Turnover of the aggregates and cross-linked products of the D1 protein generated by acceptor-side photoinhibition of photosystem II.

It is known that the reaction-center binding protein D1 in photosystem (PS) II is degraded significantly during photoinhibition. The D1 protein also cross-links covalently or aggregates non-covalently with the nearby polypeptides in PS II complexes by illumination. In the present study, we detected the adducts between the D1 protein and the other reaction-center binding protein D2 (D1/D2), the alpha-subunit of cyt b(559) (D1/cyt b(559)), and the antenna chlorophyll-binding protein CP43 (D1/CP43) by SDS/urea-polyacrylamide gel electrophoresis and Western blotting with specific antibodies. The adducts were observed by weak and strong illumination (light intensity: 50-5000 microE m(-2) s(-1)) of PS II membranes, thylakoids and intact chloroplasts from spinach, under aerobic conditions. These results indicate that the cross-linking or aggregation of the D1 protein is a general phenomenon which occurs in vivo as well as in vitro with photodamaged D1 proteins. We found that the formation of the D1/D2, D1/cyt b(559) and D1/CP43 adducts is differently dependent on the light intensity; the D1/D2 heterodimers and D1/cyt b(559) were formed even by illumination with weak light, whereas generation of the D1/CP43 aggregates required strong illumination. We also detected that these D1 adducts were efficiently removed by the addition of stromal components, which may contain proteases, molecular chaperones and the associated proteins. By two-dimensional SDS/urea-polyacrylamide gel electrophoresis, we found that several stromal proteins, including a 15-kDa protein are effective in removing the D1/CP43 aggregates, and that their activity is resistant to SDS.     

Synthèse Facile de Pyrazines Disubstituées en 2 et 31)

Corynebacterium equi IFO 3730 was found to oxidize a wide variety of sec-alcohols, including alkanols, substituted alkanols, alkenols and cyclic alcohols, in moderate to high yields. Among them, the sec-alcohols which have longer carbon chains were oxidized more smoothly than those with smaller numbers of carbon. Although both enantiomers of unsymmetrically disubstituted carbinols were oxidized, the S form of 2-dodecanol was converted to the corresponding ketone a little faster than the other enantiomer.     

Ion Efflux during a Single Action Potential of Nitella axilliformis in Medium Lacking Ca2-

Ion efflux during excitation of Nitella axilliformis was measuredconductometrically. In medium lacking Ca²⁺ but with 0.1 mM MgCl₂,the duration of the action potential and the total efflux weremuch larger than those in APW, while the efflux rate, givenas the total efflux divided by the duration, was about halfof that in APW. (Received September 4, 1986; Accepted November 25, 1986)     

Effect of forskolin on collagen production in clonal osteoblastic MC3T3-E1 cells

The effect of forskolin on collagen production in osteoblasts was investigated by using clonal osteoblastic MC3T3-E1 cells cultured in a-minimum essential medium containing 0.1% bovine serum albumin. Forskolin increased the adenylate cyclase activity in membranes pelleted from homogenates of the cell line in a dose-dependent manner. The drug caused a 13-fold stimulation at 10(-4) M, indicating that the compound directly acts on adenylate cyclase, leading to an increase in the intracellular cAMP content of the cells. Collagen accumulation in the cultures was elevated by one-day treatment with 5 X 10(-5) M forskolin to about twice that in the controls. The stimulation was mainly due to an elevation in collagen synthesis but not to an inhibition of intracellular collagen degradation because forskolin dose-dependently increased collagen synthesis; it also significantly increased the amount of low-molecular-weight hydroxyproline found in the cultures. Cells treated with forskolin produced mainly type I collagen, as found in bone matrix in situ, with only small amounts of other types of collagen. Furthermore, forskolin time-dependently inhibited DNA synthesis in the cells, indicating that the increase in type I collagen synthesis by forskolin was not due to stimulated cell proliferation. These results suggest that cAMP is closely linked to the differentiation of osteoblasts in vitro.     

Postnatal development of the ovarian bursa of the golden hamster (Mesocricetus auratus): its complete closure and morphogenesis of lymphatic stomata

The golden hamster ovarian bursa was studied by light and electron microscopy to clarify the process of its complete closure and the development of lymphatics that leads to morphogenesis of stomata. The results were as follows. 1) The bursa completely closed at 9 days of age primarily due to development of the mesotubarium superius. 2) With the closure, the ovary and bursa became closely apposed, and most of the original bursal cavity disappeared. 3) Between 9 and 12 days of age U-shaped folds of the bursal mesothelium began to invade the connective tissue of the bursa. 4) Widening of the internal angle of the U-shaped folds contributed to reappearance of the bursal cavity, and thus separation of the bursa from the ovary. It also contributed to future geometrical proximity of lymphatics to the cavity of the bursa. 5) The separation of the bursa from the ovary began as early as 12 days of age in the cephalic half of the bursa. It occurred remarkably late in the caudal half. Juxtaposition of the window portion of the bursa to the ovary remained in some adult animals. 6) Development of lymphatics in the cephalic half of the bursa was divided into two stages, before and after days 21-24 of life. In the first stage, lymphatics grew in the submesothelial connective tissue, and the framework of lymphatics was formed. In the second stage, lymphatics extended small branches to form the submesothelial plexus or lymphatic lacuna. 7) Intercellular junctions between contiguous lymphatic endothelial cells were mostly tight and desmosomelike. Open junctions were, if they occurred at all, rare. (8) A smooth-surfaced area lined with the lymphatic endothelium was found in the bursa on day 27 of life, before the initiation of ovulation. Valvelike stomal orifices were absent before the initiation of ovulation and extremely rare even after the first ovulation. They were commonly present in the bursae after the fourth ovulation, however. The process of complete closure of the ovarian bursa is very complex and may be related to the later development of the bursal mesothelium and lymphatics. Some liplike stomal orifices are of purely developmental origin. However, all valvelike stomal orifices are assumed to be formed as a result of damage to the bursal mesothelium, as well as to the submesothelial connective tissue and lymphatics, by repetition of ovulation. It is possible that liplike stomal orifices may be formed in the process of repairing the damage.     

Determination of age- and exercise-dependent changes in myoglobin contents in murine skeletal and cardiac muscles

A new method was developed to determine myoglobin (Mb) contents in as least as 5 mg of murine skeletal muscles. The method was a modification of Reynafarje's spectroscopic technique and was based on the Soret absorptions at 416 and 422 nm of the muscle extract. Mb contents in the skeletal and cardiac muscles increased with age and were widely different from muscle to muscle. The contents in most of the 13 muscles examined at the 30th week of age were less than 1.5 mg/g wet muscle, but the cardiac, soleus and gracilis muscles showed exceptionally high values of 2.2-6.0 mg/g. The relative content of one muscle to the other was the same independent of differences in age, strain and sex. There was a positive correlation between the muscle Mb contents and citrate synthase activity (r = 0.930). Young male mice (5 wk-old) were endurance-trained by a gradual load-increment program on treadmill for 10 weeks (5 days/week), but the training had no effects on the Mb contents. No substantial alteration of the contents was also observed in the limbs immobilized by plaster-fixation for 4 weeks.     

Ion Effluxes during Excitation of Characeae

Ion effluxes during excitation of Chara and Nitellopsis measuredby conductometry method were compared with results obtainedby two other analytical methods, Ag-AgCl method for Cl^–and ion chromatography method for K⁺. In both species, the averageefflux measured by the conductometry method was very close tothose of K⁺ and Cl^–. (Received May 12, 1986; Accepted June 25, 1986)     

STUDIES OF AMINES IN THE STRIATUM IN MONKEYS WITH NIGRAL LESIONS

The effects of ventromedial tegmental lesions on the biosynthesis and disposition of biogenic amines in the striatum of monkeys were investigated. The concentrations of endogenous dopamine and of the intraventricularly injected [³H]dopamine were distinctly lower in the striatum on the lesion side than on the intact side. The storage of [³H]dopamine in the caudate nucleus was impaired to a much greater extent than the storage of the newly synthesized [³H]norepinephrine. The concentrations of endogenous serotonin and of the intraventricularly injected [¹⁴C]serotonin were lower in the striatum on the lesion side than on the intact side. However following MAO inhibition, the concentration of [¹⁴C]serotonin did not differ significantly on the two sides of the caudate nucleus. The in vivo biosynthesis of dopamine from tyrosine was significantly reduced in the striatum on the lesion side. Tyrosine hydroxylase and DOPA decarboxylase activities were decreased on the lesion side of the striatum as compared with the intact side. Thus, the ventromedial tegmental lesions affect the storage and the synthesis of dopamine and serotonin in the ipsilateral striatum.     

Improved maintenance of adult rat hepatocytes in a new serum-free medium in the presence or absence of barbiturates

Summary For serum-free primary culture of adult rat hepatocytes, a synthetic medium DM-160 and rat-tail collagen were selected for the basal medium and for the culture substratum, respectively. Barbiturates, such as phenobarbital and 1-ethyl-5-isobutylbarbiturate, efficiently supported survival of hepatocytes and maintained their morphologic features at lower concentrations under the serum-free conditions than under the serum-supplemented conditions. However, the hepatocyte survival rates under the serum-free conditions were lower than those under the serum-supplemented conditions in the presence or absence of barbiturates. Supplementation of the basal medium with a combination of five groups of factors (5Fs), such as eight amino acids (Ala, Arg, Gly, Ile, Met, Phe, Pro, and Trp), two unsaturated fatty acids (linoleate and oleate), a protease inhibitor (aprotinin), three vitamins (A, C, and E), and five trace elements (Mn, Fe, Cu, Zn, and Se), improved the hepatocyte survival under the serum-free conditions in the presence or absence of barbiturates. In other words, the serum could be completely substituted by the 5Fs. Hepatocyte cultures maintained in the 5Fs-suppelemented basal medium showed excellent induction of tyrosine aminotransferase activity in response to dexamethasone in the presence or absence of barbiturates. The efficiency of the 5Fs-supplemented basal medium for maintaining hepatocytes was not inferior to those of other media in common use with hepatocytes, such as Williams' medium E and Waymouth's medium MB-752/1. In conclusion, maintenance of functional hepatocytes in serum-free primary culture could be improved by use of the new medium preparation (the 5Fs-supplemented DM-160) in the presence of barbiturates. This work was supported by a grant no. 61771923 from the Ministry of Education, Science and Culture of Japan.