Construction of transgenic Ipomoea obscura that exhibits new reddish leaf and flower colors due to introduction of β-carotene ketolase and hydroxylase genes

Ipomoea obscura, small white morning glory, is an ornamental plant belonging to the family Convolvulaceae, and cultivated worldwide. I. obscura generates white petals including a pale-yellow colored star-shaped center (flower vein). Its fully opened flowers were known to accumulate trace amounts of carotenoids such as β-carotene. In the present study, the embryogenic calli of I. obscura, were successfully produced through its immature embryo culture, and co-cultured with Agrobacterium tumefaciens carrying the β-carotene 4,4′-ketolase (crtW) and β-carotene 3,3′-hydroxylase (crtZ) genes for astaxanthin biosynthesis in addition to the isopentenyl diphosphate isomerase (idi) and hygromycin resistance genes. Transgenic plants, in which these four genes were introduced, were regenerated from the infected calli. They generated bronze (reddish green) leaves and novel petals that exhibited a color change from pale-yellow to pale-orange in the star-shaped center part. Especially, the color of their withered leaves changed drastically. HPLC-PDA-MS analysis showed that the expanded leaves of a transgenic line (T₀) produced astaxanthin (5.2% of total carotenoids), adonirubin (3.9%), canthaxanthin (3.8%), and 3-hydroxyechinenone (3.6%), which indicated that these ketocarotenoids corresponded to 16.5% of the total carotenoids produced there (530 µg g⁻¹ fresh weight). Furthermore, the altered traits of the transgenic plants were found to be inherited to their progenies by self-crossing.     

Inhibition of calpain results in impaired contraction-stimulated GLUT4 translocation in skeletal muscle

It was previously found that transgenic mice that overexpress the calpain inhibitor calpastatin (CsTg) have an approximately 3-fold increase in GLUT4 protein in their skeletal muscles. Despite the increase in GLUT4, which appears to be due to inhibition of its proteolysis by calpain, insulin-stimulated glucose transport is not increased in CsTg muscles. PKB (Akt) protein level is reduced approximately 60% in CsTg muscles, suggesting a possible mechanism for the relative insulin resistance. Muscle contractions stimulate glucose transport by a mechanism that is independent of insulin signaling. The purpose of this study was to test the hypothesis that the threefold increase in GLUT4 in CsTg would result in a large increase in contraction-stimulated glucose transport. CAMKII and AMPK mediate steps in the contraction-stimulated pathway. The protein levels of AMPK and CAMKII were increased three- to fourfold in CsTg muscles, suggesting that these proteins are also calpain substrates. Despite the large increases in GLUT4, AMPK, and CAMKII, contraction-stimulated GLUT4 translocation and glucose transport were not increased above wild-type values. These findings suggest that inhibition of calpain results in impairment of a step in the GLUT4 translocation process downstream of the insulin- and contraction-signaling pathways. They also provide evidence that CAMKII and AMPK are calpain substrates.     

Glucose metabolism via the Embden-Meyerhof pathway is not involved in ATP production during spore germination of bacillus megaterium QM B1551. A study with a mutant lacking hexokinase

In order to investigate contributions by glucose metabolism via the Embden-Meyerhof pathway and that via the direct oxidation route to gluconate to initial ATP production during spore germination, respiratory activity and RNA synthesis were compared between the mutant lacking hexokinase and the parent spores of Bacillus megaterium QM B1551. We found that time courses of those metabolic events were almost identical between those spores, thus clearly indicating that NADH formed by a spore-specific enzyme glucose dehydrogenase (EC 1.1.1.47) is solely responsible for aerobic production of ATP at this stage.     

Induction of ornithine decarboxylase in guinea-pig lymphocytes and its relation to phospholipid metabolism

Treatment of lymphocytes with exogenous phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3.) derived from Clostridium perfringens at concentrations similar to those which induced ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity produced diacylglycerol and phosphatidate. A divalent cation ionophore, A23187, and phytohemagglutinin induced not only diacylglycerol formation, but also ornithine decarboxylase activity. Dibutyryl cAMP inhibited both diacylglycerol formation and ornithine decarboxylase induction to a similar extent in phytohemagglutinin-stimulated lymphocytes, but stimulated them somewhat in ionophore A23187-activated lymphocytes. This suggests that the activation of intracellular phospholipase C and the formation of diacylglycerol is involved in ornithine decarboxylase induction in lymphocytes.     

Amniogenic band anomalies in a fifth-month fetus and in a newborn from maternal oophorectomy during early pregnancy

A fifth-month fetus and a newborn with amniogenic band anomalies were examined at autopsy. Both specimens were obtained from women who had undergone oophorectomy during early pregnancy. The dead male fetus was aborted spontaneously, and had a micrognathia, a right club foot, and a constriction ring on the left lower leg. The left fingers 2, 3, and 4 were attached to the placenta by a fibrous string. No internal anomaly was noted. In the other case, a male newborn was delivered at the 39th week of gestation and had an agenesis of the calvarium, a cleft lip with palate, an amputation of the right toe, and constriction rings on right fingers 3 and 4 and left finger 3. The placenta was attached to the left temporooccipital region of the head by a fibrous string. Also present was an atrial septum defect and a horseshoe kidney. Possible etiology is discussed in relation to the "amniogenic bands" hypotheses.     

Induction of ornithine decarboxylase in guinea pig lymphocytes by the divalent cation ionophore A 23187. Effect of dibutyryladenosine 3',5'-monophosphate

The divalent cation ionophore, A23187, at a concentration of 0.25 microgram/ml, enhanced influx of Ca2+, activity of ornithine decarboxylase and incorporation of [3H]thymidine into DNA of guinea pig lymphocytes. Combined treatment of cells with A23187 and dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) augmented these three events. A23187 at a concentration of 0.06 microgram/ml was insufficient for induction of ornithine decarboxylase stimulated neither Ca2+ influx nor [3H]thymidine incorporation, but stimulated Ca2+ efflux. A23187 (0.06 microgram/ml) in combination with Bt2cAMP caused a marked induction of ornithine decarboxylase and stimulation of [3H]thymidine incorporation into DNA. When the time of Bt2cAMP addition was delayed after A23187, the stimulation of ornithine decarboxylase activity decreased. Washout of Bt2cAMP from cell culture earlier than 4 h of incubation caused a reduction in the stimulatory effect of Bt2cAMP. These results suggest that raising concentrations of cytoplasmic Ca2+ and cellular cAMP are important to some initial events leading to induction of ornithine decarboxylase and these biochemical changes are obligatory sequential steps for stimulation of DNA synthesis.     

Intracutaneous infection with Leptospira icterohaemorrhagiae (Shibaura strain) of the guinea pig

Experimental leptospirosis with Leptospira icterohaemorrhagiae Shibaura strain was studied in guinea pigs. When the pathogen was inoculated intracutaneously to the back of the animals, localized haemorrhage was observed at the inoculated site before the appearance of general haemorrhage. The severity of the local lesion increased progressively until the 7th day of inoculation. The minimum infective dose (MID) or the 50% infective dose (ID50) of the leptospiral suspension was determined by the appearance of the macroscopic local haemorrhage 7 days after inoculation. The MID thus determined was almost comparable with the value determined by the development of general symptoms and signs by conventional ip inoculation. The number of the pathogen per ID50 varied between 6 and 35 in five experiments. The local haemorrhage was effectively protected by active or passive immunization. Microscopically, haemorrhage at the inoculated site was found mainly in the dermis, directly beneath the epidermis in particular, and accompanied with leakage of the pathogen. The pathogen was also detected abunduntly in the thickened epidermal layer covering the inoculated area as well as in the epithelial matrix of hair-follicle, probably due to the proliferation of the pathogen at the site.