressure after replacement.• Elimination of the hazard of left ventricular outflow tract obstruction with mitral valve replacement.• Decreased incidence of thromboembolization.• Abolition of possibility of ventricular septal irritation.Despite the better outlook for this valve compared with the ball valve for mitral valve substitution, the mitral valve should always be repaired whenever feasible. Repair is possible in the majority of patients.     

Alkaline serine protease produced from citric acid by Bacillus alcalophilus subsp. halodurans KP 1239

Summary Maximum production of alkaline serine protease by Bacillus alcalophilus subsp. halodurans KP 1239 was achieved after 24 h cultivation, at an initial pH of 7.6, on a medium containing 1.0% sodium citrate, 0.3% yeast extract, and 0.3% KH₂PO₄. The enzyme was purified to crystalline form from culture broth. The enzyme was most active at 60° C and at pH 11.5. The molecular weight, isoelectric point and sedimentation coefficient in water at 20° C were estimated as 29 000, 8.8 and 3.3S, respectively. The N-terminal amino acid sequence was Ala-Gln-Ser-Val-Pro-Trp-Gly-Ile-Ser-Arg-Val-Gln-Ala-Pro-Ala-Ala-His-Asn-Arg-Gly-. The enzyme shared its antigenic determinants with B. alcalophilus ATCC 21522 serine protease, but not with the subtilisins Carlsberg and BPN. Offprint requests to: Yuzuru Suzuki     

Characterization of a Dopamine-Releasing Action of 6R-l-erythro-Tetrahydrobiopterin: Comparison with a 6S-Form

Abstract: 6R-l -erythro-Tetrahydrobiopterin (6R-BH₄) is a cofactor for aromatic l -amino acid hydroxylases and nitric oxide synthase. Recently, we have reported that independently of its cofactor activities, 6R-BH₄ acts from the outside of neurons in the brain to enhance the release of monoamine neurotransmitters such as dopamine. To characterize the pharmacological properties of the action, we examined the effects of 6S-BH₄, a diastereoisomer of 6R-BH₄, on dopamine release in the rat striatum by using brain microdialysis and compared its effects with those of 6R-BH₄. Perfusion of 6S-BH₄ or 6R-BH₄ through the dialysis probe increased extracellular dopamine levels (an index of in vivo dopamine release) concentration dependently; the maximal increase by 6S-BH₄, was one-sixth of that by 6R-BH₄. 6S-BH₄ increased extracellular DOPA levels in the presence of NSD 1015, an inhibitor of aromatic l -amino acid decarboxylase (an index of in vivo tyrosine hydroxylase activity), to an extent similar to the increase induced by 6R-BH₄. The increase in the DOPA levels induced by either of the pteridines was abolished after pretreatment of rats with α-methyl-p-tyrosine (an inhibitor of tyrosine hydroxylase). Under the same conditions, the 6S-BH₄-induced dopamine release was abolished, but most of the 6R-BH₄-induced increase persisted. Coadministration of 6S-BH₄ with 6R-BH₄ inhibited the increase in dopamine release induced by 6R-BH₄ alone. These results show that 6R-BH₄ stimulates dopamine release by acting at the specific recognition site on the neuronal membrane, and that 6S-BH₄ acts as an antagonist of 6R-BH₄ at this site, although it has cofactor activities.     

Putative morphogen, DIF, of Dictyostelium discoideum induces apoptosis in rat pancreatic AR42J cells

We have recently shown that differentiation-inducing factor-1 (DIF-1) of Dictyostelium discoideum is capable of raising intracellular calcium concentration ([Ca²⁺]_i) and suppressing cell proliferation of rat pancreatic AR42J cells in a dose-dependent manner, and that DIF-1 at a concentration of 40 μmol/L is toxic to the cells. In this study, we have further characterized the cytotoxic effect of DIF-1 on AR42J cells and have analyzed the effect of DIF-1 on [Ca²⁺]_i. In the presence of 40 μmol/L DIF-1, cells began to bleb after approximately 6 h, and most had died within 48 h. Biochemical analysis revealed that DNA fragmentation was accompanied by cell death. Monitoring the changes in [Ca²⁺]_i induced by DIF-1, it was found that cells were able to adapt to stimulation with DIF-1 so that they did not respond to subsequent stimulation by DIF-1. These results indicate that DIF-1 induced apoptosis in AR42J cells probably via a cell signaling system.     

Phase transitions of phospholipid bilayers from an unsaturated fatty acid auxotroph of Escherichia coli

Total phospholipids were extracted from cells of temperature sensitive unsaturated fatty acid auxotrophs of Escherichia coli (K-12 UFA^ts) grown at 28°C (PL28), and at 42°C in the presence of 2% KCl as an osmotic stabilizer (PL42 (KCl)). From the analysis of fatty acids, it was shown that the content of unsaturated fatty acids of PL42 (KCl) is only 9% of the total fatty acids, while that of PL28 is 54%. The thermal phase transitions of the bilayers prepared from the phospholipid fractions were studied by proton magnetic resonance. The line widths of the methylene signals and the sums of the methylene and methyl signal intensities were plotted against reciprocal values of absolute temperature 1/T or temperature itself. From the plots phase transitions were detected at about 19°C for PL28 and at 43°C for PL42 (KCl). In spite of its complex composition of fatty acids a highly cooperative transition was observed in the case of PL42 (KCl). It was also suggested that the phospholipids bilayers in the biomembranes of this strain at the growth temperature (42°C) are in the state where the gel and liquid crystalline phases coexist.     

Production of L-aspartic acid from fumaric acid by a fumaric acid-assimilating obligate thermophile,Bacillus stearothermophilus KP 1041

Summary A fumaric acid-assimilating obligate thermophile having a high aspartase activity was isolated from soil. The isolate (KP 1041) that grew at 45 to 68 °C was assigned to a strain of Bacillus stearothermophilus. The cell suspensions produced L-aspartate from fumarate and ammonium ion, with the rapidest initial rate at 65 °C and pH 9.5. The Michaelis constant for fumarate was 0.2 M. The cellular aspartase was relatively stable for 18 h at and below 50 °C over a pH range 6.7–8.3 in the presence of ammonium fumarate; this substance protected the enzyme from heat inactivation. The best yield in L-aspartic acid production was achieved at 6 h incubation at 53 °C and pH 8.5, using 0.88 M fumarate, 3.1 M ammonium ion, and the cells at 53 mg dry weight per ml. In this case, 85% of fumarate added was converted into aspartic acid. The structure of the product was determined from its infrared spectrum, specific rotation, melting point and ultimate analysis.Presented at the Annual Meeting of the Agricultural Chemical Society of Japan, Yokohama, April 2, 1977     

Interrelationship between serum muscle enzymes and a low T3 in anorexia nervosa

To determine the interrelationship between muscle dysfunction and a low T3 state, both seen in anorexia nervosa, we studied the relationship between the degree of muscle involvement, as assessed by the circulating concentration of the three muscle indicators (CPK, GOT and LDH), and serum T3 in thirty-three patients when they were admitted to the hospital. We also studied the malnutritional state, as assessed by their body weight or serum GH, serum potassium and the degree of hyperactivity exhibited. Additionally, another twelve patients were studied in order to explore the mounding phenomenon which is typically elicited in hypothyroidism. The logarithms of serum CPK and GOT correlated only with the serum T3 concentration (r = -0.35, p less than 0.05; r = -0.41, p less than 0.05; respectively). The logarithm of serum LDH highly correlated with serum T3, the percentage of ideal body weight, and the logarithm of serum GH (r = -0.55, p less than 0.01; r = -0.66, p less than 0.001; r = 0.43, p less than 0.05; respectively). The mounding phenomenon was elicited in ten out of twelve patients. In conclusion, it was implied that a low T3 state was associated with an increase in serum muscle indicators and thus with muscle dysfunction encountered in anorexia nervosa.