Oxidation Rate of Monomeric Radish Cytochrome f with Plastocyanin

The reaction rate of reduced monomeric cytochrome f with oxidizedplastocyanin, both purified from Japanese radish, was determinedby a stopped-flow method. The oxidation rate constant was 6.0x 10⁷ M¹sec¹ at pH 7.0 and 25°C, which is slightly higherthan the value reported by Wood [(1974) Biochim. Biophys. Acta357 : 370] for oligomeric parsley cytochrome f Thermodynamicparameters also were determined to be 56 KJ M^–1 for activationenthalpy and 90 J M^–1 K^–1 for activation entropy.Neither a pH from 6 to 9 nor the addition of NaCl, polylysine,histone or polyaspartate affected the rate constant. ¹Present address: The National Institute for Environmental Studies,Yatabe, Ibaraki 305, Japan. (Received October 9, 1980; Accepted November 17, 1980)     

Purification of cytochrome f from Japanese-radish leaves

Cytochrome f was purified (A_420.5/A₂₇₃, 2.0) from acetone extractsof Japaneseradish leaves without use of detergent. By gel filtrationwidi Sephadex G-100 the molecular weight was estimated to be33,000 daltons. (Received September 17, 1974; )     

Incidence of trauma in a breeding and rearing colony of cynomolgus monkeys (Macaca fascicularis)

A statistical survey was performed in 1982 to ascertain the incidence of various diseases, especially trauma, in the cynomolgus monkey colony at Tsukuba Primate Center for Medical Science. In total, 1,029 cases of disease, occurred of which 866, 89 and 74 were surgical, internals and reproductive disease cases, respectively (Table 1). Among the 866 surgical cases, trauma (746 cases) was most often observed (Table 2). It was shown that trauma occurred most frequently during the group feeding of juvenile monkeys (Table 3). Analysis of the factors influencing the incidence of trauma during group feeding was carried out. As a result, it was revealed that the number of days after beginning group feeding and the age at which group feeding was begun had marked influences on the incidence of trauma (Figs. 1 and 2). However, there was no significant relationship between the incidence of trauma and the number of animals per group or the sex-ratio in a group (Tables 4 and 5). From the present survey results, a prospective rearing system should be established to reduce the incidence of trauma in the cynomolgus breeding and rearing colony.     

Purification and biochemical properties of an N-hydroxyarylamine O-acetyltransferase from Escherichia coli

The N-hydroxyarylamine O-acetyltransferase of Escherichia coli has been expressed as a histidine tagged fusion protein and purified using immobilized nickel column chromatography. The molecular mass of the histidine tagged N-hydroxyarylamine O-acetyltransferase was estimated to be 60.0 kDa by gel filtration and 34.0 kDa by SDS–PAGE and DNA sequence, suggesting that the native enzyme exists as homo dimer. The catalytic properties were investigated using o-aminobenzoic acid as a substrate. No difference in acetyltransfer activity was observed between histidine tagged protein and untagged enzyme. Kinetic studies indicated a ping-pong bi bi mechanism of the catalysis. Inhibition by N-ethylmaleimide and salicylic acid was competitive with o-aminobenzoic acid and non-competitive with acetyl-CoA.     

Spontaneous diabetes mellitus in cynomolgus monkeys (Macaca fascicularis)

Glucose tolerance tests were performed with fourteen cynomolgus monkeys. They were divided into two groups with regard to the serum glucose level at the time of routine health-examination. Nine of them had normal glucose level (below 123 mg/dl, the normal group) and the other five monkeys exhibited hyperglycemia (the abnormal group). Fifty per cent glucose solution was administered into the saphenous vein at a dose of 4 ml/head. Blood samples were taken just before and 5, 10, 20, 30, 60 and 120 minutes after the glucose administration. K-value (K = 0.693/t 1/2 X 100) as the decreasing rate of serum glucose during from 5 to 60 minutes after the administration was calculated. Average K-value for eight monkeys of the normal group was 3.12 +/- 0.48. Both immunoreactive insulin level (IRI) and C-peptide immunoreactivity (CPR) increased just after the glucose administration and began to decrease 5 to 30 minutes after the administration in all the eight animals. Remaining one animal (No. 009) of the normal group showed 1.03 in K-value. For the abnormal group, K-value averaged 0.75 +/- 0.25. IRI was slightly higher in this group than in the 8 monkeys of the normal group. Furthermore, the abnormal group did not show any definite change of a certain trend in IRI and CPR. In conclusion, the former 8 monkeys were judged to be normal in the function of pancreatic beta-cells, and the latter 5 monkeys and No. 009 monkey were judged to be suffering from type II (noninsulin dependent) diabetes mellitus at different stages of the disease.     

Murine forebrain and midbrain crest cells generate different characteristic derivatives in vitro

Neural crest (NC) is a transient structure that gives rise to various types of tissues. Many NC cells are pluripotent in the sense that their progeny can generate more than one derivative. However, the potentiality to differentiate into certain derivatives, such as cartilage and bone, seems to be specified with respect to the neuraxial levels at which the NC generates. In order to compare the differentiation potentiality of different regions of head NC, the derivatives of forebrain and midbrain mouse NC have been investigated in vitro using explant cultures of neuroepithelial fragments. From morphology and expression of specific markers, the midbrain crest cultures obviously generated earlier and were greater in number of neuronal cells than were the forebrain ones. Moreover, collagen type II positive cells were detected in the midbrain but not in the forebrain crest cultures. Finally, pigment cells were only observed in the forebrain cultures. The results suggest that the forebrain and midbrain crest cells have a different potentiality to differentiate.     

Cloning of the Promoter Regions of Mouse TGF-{beta} Receptor Genes by Inverse PCR with Highly Overlapped Primers

In order to isolate promoters of mouse TGF-ß receptorgenes, we used inverse PCR with highly overlapped primers correspondingto the 5' sequence of the receptor cDNAs. Nested primer setsonly covered a 30- to 40-base region of the sequences. HinfI-digestedand self-ligated mouse genomic DNA was used as a PCR template.Only one band for each receptor was seen after PCR. The amplifiedDNA fragments could direct luciferase production when the luciferasecoding sequence was ligated after the fragments. The sequenceof the fragment which correspond to the type II receptor showedpartial homology with the promoter region of the human TGF-ßtype II receptor. Thus, the inverse PCR with highly overlappedprimers could be an easy way to isolate the promoter regionsof many genes.     

Two types of surface amine receptors modulating the feeding response inHydra japonica: the depressing action of dopamine and related amines

The effect of amines on the feeding response of Hydra japonicawas determined using the tentacle ball formation assay. Manyaliphatic and aromatic amines depressed the response immediatelyupon addition to the bathing medium at final concentrationsranging from 1 nmol/l to 100 µmol/l. Examining the depressingeffect of various amines, we concluded that two kinds of aminereceptors were located on the surface of Hydra. One perceiveddopamine and D-noradrenaline, and depressed only the responseelicited by weak stimuli. Dopamine depressed the response at1 nmol/l. D,L-Propranolol was a competitive antagonist againstthis receptor. The other perceived tyramine and most of theamines examined, and depressed the response elicited by bothweak and strong stimuli. Tyramine depressed the response at1 µmol/l. D,L-Propranolol did not block its action. Afterthe animals were treated with dibenamine, both dopamine andtyramine became ineffective in depressing the response withoutinfluence on the response of the animal elicited by S-methylglutathione. ^*Present address: Tokyo Laboratory of Japan Synthetic RubberCo. Kawasaki 215, Japan     

Manganese binding to sodium cyanide-treated chloroplasts: Effects of light and redox-potentials on the binding

Mn²⁺-binding to Mn-depleted chloroplasts by the treatment withcyanide was inhibited by light. Quantitative study indicatesthat only the binding with a high affinity constant was inhibitedby light. This photoinhibition required electron transport activityand disappeared on addition of 2,6-dichlorophenolindophenol.The binding of Mn²⁺ was also inhibited by addition of reductantin the dark. The high affinity binding at various redox potentialsindicates the participation of a two electrons-transfer componenthaving a midpoint potential of 336 mV. Mn²⁺-binding was decreasedon treatment of chloroplasts with proteolytic enzyme in thedark but not in the light. We propose that the high affinitybinding site is concealed in the membrane matrix of chloroplastsat low oxidation-reduction potential. (Received December 14, 1976; )     

A microcomputer (PC 9801/MS mouse) system to record and analyze time-intensity curves of sweetness

The sweetness of sugar, glucose, xylose, saccharin, aspartame,etc. was assessed by magnitude estimation, with sensory intensitiesof 5, 10 and 20 defined as being equivalent to 2.5, 5.0 and10.0% concentrations of sugar in tap water. Sensory magnitudewas determined as being the input through a ‘mouse’to a microcomputer, PC 9801F2, which digitized the time intoseconds, while intensity was expressed in terms of tenths ofthe sensory magnitude 1.0. The two-dimensional information wasregistered on floppy disc memory and retrieved to calculatethe average time-intensity curves for each subject, or the averagefor several subjects. With this technique, it is easy to measure:(i) time of maximum intensity; (ii) height of maximum intensity;(iii) amount of adaptation; (iv) amount of after-taste; or (v)total amplitude (the sum of areas under the curve during andafter stimulation). The subjects were seven male students. Witha little training, they produced highly reliable curves forthe replicates of a sweetener at one concentration. Multidimensionalscaling of these curves did not disclose any clusterings ofnatural versus synthetic sweeteners.