Quantification of metabolic activity of cultured plant cells by vital staining with fluorescein diacetate

The metabolic activity of suspension cultures of Sonneratia alba cells was quantified by measurement of the hydrolysis of fluorescein diacetate (FDA). FDA is incorporated into live cells and is converted into fluorescein by cellular hydrolysis. Aliquots (0.1–0.75 g) of S. alba cells were incubated with FDA at a final concentration of 222 μg/ml suspension for 60 min. Hydrolysis was stopped, and fluorescein was extracted by the addition of acetone and quantified by measurement of absorbance at 490 nm. Fluorescein was produced linearly with time and cell weight. Cells of S. alba are halophilic and proliferated well in medium containing 50 and 100 mM NaCl. Cells grown in medium containing 100 mM NaCl showed 2- to 3-fold higher FDA hydrolysis activity than those grown in NaCl-free medium. When S. alba cells grown in medium supplemented with 50 mM NaCl were transferred to fresh medium containing 100 mM mannitol, cellular FDA hydrolysis activity was down-regulated after 4 days of culture, indicating that the moderately halophilic S. alba cells were sensitive to osmotic stress. Quantification of cellular metabolic activity via the in vivo FDA hydrolysis assay provides a simple and rapid method for the determination of cellular activity under differing culture conditions.     

Amygdaloid-induced jaw opening and facilitation or inhibition of the trigeminal motoneurons in the rat

1. Repetitive stimulation of the rat'se central amygdaloid (CAm) nucleus induced rhythmic masticatory jaw movements or continuous jaw opening. Both types of jaw movements were accompanied by coincidental activities of the mylohyoid (Myl) nerve. 2. The effects of CAm stimulation were examined on activities of bilateral Myl and masseteric (Mass) nerves or their motoneurons (Myl-Dig and Mass, respectively). 3. CAm stimulation induced contralaterally dominant facilitation of the Myl nerve activity as well as Myl-Dig motoneurons. These facilitatory effects were caused by EPSPs seen in Myl-Dig motoneurons. 4. One third of the Mass motoneurons were inhibited or hyperpolarized by contralateral CAm stimulation, while a few were facilitated and the majority unaffected.     

Amygdaloid or cortical facilitation of antidromic activity of trigeminal motoneurons in the rat

1. Electrical stimulation of the rat's contralateral central amygdaloid (CAm) nucleus or the contralateral frontal cortex markedly augmented the antidromic field potential evoked by stimulation of mylohyoid (Myl) nerve. 2. This facilitation was shown to be due to EPSPs of the mylohyoid-anterior digastric (Myl-Dig) motoneurons. 3. In a few motoneurons, cortical EPSPs had fixed short latencies following high-frequency double stimuli and this is believed to be due to a monosynaptic pathway. 4. The amygdaloid or cortically evoked EPSPs relieved IS-SD blockade in a few motoneurons and also facilitated antidromic discharge in others which did not show any IS or M spike response to the same subthreshold antidromic stimulation. The underlying mechanisms are discussed.     

Changes in the secretion of inhibin and steroid hormones during induced follicular atresia after hypophysectomy in the rat

Sequential changes in the function of antral follicles during the period of follicular atresia were investigated after hypophysectomy (Hypox) at 1100 hr on proestrus. Within 6 hours after Hypox, concentrations of progesterone (P), testosterone (T) and estradiol-17 beta (E) decreased abruptly in ovarian venous plasma (OVP) and follicles showed a reduced ability to ovulate. Six hours after Hypox, ovulation was still induced by human chorionic gonadotropin (hCG) in all animals but with significantly fewer number of oocytes compared to the group given hCG at 1100 hr on the day of proestrus. Nine hours after Hypox, several granulosa cells of all large follicles (greater than 400 microns in diameter) exhibited morphological signs of atresia. Twelve hours after Hypox, all large and medium sized (200-400 microns in diameter) follicles showed advanced stages of atresia and almost all follicles failed to ovulate in response to hCG. Inhibin activity in OVP declined more slowly compared to the profiles of steroid hormones and 53% of the initial inhibin activity was still maintained at 18 hours after the operation. Inhibin activity further decreased to 7% of the initial level at 24 hours and was undetectable by 48 hours after Hypox. These results suggest that fully developed Graafian follicles gradually lose their ability to secrete inhibin in contrast to the rapid decrease in secretion of steroid hormone during the process of atresia.     

Glyceraldehyde-derived advanced glycation end-products having pyrrolopyridinium-based crosslinks

Reducing sugars and reactive aldehydes, such as glyceraldehyde, non-enzymatically react with amino or guanidino groups of proteins to form advanced glycation end-products (AGEs) by the Maillard reaction that involves Schiff base formation followed by Amadori rearrangement. AGEs are found relatively in abundance in the human eye and to accumulate at a higher rate in diseases that impair vision such as cataract, diabetic retinopathy or age-related macular degeneration. We identified two novel AGEs of pyrrolopyridinium lysine dimer derived from glyceraldehyde, PPG1 and PPG2, in the Maillard reaction of N^α-acetyl-l-lysine with glyceraldehyde under physiological conditions. Having fluorophores similar to that of vesperlysine A, which was isolated from the human lens, PPGs were found to act as photosensitizers producing singlet oxygen in response to blue light irradiation. Moreover, PPG2 interacts with receptor for AGE (RAGE) in vitro with a higher binding affinity than GLAP, a well-known ligand of the receptor. We also proposed a pathway to form PPGs and discussed how they would be formed in vitro. As glyceraldehyde-derived AGEs have been studied extensively in connection with various hyperglycemia-related diseases, further studies will be required to find PPGs in vivo such as in the lens or other tissues.     

Immunoreactive inhibin levels in peripheral blood during the breeding season in the female Japanese monkey

Serum levels of immunoreactive inhibin as well as FSH, LH, estradiol-17 beta, and progesterone were measured by RIA in four mature female Japanese monkeys (Macaca fuscata fuscata) during the breeding season and subsequent transition into the nonbreeding season. During the breeding season, each monkey showed 2-6 ovulations, which were inferred from underlying endocrine events. The concentrations of serum inhibin increased during the luteal phase, but were low during the follicular phase. Such changes in serum inhibin levels correlated positively with those in serum progesterone levels. Basal levels of serum inhibin also increased during the breeding season, decreased during transition from the breeding season, and were low during the nonbreeding season. The parallel change in serum levels of inhibin and progesterone together with the increased basal levels of serum inhibin during this period suggests that both the CL and antral follicles are sources of circulating inhibin. Decreases in serum FSH levels during the luteal phase suggest that secretion of FSH is controlled by an inhibitory action of ovarian inhibin in addition to steroid hormones.     

Allelopathic activities of three carotenoids,neoxanthin, crocin and β-carotene,assayed using protoplast co-culture method with digital image analysis

Allelopathic activities of three carotenoids of a natural pigment group, neoxanthin, crocin and β-carotene, were assayed by the protoplast co-culture method with digital image analysis (DIA-PP method). Effects on three different growth stages of lettuce protoplasts, i.e., cell wall formation, cell division, and yellow pigment accumulation, were investigated using 96-well culture plates. Cell division was inhibited 65–95% by all three carotenoids at 33–100 µM. Inhibition of cell division stage was stronger than at the cell wall formation stage in neoxanthin, and the water-soluble carotenoid, crocin, whose yellow pigment was incorporated into the vacuole of lettuce protoplasts. Neoxanthin at 33 µM and crocin at higher than 100 µM inhibited more than 100% of the yellow pigment accumulation. By contrast, at low concentrations (0.01–1 µM) β-carotene stimulated growth at the cell division stage. At high concentrations of β-carotene (100–500 µM), inhibition was prominent at all three stages, and also in neighboring wells of zero control, which suggested emission of a volatile compound by β-carotene. They were compared with the report of the volatile compound, tulipalin A. Differences in patterns of inhibition of carotenoids on lettuce protoplast growth were compared with reports of another natural pigment, anthocyanin, and anthocyanin-containing red callus cultured in the light, and with that of neoxanthin-containing yellow callus cultured in the dark.