The regulation of circadian period by phototransduction pathways in Arabidopsis.

Transgenic Arabidopsis plants expressing a luciferase gene fused to a circadian-regulated promoter exhibited robust rhythms in bioluminescence. The cyclic luminescence has a 24.7-hour period in white light but 30- to 36-hour periods under constant darkness. Either red or blue light shortened the period of the wild type to 25 hours. A phytochrome-deficient mutation lengthened the period in continuous red light but had little effect in continuous blue light, whereas seedlings carrying mutations that activate light-dependent pathways in darkness maintained shorter periods in constant darkness. These results suggest that both phytochrome- and blue light-responsive photoreceptor pathways control the period of the circadian clock.

Conditional circadian dysfunction of the Arabidopsis early-flowering 3 mutant.

Photoperiodic responses, such as the daylength-dependent control of reproductive development, are associated with a circadian biological clock. The photoperiod-insensitive early-flowering 3 (elf3) mutant of Arabidopsis thaliana lacks rhythmicity in two distinct circadian-regulated processes. This defect was apparent only when plants were assayed under constant light conditions. elf3 mutants retain rhythmicity in constant dark and anticipate light/dark transitions under most light/dark regimes. The conditional arrhythmic phenotype suggests that the circadian pacemaker is intact in darkness in elf3 mutant plants, but the transduction of light signals to the circadian clock is impaired.

Orchestrated transcription of key pathways in Arabidopsis by the circadian clock.

Like most organisms, plants have endogenous biological clocks that coordinate internal events with the external environment. We used high-density oligonucleotide microarrays to examine gene expression in Arabidopsis and found that 6% of the more than 8000 genes on the array exhibited circadian changes in steady-state messenger RNA levels. Clusters of circadian-regulated genes were found in pathways involved in plant responses to light and other key metabolic pathways. Computational analysis of cycling genes allowed the identification of a highly conserved promoter motif that we found to be required for circadian control of gene expression. Our study presents a comprehensive view of the temporal compartmentalization of physiological pathways by the circadian clock in a eukaryote.

Novel features of drosophila period Transcription revealed by real-time luciferase reporting.

The rapid turnover of luciferase and the sensitive, non-invasive nature of its assay make this reporter gene uniquely situated for temporal gene expression studies. To determine the in vivo regulatory pattern of the Drosophila clock gene period (per), we generated transgenic strains carrying a luciferase cDNA fused to the promoter region of the per gene. This has allowed us to monitor circadian rhythms of bioluminescence from pacemaker cells within the head for several days in individual living adults. These high time-resolution experiments permitted neuronal per transcription and opens the door to vastly simplified experiments in general chronobiology and studies of temporally regulated transcription in a wide range of experimental systems.

Long-term monitoring of circadian rhythms in c-fos gene expression from suprachiasmatic nucleus cultures.

BACKGROUND: The AP-1 family of transcription factors has been implicated in the control of the expression of many genes in response to environmental signals. Previous studies have provided temporal profiles for c-fos expression by taking measurements from many animals at several points in time, but these studies provide limited information about dynamic changes in expression. Here, we have devised a method of continuously measuring c-fos expression. RESULTS: A transgenic mouse line expressing the human c-fos promoter linked to the firefly luciferase reporter gene (fos/luc) was generated to continuously monitor c-fos gene expression. A second transgenic mouse line expressing luciferase under the control of the cytomegalovirus promoter (CMV/luc) served as a control. Luminescence originating from identifiable brain regions was imaged from fos/luc brain slice cultures. Expression of the fos/luc transgene accurately reflected transcriptional responses of the endogenous c-fos gene. Dynamic changes in fos/luc expression in suprachiasmatic nuclei (SCN) explant cultures were monitored continuously, and luminescence showed almost 24 hour rhythms lasting up to five circadian cycles. In contrast, bioluminescence monitored from CMV/luc SCN explant cultures was not rhythmic. CONCLUSION: The fos/luc transgenic mouse will be useful for long-term, non-invasive monitoring of c-fos transcriptional responses to the changing cellular environment. Circadian rhythms in c-fos expression can be monitored non-invasively in real time from the SCN, clearly demonstrating that c-fos transcription is regulated by the circadian clock.

Decrease in beta-cell mass leads to impaired pulsatile insulin secretion, reduced postprandial hepatic insulin clearance, and relative hyperglucagonemia in the minipig.

Most insulin is secreted in discrete pulses at an interval of approximately 6 min. Increased insulin secretion after meal ingestion is achieved through the mechanism of amplification of the burst mass. Conversely, in type 2 diabetes, insulin secretion is impaired as a consequence of decreased insulin pulse mass. beta-cell mass is reported to be deficient in type 2 diabetes. We tested the hypothesis that decreased beta-cell mass leads to decreased insulin pulse mass. Insulin secretion was examined before and after an approximately 60% decrease in beta-cell mass achieved by a single injection of alloxan in a porcine model. Alloxan injection resulted in stable diabetes (fasting plasma glucose 7.4 +/- 1.1 vs. 4.4 +/- 0.1 mmol/l; P < 0.01) with impaired insulin secretion in the fasting and fed states and during a hyperglycemic clamp (decreased by 54, 80, and 90%, respectively). Deconvolution analysis revealed a selective decrease in insulin pulse mass (by 54, 60, and 90%) with no change in pulse frequency. Rhythm analysis revealed no change in the periodicity of regular oscillations after alloxan administration in the fasting state but was unable to detect stable rhythms reliably after enteric or intravenous glucose stimulation. After alloxan administration, insulin secretion and insulin pulse mass (but not insulin pulse interval) decreased in relation to beta-cell mass. However, the decreased pulse mass (and pulse amplitude delivered to the liver) was associated with a decrease in hepatic insulin clearance, which partially offset the decreased insulin secretion. Despite hyperglycemia, postprandial glucagon concentrations were increased after alloxan administration (103.4 +/- 6.3 vs. 92.2 +/- 2.5 pg/ml; P < 0.01). We conclude that an alloxan-induced selective decrease in beta-cell mass leads to deficient insulin secretion by attenuating insulin pulse mass, and that the latter is associated with decreased hepatic insulin clearance and relative hyperglucagonemia, thereby emulating the pattern of islet dysfunction observed in type 2 diabetes.

Quantitative analysis of Drosophila period gene transcription in living animals.

To determine the in vivo regulatory pattern of the clock gene period (per), the authors recently developed transgenic Drosophila carrying a luciferase cDNA fused to the promoter region of per. They have now carried out noninvasive, high time-resolution experiments allowing high-throughput monitoring of circadian bioluminescence rhythms in individual living adults for several days. This immediately solved several problems (resulting directly from individual asynchrony within a population) that have accompanied previous biochemical experiments in which groups of animals were sacrificed at each time point. Furthermore, the authors have developed numerical analysis methods for automatically determining rhythmicity associated with bioluminescence records from single flies. This has revealed some features of per gene transcription that were previously unappreciated and provides a general strategy for the analysis of rhythmic time series in the study of molecular rhythms.

Growth hormone (GH) receptor blockade with a PEG-modified GH (B2036-PEG) lowers serum insulin-like growth factor-I but does not acutely stimulate serum GH.

B2036-PEG, a GH receptor (GH-R) antagonist, is an analog of GH that is PEG-modified to prolong its action. Nine mutations alter the binding properties of this molecule, preventing GH-R dimerization and GH action. A potential therapeutic role of B2036-PEG is to block GH action, e.g. in refractory acromegaly. A phase I, placebo-controlled, single rising-dose study was performed in 36 normal young men (ages, 18-37 yr; within 15% ideal body weight). Four groups received a single s.c. injection of either placebo (n = 3 in each group, total n = 12) or B2036-PEG (0.03, 0.1, 0.3, or 1.0 mg/kg; n = 6 each dose). B2036-PEG and GH concentrations were measured 0, 0.25, 0.5, 1, 3, 6, 9, 12, 24, 36, 48, 72, 96, 120, and 144 h after dosing. Serum insulin-like growth factor-I was measured before and 1-7 days after dosing. All doses were well tolerated, with no serious or severe adverse reactions. B2036-PEG, at 1.0 mg/kg, reduced insulin-like growth factor-I by 49 +/- 6% on day 5 (P < 0.001 vs. placebo). GH was measured by two independent methods: 1) modified Nichols chemiluminescence assay (empirically corrected for B2036-PEG cross-reactivity); and 2) direct GH two-site immunoassay, using monoclonal antibodies that did not react with B2036-PEG. There was good agreement between the two methods. GH did not change substantially at any B2036-PEG dose, suggesting that B2036-PEG does not interact with hypothalamic GH-Rs to block short-loop feedback. B2036-PEG may thus block peripheral GH action without enhancing its secretion.

Episodes of severe hypoglycemia in type 1 diabetes are preceded and followed within 48 hours by measurable disturbances in blood glucose.

This study quantifies blood glucose (BG) disturbances occurring before and after episodes of severe hypoglycemia (SH). For 6-8 months, 85 individuals with type 1 diabetes and a history of SH (age, 44+/-10 yr; 41 women and 44 men; duration of diabetes, 26+/-11 yr; hemoglobin A1c, 7.7+/-1.1%) used Lifescan One Touch BG meters for self-monitoring three to five times daily and recorded the date and time of SH episodes in diaries. For each subject, the timing of SH episodes was located in the temporal stream of SMBG readings recorded by the meter, and characteristics, including the Low BG index (LBGI), were computed in 24-h increments. In the 24-h period before the SH episode LBGI rose (P < 0.001), average BG was lower (P = 0.001), and BG variance increased (P = 0.001). In the 24 h after SH, LBGI and BGvariance remained elevated (P < 0.001), but average BG returned to baseline. These disturbances disappeared in 48 h. On the basis of LBGI we identified subjects at low, moderate, and high risk of SH, who reported, on the average, 1.7, 3.4, and 7.4 SH episodes (P < 0.005) during the study. In addition, we designed an algorithm that predicted 50% of all SH episodes that occurred in this subject group. We conclude that episodes of SH are preceded and followed by quantifiable BG disturbances, which could be used to devise warnings of imminent SH.

Enhanced sensitivity growth hormone (GH) chemiluminescence assay reveals lower postglucose nadir GH concentrations in men than women.

Modifications were made to a commercially available human (h) GH chemiluminescence assay (Nichols Luma Tag hGH assay), which improved its sensitivity to 0.002 micrograms/L. The results of this assay had a high correlation with those of the Nichols hGH immunoradiometric assay (IRMA; r = 0.91; P < 0.001). The addition of recombinant hGH-binding protein (0.1-10 nmol/L) to standards and serum samples caused a dose-responsive reduction in measured GH in both the chemiluminescence assay and the IRMA; at physiological concentrations of hGH-binding protein, a 10-20% reduction was observed. Fifteen normal young adults (nine men and six women) underwent a standard 100-g oral glucose tolerance test, and plasma GH was measured from 30 min before until 5 h after glucose ingestion. GH was measurable in all samples with the chemiluminescence assay, but fell below the sensitivity of the IRMA in 59% of the samples. There was no difference between baseline or peak glucose levels in male and female subjects, but serum GH concentrations (mean +/- SD) measured by the enhanced sensitivity chemiluminescence assay were lower in male than female subjects at both baseline (0.12 +/- 0.08 vs. 2.3 +/- 2.3 micrograms/L; P < 0.01) and the postglucose GH nadir (0.029 +/- 0.014 vs. 0.25 +/- 0.23 micrograms/L; P < 0.01). The high correlation between baseline and nadir GH (r = 0.82; P < 0.001) and the equivalent fractional decline in mean GH levels in men and women after glucose administration (67 +/- 17% vs. 84 +/- 8%; P = 0.06) suggest that the lower GH levels in men after glucose treatment are due to lower baseline values and not to a greater suppressive effect of glucose.

Nuclear scintigraphic assessment of intestinal dysfunction after combined treatment with 9-amino-20(S)-camptothecin (9-AC) and irradiation.

PURPOSE: The camptothecins (CPTs) are potent radiosensitizers of malignant tumors in vivo. The extent of normal tissue damage after combined CPT and radiation treatment is unknown. In this article, a jejunal absorption assay with (99m)Tc- pertechnetate (Na[(99m)TcO(4)]) was used to assess C3H/Kam mice given total body irradiation (TBI) of 4 Gy, 6 Gy, and 8 Gy, 2 mg/kg single intramuscular injection of 9-AC or a combination of 2 mg/kg 9-AC + 4 Gy TBI. We also correlated the absorption data with morphologic changes in the jejunal mucosa. MATERIALS AND METHODS: ((99m)TcO(4))(-) absorption from the intestinal lumen into the circulation was studied with dynamic gamma-scintigraphy combined with a multichannel analyzer to record the radiometry data in a time-dependent fashion. Jejunal cross sections were scored for the number of cells per villus and the percentage of apoptotic and mitotic cells in the crypt compartment. The jejunal microcolony assay was used to quantify jejunal crypt survival. RESULTS: A dose-dependent decrease in the absorption function was observed 3.5 days following TBI. The mean absorption rate was reduced to 89 +/- 16% of control in response to a sublethal 4 Gy TBI and dropped to 47. 5 (9.8% in response to 8 Gy TBI. The mean rate of intestinal absorption was delayed by single sublethal 2 mg/kg 9-AC injection to 62 (11% in comparison with control values. The combination of a single 4 Gy TBI with a 9-AC treatment decreased the ((99m)TcO(4))(-) jejunal absorption in an additive fashion producing absorption lifetime values more than twofold longer than controls. The decrease in ((99m)TcO(4))(-) absorption at 3.5 days after irradiation, 9-AC treatment or the combination of the two agents correlates with the number of cells per villus and the percentage of apoptotic cells in the crypt compartment. CONCLUSION: Dynamic enteroscintigraphy with (99m)Tc-pertechnetate is a sensitive functional assay for rapid evaluation of radiation and chemotherapy induced intestinal damage. Reduced intestinal absorptive function has a cellular basis and correlates directly with the numbers of cells lost per villus in a treatment-dependent manner.

Nuclear scintigraphic assessment of radiation-induced intestinal dysfunction.

Radiation-induced damage to the intestine can be measured by abnormalities in the absorption of various nutrients. Changes in intestinal absorption occur after irradiation because of loss of the intestinal absorptive surface and a consequent decrease in active transport. In our study, the jejunal absorption of (99m)Tc-pertechnetate, an actively transported gamma-ray emitter, was assessed in C3H/Kam mice given total-body irradiation with doses of 4, 6, 8 and 12.5 Gy and correlated with morphological changes in the intestinal epithelium. The absorption of (99m)Tc-pertechnetate from the intestinal lumen into the circulation was studied with a dynamic gamma-ray-scintigraphy assay combined with a multichannel analyzer to record the radiometry data automatically in a time-dependent regimen. The resulting radioactivity-time curves obtained for irradiated animals were compared to those for control animals. A dose-dependent decrease in absorptive function was observed 3.5 days after irradiation. The mean absorption rate was reduced to 78.8 +/- 9.3% of control levels in response to 4 Gy total-body irradiation (mean +/- SEM tracer absorption lifetime was 237 +/- 23 s compared to 187 +/- 12 s in nonirradiated controls) and to 28.3 +/- 3.7% in response to 12.5 Gy (660 +/- 76 s). The decrease in absorption of (99m)Tc-pertechnetate at 3.5 days after irradiation correlated strongly (P < 0.001) with TBI dose, with the number of cells per villus, and with the percentage of cells in the crypt compartment that were apoptotic or mitotic. A jejunal microcolony assay showed no loss of crypts and hence no measured dose-response effects after 4, 6 or 8 Gy TBI. These results show that dynamic enteroscintigraphy with sodium (99m)Tc-pertechnetate is a sensitive functional assay for rapid evaluation of radiation-induced intestinal damage in the clinically relevant dose range and has a cellular basis.

Seizures induce phase shifts of rat circadian rhythms.

RATIONALE: Epileptic seizures may alter neuroendocrinological cycles. Light pulses induce phase shifts in circadian rhythms. Using hippocampal-kindled rats to ensure maximal clinical expression, we determined if seizures likewise induce phase shifts. METHODS: We monitored the circadian rhythm of temperature (CRT) with intraperitoneal radiotelemetry in rats (n=21) isolated from time cues and light for 3-week trials. Seizures were triggered with hippocampal electrical stimulation at different circadian phases. Optimized, least-error phase shifts were calculated from preictal and postictal CRTs. Induced seizures were referenced to CRT (t(max)=00:00, 24-h circadian cycle). RESULTS: Phase shifts (individual responses=57) differed across the circadian cycle. Rather than forming a clear phase-response curve, phase shifts were especially variable between 00:00 and 06:00 h. CONCLUSIONS: This study demonstrates that electrically-induced seizures induce advances and delays in CRT in a phase-dependent fashion but in a pattern different from typical light-induced phase shifts. Disorders of circadian regulation may contribute to some of the altered endogenous cycles associated with epilepsy.

Circadian rhythms in mouse suprachiasmatic nucleus explants on multimicroelectrode plates.

The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus functions as a circadian pacemaker. This study used multimicroelectrode plates to measure extracellular action potential activity simultaneously from multiple sites within the cultured mouse SCN. Neurons within the isolated mouse SCN expressed a circadian rhythm in spontaneous firing rate for weeks in culture.

Circadian rhythms of locomotor activity in zebrafish.

As part of an effort to characterize the circadian system of the zebrafish, we examined the circadian regulation of locomotor activity in adult males and females. Gross locomotor activity was measured using infrared movement detectors. The effects of light, dark, and temperature on the amplitude, phase, and free-running periods of locomotor rhythms were determined. When zebrafish were maintained in a 12-h light:12 h dark cycle at 25 degrees C, 86% of the fish were most active during the light phase of the cycle. The phases of free-running rhythms measured after transfer of fish from light cycles to constant conditions indicate that this diurnal activity profile reflects entrained circadian rhythmicity. When animals were maintained in constant conditions, the proportion that expressed significant circadian rhythmicity depended on ambient temperature. At 21 degrees C, 73% of the animals were rhythmic in constant darkness, and 65% were rhythmic in constant light. Fewer (28-59%) were rhythmic at 18 degrees, 25 degrees, and 28.5 degrees C. The free-running period of rhythmic animals was not affected by temperature within this range. The average period was shorter in constant light (LL; 12 lx) than in constant darkness (DD) in all but one experiment, and the difference was statistically significant for animals held at 21 degrees C. These data indicate that zebrafish locomotor activity is regulated by a circadian clock that is temperature compensated. Because rhythmicity is most robust at 21 degrees C, this would be the optimal temperature for future studies of the physiological basis of zebrafish behavioral rhythms.

Modulation of dose intensity in aerodigestive tract cancers: strategies to reduce toxicity.

Advances in diagnostic and therapeutic radiology and a better understanding of cell biology are being applied in practical ways to modulate treatment morbidity. Conformal radiotherapy targets the cancer precisely and can be combined with new systemically administered radiosensitizers. The successes of conventional chemoradiation programs support continued study of newer ways to deliver systemic radiosensitizing chemotherapy. However, chemoradiation creates a narrower therapeutic window compared to irradiation alone and increased treatment intensity, even with conformal chemoradiation techniques, can potentially result in frequent complications, detrimental treatment delays, and decreased quality of life. Treatment schedules employing a "best tolerated time" modelfor systemic administration of radiosensitizing chemotherapy, based on the concept of chronotolerance, offer attractive ways to address the challenging problem of normal tissue toxicity associated with conformal chemoradiation. This approach may be beneficial in the elderly and those medically unfit to tolerate traditional dose-intense combined-modality schedules. Further evaluation of this concept is warranted, based on existing data.