Transforming growth factor-ß decreases the cancer-initiating cell population within diffuse-type gastric carcinoma cells.

Stem cells in normal tissues and cancer-initiating cells (CICs) are known to be enriched in side population (SP) cells. However, the factors responsible for the regulation of expression of ABCG2, involved in efflux of dyes, in SP cells have not been fully investigated. Here, we characterized the SP cells within diffuse-type gastric carcinoma, and examined the effects of transforming growth factor-ß (TGF-ß) on SP cells. Diffuse-type gastric carcinoma cells established from four independent patients universally contained SP cells between 1 and 4% of total cells, which displayed greater tumorigenicity than non-SP cells did. TGF-ß repressed the transcription of ABCG2 through direct binding of Smad2/3 to its promoter/enhancer, and the number of SP cells and the tumor-forming ability of cancer cells were decreased by TGF-ß, although ABCG2 is not directly involved in the tumor-forming ability of SP cells. Cancer cells from metastatic site expressed much higher levels of ABCG2 and included a greater percentage of SP cells than parental cancer cells did. SP cells are thus responsible for the progression of diffuse-type gastric carcinoma, and TGF-ß negatively contributes to maintain the CICs within the cancer.

Bone morphogenetic protein signaling enhances invasion and bone metastasis of breast cancer cells through Smad pathway.

Transforming growth factor (TGF)-beta is known to promote tumor invasion and metastasis. Although bone morphogenetic proteins (BMPs), members of the TGF-beta family, are expressed in a variety of human carcinoma cell lines, their roles in tumor progression have not been fully clarified. In this study, we sought to determine the roles of BMPs in the progression of breast cancer bone metastasis using human breast cancer samples and a mouse xenograft model. Immunohistochemical analysis of samples from breast cancer patients as well as a mouse xenograft model of MDA-231-D, highly metastatic human breast cancer cells, revealed phospho-Smad2 and phospho-Smad1/5/8 staining in the nuclei of cancer cells in primary tumor and/or bone metastasis. Using a functional in vivo bioluminescence imaging system, we showed that TGF-beta- and BMP-induced transcriptional pathways are active in bone metastatic lesions in vivo. In addition, both TGF-beta3 and BMP-2 promoted the motility and invasiveness of the MDA-231-D cells in vitro. Moreover, expression of dominant-negative receptors for TGF-beta and/or BMPs in the MDA-231-D cells inhibited invasiveness in vitro and bone metastasis in the xenograft model. These results suggest that BMPs as well as TGF-beta promote invasion and bone metastasis of breast cancer.

Apoptosis induction associated with cell cycle dysregulation by rice bran agglutinin.

Effects of rice bran agglutinin (RBA) on human monoblastic leukemia U937 cells were examined in comparison with those of wheat germ agglutinin (WGA) and Viscum album agglutinin (VAA). These lectins inhibit cell growth, and several lines of evidence indicate that the growth inhibition is caused by the induction of apoptosis. We observed that RBA induces chromatin condensation, externalization of membrane phosphatidylserine, and DNA ladder formation, features of apoptosis. DNA ladder formation was inhibited by a general inhibitor against caspases, which are known to play essential roles in apoptosis. Flow cytometric analysis revealed that RBA and WGA cause G2/M phase cell cycle arrest with increased expression of Waf1/p21, while cell cycle arrest was not observed for VAA. These data indicate that RBA induces apoptosis associated with cell cycle arrest in U937 cells, and suggest that the induction mechanism for RBA is similar to that for WGA, but different from that for VAA.

Apoptosis-inducing activity of a driselase digest fraction of green tea residue.

We enzymatically digested green tea residue with Driselase, a crude preparation containing cellulase, pectinase and proteases, in order to examine the potential usefulness of the residue. A fraction of the digest soluble in 70% ethanol was found to induce the death of U937 human histiocytic lymphoma cells by apoptosis. Other enzyme preparations gave similar products with cell death-inducing activity of varing potency. The green tea residue may therefore be a useful source of potential agents with anti-cancer activity.

Continuous measurement of targeted promoter activity by a secreted bioluminescence reporter, Vargula hilgendorfii luciferase.

The promoter activity of growth hormone (GH) was continuously monitored in rat pituitary adenoma cells (GH3) by a secreted bioluminescence reporter, Vargula hilgendorfii (Vh) luciferase. The sensitivity of the reporter was approximately 60-times higher than that of the firefly luciferase. GH3 cells were transfected with a plasmid containing a DNA sequence of the GH promoter (1.8 kb) and a full length of the Vh luciferase cDNA. Using the stable transformants, the Vh luciferase activity was monitored in the plate culture through the bioluminescence of Vh luciferase secreted into the culture medium. The reporter activity was well correlated with GH mRNA as well as GH when the GH promoter activity was activated by thyroid hormone. To develop a continuous monitoring system of the promoter, the reporter activity was sequentially measured in the perfusion system. When the promoter activity of the stable transformants was suppressed by a transcription inhibitor, the reporter activity and GH in the perfusate were simultaneously decreased. The Vh luciferase reporter is a sensitive and convenient tool for continuous and prolonged measurement of promoter activity in living cell culture systems.

Synchronization of circadian firing rhythms in cultured rat suprachiasmatic neurons.

The circadian clock in mammals is located in the suprachiasmatic nucleus (SCN) which consists of multiple oscillating neurons. Integration of the cellular oscillations is essential for the generation of a single circadian period in the SCN. By using a multielectrode dish (MED), we measured circadian firing rhythms in individual SCN neurons for more than 2 weeks continuously, and examined the involvement of synaptic communication in the synchronization of circadian rhythms. Cross-correlation analysis of spontaneous action potentials revealed that a neuron pair was functionally connected by synapses when their circadian rhythms were synchronized. No correlation was found between the paired neurons whose circadian rhythms were not synchronized. Calcium (Ca2+)-dependent synaptic transmission in the cellular communication was indicated by dose-dependent lengthening of an intercellular spike interval and loss of spike correlation with a Ca2+ channel blocker. Approximately 60% of the SCN neurons in culture were immunoreactive to antibodies against gamma-aminobutyric acid (GABA) or glutamic acid decarboxylase (GAD). Spontaneous firing of all the neurons tested was either increased or decreased by bicuculline, the GABAA receptor antagonist. These findings indicate that synaptic communication plays a critical role in the synchronization of circadian rhythms in individual SCN neurons and the GABAergic transmission is involved in the synchronization mechanism.

Circadian release of excitatory amino acids in the suprachiasmatic nucleus culture is Ca(2+)-independent.

We have previously reported that spontaneous release of excitatory amino acids (aspartate and glutamate) show remarkable circadian rhythms in the organotypic slice culture of rat suprachiasmatic nucleus (SCN). Here we showed effects of extracellular Ca(2+) removal and of L-trans-pyrrolidine-2,4-dicarboxylic acid, a glutamate/aspartate uptake inhibitor on the circadian release of excitatory amino acids in the SCN culture. Amino acids were measured by high-performance-liquid-chromatography. Removal of extracellular Ca(2+) exerted no effect on the spontaneous release of the excitatory amino acids, while it blocked high K(+)-evoked release of the amino acids. Neither the period nor the amplitude of the spontaneous circadian release of amino acids in Ca(2+)-free medium was different from those in the Ca(2+)-containing medium. On the other hand, L-trans-pyrrolidine-2,4-dicarboxylic acid increased the excitatory amino acid levels without affecting the amplitude of excitatory amino acid rhythms. These results indicated that the circadian release of excitatory amino acids in the SCN is Ca(2+)-independent and L-trans-pyrrolidine-2,4-dicarboxylic acid- insensitive. Therefore, Ca(2+)-dependent chemical synaptic transmission may not be involved in the circadian rhythm generation in the SCN.

Circadian rhythms of arginine vasopressin and vasoactive intestinal polypeptide do not depend on cytoarchitecture of dispersed cell culture of rat suprachiasmatic nucleus.

Dispersed cells of rat suprachiasmatic nucleus were cultured for more than a month with chemically defined medium. Arginine vasopressin and vasoactive intestinal polypeptide in the culture medium showed robust circadian rhythms starting 24 h after the cell dissociation. The two rhythms had similar periods, with a phase-lead of the vasoactive intestinal polypeptide peaks to the arginine vasopressin peak of about 1 h. The two rhythms remained two weeks later, with both peaks appearing at almost the same time, suggesting the synchronization of the two rhythms. Significant differences in cell architecture were detected depending on precoating matrices of culture dishes, which did not affect the circadian rhythms of arginine vasopressin and vasoactive intestinal polypeptide. Antimitotic treatment at the beginning of the culture not only reduced the number, but also changed the type of glial cells developed. The treatment did not interrupt the synchronized arginine vasopressin and vasoactive intestinal polypeptide rhythms until day 31. Early appearance of circadian rhythms indicates that neural networks in the suprachiasmatic nucleus are not necessary for the synchronous release of arginine vasopressin and vasoactive intestinal polypeptide. Glial proliferation is not essential for the generation, expression and synchronization of arginine vasopressin and vasoactive intestinal polypeptide rhythms in the dispersed suprachiasmatic nucleus cell culture.

Circadian periods of single suprachiasmatic neurons in rats.

Neuronal activity of a single neuron was monitored continuously for more than 5 days by means of a multi-electrode dish in dispersed cell culture of the rat suprachiasmatic nucleus (SCN). Sixty-seven out of 88 neurons showed a robust circadian rhythm in firing rate. The mean circadian period was 24.2 h, which was almost identical to that of the locomotor activity rhythm in 114 weanling rats blinded on the day of birth. However, the circadian period in individual SCN neurons was scattered from 20.0 to 28.3 h (SD, 1.4 h), while the period of activity rhythm clustered from 24.0 to 24.8 h (SD, 0.2 h). It is concluded that a large number of SCN neurons contain the circadian oscillator, the period of which is more variable than the circadian period of the SCN as a whole. It is suggested that the circadian rhythms in individual SCN neurons are capable of synchronizing to each other and are integrated to constitute a multiple oscillator system(s) within the SCN.

Circadian release of amino acids in the suprachiasmatic nucleus in vitro.

Temporal patterns of release of aspartate, glutamate and glycine, which are related to excitatory amino acidergic transmission, were examined in organotypic slice cultures of rat suprachiasmatic nucleus over a 60 h period. Vasopressin release in the same culture was measured simultaneously to compare the temporal pattern with that of the amino acids. Amino acids and vasopressin were measured by high performance liquid chromatography and enzyme immunoassay, respectively. Robust circadian rhythms were detected in release of aspartate, glutamate and glycine. Glycine levels were about 10 times higher than those of aspartate and glutamate in the culture. Vasopressin also showed a clear circadian rhythm and the phase angle difference between each amino acid and AVP was not significantly different. The results indicate that cultured SCN cells release these amino acids and the release is under the control of the circadian pacemaker.

Aging affects development and persistence of feeding-associated circadian rhythm in rat plasma corticosterone.

Effects of aging on development and persistence of feeding-associated circadian rhythm were examined with respect to the prefeeding plasma corticosterone peak under restricted feeding. Old (20 mo), middle-aged (12 mo), and young (2.5 mo) male rats were subjected to a restricted feeding schedule for 21 days in which free access to food was limited to 2 h/day in the light phase. Prefeeding corticosterone peak was detected 1 wk after the start of the schedule in the young and 3 wk after in the old and middle-aged rats. After the restricted feeding, a feeding/ fasting cycle composed of 7 days of ad libitum feeding and 3 days of fasting was imposed four times. The persistence of feeding-associated rhythm was evaluated by examining the reappearance of the corticosterone peak at the previous meal time during each fasting period of the feeding/fasting cycle. The corticosterone peak was not detected in the old and middle-aged rats even at the first cycle, but it was still distinct at the third cycle in the young rats. In conclusion, aging impaired the development and persistence of feeding-associated circadian rhythm in rats.

Circadian rhythm and response to light of extracellular glutamate and aspartate in rat suprachiasmatic nucleus.

Extracellular concentrations of glutamate and aspartate were measured in the vicinity of rat suprachiasmatic nucleus (SCN) by means of in vivo microdialysis. The concentrations of both excitatory amino acids (EAAs) were higher during the dark phase than during the light under the light-dark cycle, showing pulsatile fluctuations throughout the day. When rats were released into the complete darkness, the 24-h pattern in the aspartate continued for at least one cycle, whereas that in the glutamate disappeared. The nocturnal increases in the EAA levels were not due to the increase of locomotor activity during the nighttime, because the 24-h rhythms were also detected in animals under urethan anesthesia. The patterns of extracellular EAA levels were changed when rats were released into the continuous light. Circadian rhythm was not detected in the glutamate, whereas the 24-h pattern was maintained in the aspartate with the levels increased to various extents. A 30-min light pulse given either at zeitgber time (ZT) 1 or ZT 13 elevated the EAA levels during the latter half of the light pulse, except glutamate by a pulse at ZT 1. The extracellular EAA levels in the vicinity of the rat SCN showed the circadian rhythm with a nocturnal peak and increased in response to the continuous light and a brief light pulse. The aspartate level is considered to be regulated by the endogenous circadian rhythm, but the glutamate levels seems to be modified by the light-dark cycle.

Persistence of circadian oscillation while locomotor activity and plasma melatonin levels became aperiodic under prolonged continuous light in the rat.

In order to examine the mechanism for a loss of circadian rhythms in several functions under prolonged continuous light (LL), rats were blinded following LL over 5 months, and the mode of reappearance of circadian rhythms were analyzed in locomotor activity and plasma melatonin levels. Locomotor activity and plasma melatonin levels in individual rats became aperiodic after the exposure to LL. On the day of blinding, plasma melatonin levels showed circadian rhythms having a peak coincided with the activity time of locomotor rhythm which was restored after blinding. The time of melatonin peak was not related to the time of blinding (onset of darkness) nor to the initial time of blood sampling. Circadian rhythm in plasma melatonin levels reappeared faster than those in locomotor activity. The findings suggest that aperiodism developed in these functions under prolonged LL is not due to disruption of the circadian oscillation but to uncoupling of overt functions from the circadian pacemaker.

Dissociation of paraventricular NPY release and plasma corticosterone levels in rats under food deprivation.

Extracellular neuropeptide Y (NPY) in the vicinity of the paraventricular nucleus (PVN) as well as NPY concentrations in the PVN were measured in rats under ad libitum feeding and 2-day and 10-day food deprivation. Plasma corticosterone levels were not changed by 2-day food deprivation but were increased by subsequent refeeding. In contrast, the extracellular NPY levels were increased by 2-day food deprivation and were decreased rapidly by refeeding. The NPY concentrations were also increased and increased further by refeeding. On the other hand, plasma corticosterone levels were elevated by 10-day food deprivation and were decreased by subsequent refeeding. The extracellular NPY levels were also increased by food deprivation and decreased gradually after refeeding. However, the postprandial levels were still elevated when plasma corticosterone levels were returned to the basal levels. The NPY concentrations were also increased and increased further by refeeding. The amount of food intake after refeeding was positively correlated with the extracellular NPY levels. It is concluded that extracellular NPY levels in the PVN do not necessarily covariate with plasma corticosterone levels in rats under food deprivation.

Overproduction of D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 in Escherichia coli and its purification.

We constructed the high-expression plasmid for D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6. The appropriate Shine-Dalgarno sequence (AAGGAG) was introduced to the eight bases upstream of start codon (ATG) of D-aminoacylase structural gene by site-directed mutagenesis, and then the 1.75-kb DNA fragment including the open reading frame was inserted into the downstream of the tac promoter of plasmid vector pKK223-3. The resultant plasmid, which was named pKNSD2, showed a high D-aminoacylase activity in Escherichia coli JM109 cells transformed with it. The enzyme was purified to homogeneity in only two steps with a final yield of 24% (sp act, 2023 U/mg).

Cloning and sequencing of a gene encoding D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 and expression of the gene in Escherichia coli.

The gene encoding the D-aminoacylase of Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was cloned and its complete nucleotide sequence was identified. The D-aminoacylase structural gene consists of 1452 nucleotides and encodes 484 amino acid residues. The molecular weight of D-aminoacylase was calculated to be 51,918. This value agreed well with the apparent molecular weight of 52,000 found for the purified enzyme from Alcaligenes A-6 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The N-terminal amino acid sequence (NH2-SQSDSQPFDLLRAG-) predicted by the nucleotide sequence exactly matched those of the purified D-aminoacylase both from Alcaligenes A-6 and from cloned Escherichia coli (E. coli), with the exception of the removal of the N-terminal methionine processed after translation. The purified recombinant enzyme showed almost the same enzymatic properties as the native enzyme from Alcaligenes A-6. Alcaligenes A-6 D-aminoacylase showed 25-29% homology with L-aminoacylases from Bacillus stearothermophilus, porcine and humans.

Two distinct oscillators in the rat suprachiasmatic nucleus in vitro.

In the rat suprachiasmatic nucleus slice culture, circadian rhythms in the release of arginine vasopressin and vasoactive intestinal polypeptide were measured simultaneously and longitudinally. The phase relationship between the two peptide rhythms was relatively constant in the culture without a treatment of antimitotic drugs but became diverse by an introduction of antimitotics, which is generally used to reduce the number of glial cells. By monitoring the two rhythms continuously for 6 days, different periods were detected in culture with the antimitotic treatment. Furthermore, N-methyl-D-aspartate shifted the phase of the two peptide rhythms in the same culture differently. These results indicate that the arginine vasopressin and vasoactive intestinal polypeptide release are under control of different circadian oscillators.

[Changes in biological rhythm and sleep structure during the menstrual cycle in healthy women].

It is well known that clinical symptoms such as psychosis, epileptic seizures and sleep disturbances aggravate around the time of menstruation. In some healthy women, subjective sleep feelings or moods have been reported to change throughout the menstrual cycle, which suggests that sleep structure and sleep-wake rhythm may change during the menstrual cycle. We investigated a circadian pattern of plasma melatonin, sleep-wake rhythm and sleep characteristics in the different phases of the menstrual cycle in young healthy women under controlled environmental conditions. The subjects were seven healthy women, aged 18 to 19, with regular menstrual cycles. They stayed in an experimental facility, where temperature and humidity were kept within a narrow range, for three days in successive five weeks. They got up and went to bed at their preferable time. Polysomnography was performed using ambulatory cassette EEG system on the first and second night. Sleep stages were scored according to Rechtshaffen and Kales' criteria. Plasma melatonin was measured at 1-hour intervals for 24 hours on the third day. The menstrual cycle was divided into four phases (menstruation, follicular, early luteal and late luteal). Although the plasma melatonin level in the late luteal phase tended to be higher than in other phases, no significant difference was observed across four phases. The phase in plasma melatonin level did not change. As for sleep-wake rhythm, the time of getting up on the first day was significantly late in the late luteal phase (p < 0.05), although it showed no significant change on the second day. The time of going to bed did not change. Sleep characteristics changed during the menstrual cycle. There was a significant difference in the amount of stage 3 + 4, slow wave sleep (p < 0.05), which was more abundant in the follicular phase than in the luteal phase. TIB (time in bed), SPT (sleep period time), TST (total sleep time) seemed to increase in the menstrual and follicular phases, while stage W increased in the early and late luteal phases. However, there were no significant differences in these parameters. The other parameters did not show any changes. The changes in amount of stage 3 + 4 throughout the menstrual cycle seem to be due to endogenous factors, because environmental factors were controlled in this experiment. It is possible that the menstrual cycle also affect the plasma melatonin level and sleep-wake rhythm.

Immediate response to light of rat pineal melatonin rhythm: analysis by in vivo microdialysis.

Melatonin in the extracellular space of the pineal gland was measured continuously for 4 consecutive days from single, freely moving rats by means of in vivo microdialysis. A robust circadian rhythm was observed in the pineal extracellular melatonin under both light-dark (LD) and continuous dark (DD) conditions, the patterns of which were almost identical for 4 days within individuals but varied substantially among individuals. The offset phase of melatonin rhythm was more stable than the onset phase. Light-induced phase shift of melatonin rhythm was measured in individual rats, which had been entrained to LD and subsequently released into DD. On the 1st day in DD, a 3-min light pulse of 200 lx was applied either at circadian time (CT) of 17 or 22 h (5 and 10 h after the dark onset, respectively). The light pulse rapidly suppressed the nocturnal melatonin level. The rate as well as the level of melatonin suppression was significantly greater by the pulse at CT22 than at CT17. A phase shift of the melatonin rhythm was calculated on the 2nd and 3rd days in DD. Significant phase delay shift was observed after the pulse at CT17 and advance shift after the pulse at CT22 of approximately 1 h in either case. Because the amount of phase shift was not different between the 2nd and 3rd days in DD, the phase shift of pineal melatonin rhythm by single light pulse seems to be completed immediately.

Circadian rhythms in the release of vasoactive intestinal polypeptide and arginine-vasopressin in organotypic slice culture of rat suprachiasmatic nucleus.

Temporal profiles of the amount of vasoactive intestinal polypeptide (VIP) were examined in the medium of organotypic suprachiasmatic nucleus (SCN) slice cultures over a 2-day period. Arginine-vasopressin (AVP) level was also measured in the same medium. The slices of the SCN were obtained from 7-8-day-old rats and cultured individually in tubes on a roller drum for 14 days. The VIP amount in the medium of SCN culture showed a circadian rhythm with a approximately 22-h period. Circadian rhythms with identical periods were also observed in AVP amount of the same culture. However, the peak time of the VIP rhythm was slightly ahead of that of the AVP rhythm. Furthermore, the total VIP amount in the medium over a 24-h period was six times as large as that of AVP. These results suggest that there is a circadian rhythm of VIP which is released from the ventrolateral SCN.