Prostaglandin F2 alpha stimulates proliferation of clonal osteoblastic MC3T3-E1 cells by up-regulation of insulin-like growth factor I receptors.

Prostaglandin F2 alpha (PGF2 alpha) stimulates proliferation of clonal osteoblastic MC3T3-E1 cells mainly via the stimulation of phospholipase C. These cells constitutively produced and secreted insulin-like growth factor I (IGF-I). In addition, a neutralizing anti-IGF-I antibody completely abolished DNA synthesis stimulated by PGF2 alpha in MC3T3-E1 cells, suggesting that IGF-I indeed mediates the PGF2 alpha effect. However, PGF2 alpha decreased the expression of IGF-I mRNA and the secretion of immunoreactive IGF-I into the medium, whereas progression activity in the conditioned medium was not affected by PGF2 alpha. Although IGF-I alone did not stimulate DNA synthesis in MC3T3-E1 cells, when PGF2 alpha was added to the cultures, IGF-I stimulated their proliferation. Thus, PGF2 alpha may potentiate the action of IGF-I. At the same time, PGF2 alpha increased the number of high affinity binding sites (molecular mass of 130 kDa) for IGF-I in a dose-dependent manner. The increase in IGF-I-binding site number preceded the elevation of DNA synthesis by approximately 3 h. Furthermore, MC3T3-E1 cells secreted at least three species of IGF-binding proteins (IGFBPs) with molecular masses of 24, 30, and 34 kDa. In the early period of PGF2 alpha exposure, PGF2 alpha attenuated the secretion of all of these IGFBPs, whereas thereafter, it markedly increased their secretion, especially that of the 34-kDa IGFBP, suggesting a modulation of metabolism and action of IGF-I. These effects of PGF2 alpha on IGF-I receptor number and IGFBP secretion may play a role in the synergism between PGF2 alpha and IGF-I that results in the stimulation of DNA synthesis in MC3T3-E1 cells.     

Distribution of Ankyrin Isoforms and Their Proteolysis After Ischemia and Reperfusion in Rat Brain

Abstract: The distribution of brain-type ankyrin (ankyrin_B, 212 kDa) and erythrocyte-type ankyrin (ankyrin_R, 239 kDa) was investigated in the subcellular fractions of rat forebrain (P1, 1,000 g pellet; P2, 15,000 g pellet; P3, 100,000 g pellet; S, 100,000 g supernatant) by immunoblotting using specific antibodies. The P2 fraction contained ∼40% of the 212- and 163-kDa isoforms of ankyrin_B and the 239-kDa isoform of ankyrin_R. Further subfractionation of the P2 by Percoll gradient centrifugation followed by separation of myelin showed association of the three ankyrin isoforms with the synaptosome-rich fraction but not with the myelin-rich fraction. The plasma membrane-rich P3 fraction contained a concentration of ankyrin isoforms similar to that in the P2 fraction. In vitro proteolysis of ankyrin in the P2 fraction with calpain showed that the 212-kDa ankyrin_B was more susceptible to calpain than was ankyrin_R. In the two-vessel occlusion model, ischemia for 30 min generated the 160-kDa fragment of ankyrin_R, and reperfusion for 60 min after 30 min of ischemia remarkably increased the 160-kDa fragment. The reperfusion also significantly decreased the 212-kDa isoform of ankyrin_B. Both ischemia-reperfusion and in vitro proteolysis with calpain generated the 160-kDa fragment of ankyrin_R, suggesting the involvement of calpain.     

The rare inversion with a P element at the breakpoint maintained in a natural population of Drosophila melanogaster

We found a rare inversion which is superimposed on the In (2R) NS chromosome. It has been maintained for several years in the Raleigh, N. C., USA population and possibly for more than ten years in the southern populations of the USA. Both the breakpoint of the rare inversion and the corresponding site of the In (2R) NS chromosome have P elements and therefore, the rare inversion might be induced by P element activity in a natural population.     

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.     

A New Keto Acid, 5-Acetoxy-4-ketohexanoic Acid,Formed from Ethanol by Yeast

Of five strains of yeast tested, three which produced 5-hydroxy-4-ketohexanoic acid (HKH) were also found to form a new keto acid, when grown on medium containing ethanol as the sole carbon source. The new product was isolated in pure form and the structure was investigated by elementary analysis, infrared spectral analysis, NMR analysis and determination of its degradation product.

The compound was shown to be 5-acetoxy-4-ketohexanoic acid. 5-Acetoxy-4-keto-hexanoic acid (AKH) was synthesized and found to be identical with the isolated product.     

Early changes in mitochondrial monoamine oxidase activity of rat liver during 2-acetylaminofluorene feeding

The activities of mitochondrial type A and B monoamine oxidase were determined in the liver of rats fed a diet containing 2-acetylaminofluorene (AAF). Three days after the initiation of AAF-feeding, there was a significant decrease of type B monoamine oxidase activity without affect on type A enzyme. The decreased activity of type B monoamine oxidase, which reached a minimum after three weeks, was sustained for as long as AAF-feeding was continued. Sex-related difference in response to AAF was seen in the rat with respect to the onset and the intensity of the decreased type B monoamine oxidase activity, male rats being more sensitive to the carcinogen than female rats. In contrast to the in vivo effect, AAF showed a potent inhibitory effect on type A monoamine oxidase, rather than on type B enzyme, when added in vitro. The pI₅₀ values were estimated to be 7.5 against type A monoamine oxidase and 4.1 against type B enzyme, respectively. The in vitro inhibition of both types of monoamine oxidase by AAF was competitive. The K_i values for AAF were calculated to be 9.51 · 10^?9 M for type A monoamine oxidase and 1.30 · 10^?5 M for type B enzyme, respectively. In accordance with the potent inhibitory effect of AAF on type A monoamine oxidase in vitro, a single administration of the carcinogen, at a dose of 50 mg/kg, resulted in a marked and temporal decrease of the enzyme activity in the mitochondria of male rat liver. Recovery of the decreased type B monoamine oxidase activity was slow, and the enzyme activity did not return to control levels, even if rats were fed the basal diet for 2 or 4 weeks after the cessation of AAF-feeding.     

Toxicity of gentamicin in organ culture of fetal rat intestine.

The effect of gentamicin in the culture of fetal rat intestine was studied. Fetal rat intestine was cultured with gentamicin or kanamycin at the concentration between 4 to 200 micrograms/ml. Kanamycin did not have influence on lactase, maltase and ALP activities. On the other hand, gentamicin caused decrease of lactase and ALP activities at the concentration of 40 and 200 micrograms/ml compared with the activities at 4 micrograms/ml. Maltase activities did not decrease with gentamicin. Our data suggest that gentamicin could affect lactase and ALP activities and lower concentration should be used in the culture.     

Glass Capillary Gas Chromatography of Secondary Amines in Foods with Flame Photometric Detection after Derivatization with Benzenesulfonyl Chloride

A simple and selective gas chromatographic method was established for determining naturally occurring secondary amines. Secondary amines separated from foods by extraction with dichloro-methane and reextraction with hydrochloric acid were readily converted into the corresponding sulfonamides by reaction with benzenesulfonyl chloride under alkaline condition. Gas chromatography was carried out with a capillary coated with OV-101 and a flame photometric detector. Column temperature was programmed from 170 to 230°C at a rate of 5°C/min.

The obtained sulfonamides were separated from one another within 40 min.

Eleven secondary amines examined were added to 5 kinds of foods and recovered from them. The mean recovery rates were in the range 71.3% (dimethylamine)–99.8% (piperidine).

The limits of detection varied from 0.002 ppm (dimethylamine) to 0.01 ppm (morpholine).