Light-independent role of CRY1 and CRY2 in the mammalian circadian clock.

Cryptochrome (CRY), a photoreceptor for the circadian clock in Drosophila, binds to the clock component TIM in a light-dependent fashion and blocks its function. In mammals, genetic evidence suggests a role for CRYs within the clock, distinct from hypothetical photoreceptor functions. Mammalian CRY1 and CRY2 are here shown to act as light-independent inhibitors of CLOCK-BMAL1, the activator driving Per1 transcription. CRY1 or CRY2 (or both) showed light-independent interactions with CLOCK and BMAL1, as well as with PER1, PER2, and TIM. Thus, mammalian CRYs act as light-independent components of the circadian clock and probably regulate Per1 transcriptional cycling by contacting both the activator and its feedback inhibitors.

Group II intron ribozymes that cleave DNA and RNA linkages with similar efficiency, and lack contacts with substrate 2'-hydroxyl groups.

BACKGROUND: Group II introns are self-splicing RNAs that have mechanistic similarity to the spliceosome complex involved in messenger RNA splicing in eukaryotes. These autocatalytic molecules can be reconfigured into highly specific, multiple-turnover ribozymes that cleave oligonucleotides in trans. We set out to use a simplified system of this kind to study the mechanism of cleavage. RESULTS: Unlike other catalytic RNA molecules, the group II ribozymes cleave DNA linkages almost as readily as RNA linkages. One ribozyme variant cleaves DNA linkages with an efficiency comparable to that of restriction endonuclease EcoRI. Single deoxynucleotide substitutions in the substrate showed that the ribozymes bind substrate without engaging 2'-hydroxyl groups. CONCLUSIONS: The ribose 2'-hydroxyl group at the cleavage site has little role in transition-state stabilization by group II ribozymes. Substrate 2'-hydroxyl groups are not involved in substrate binding, suggesting that only base-pairing is required for substrate recognition.

Characterization of mutagenic activity in cooked-grain-food products.

Wheat gluten or flour from several plant sources heated at 210 degrees C for 1 hr produced 0-1800 revertant colonies/g in the Ames/Salmonella test using strain TA98 with metabolic activation. Baked or toasted foods and a heated grain beverage showed a mutagenic response in all cases from 2 to 320 revertants/g, with higher values seen when overcooked. Fried meat-substitute patties showed 0-23 revertants/g when fried at 210 degrees C. A greater mutagenic response in bacterial strain TA98 than in strain TA100 and a requirement for metabolic activation suggests that one or more aromatic amine mutagens are formed at normal cooking temperatures, but the mutagenic activity measured cannot be accounted for by the known heterocyclic amines commonly found in cooked meats. We conclude that grain products from aromatic amine chemicals during heating that are mutagenic in bacterial mutation tests.

Formation of mutagenic activity from amino acids heated at cooking temperatures.

To investigate the formation of aromatic amine-like mutagenic activity in cooked grain foods, amino acids were heated alone or in binary combinations at either 150 or 210 degrees C. About half of the binary mixtures of arginine heated with other amino acids produced potent mutagenic responses in the Ames/Salmonella assay, but only cysteine produced mutagenic products when heated alone. One-to-one molar ratios of arginine heated with threonine, valine, cystine, cysteine or tryptophan produced reaction products that gave 1200-3200 revertants/mmol in Salmonella strain TA98 with metabolic activation. 1-Methylguanidine, a fragment of arginine, produced a mutagenic response when heated alone or in binary mixtures with all amino acids tested. Analysis of reaction product extracts by solid-phase extraction and HPLC failed to find the known heterocyclic amines commonly found in cooked meats that would explain the measured mutagenic activity. As judged by biological and chemical characterization, several new aromatic amine mutagens are formed by heating some simple amino acids combined with arginine, and these reactants may be the source of the mutagenic products detected in the extracts of some cooked grain-based foods.

Anthracycline binding to synthetic and natural membranes. A study using resonance energy transfer.

The binding of adriamycin and its two analogues 4'-epidoxorubicin and 4'-deoxydoxorubicin to synthetic and mitochondrial membranes was investigated by using resonance energy transfer between these drugs and two fluorescent probes, diphenylhexatriene (DPH) and tryptophan. The fluorescence of the lipid probe DPH in both types of membranes and tryptophan in mitochondria was quenched by the anthracyclines in a dose-dependent manner. In sonicated, fluid-phase dimyristoyl-L-alpha-phosphatidylcholine (DMPC) vesicles, the half-quenching concentration (K50) of adriamycin was 17 +/- 1 microM, whereas in bilayers containing a 1:1 molar ratio of DMPC to cardiolipin (CL), the value was 8 +/- 1 microM. In liver and heart mitochondria, the K50 values were 8 +/- 2 and 11 +/- 3 microM, respectively. Similar results were obtained for the other two drugs. Replacing a nonionic with an ionic medium or decreasing the pH from pH 7.7 to pH 6.9 increased the K50 value of adriamycin for DPH in DMPC/CL (1:1 molar) liposomes and in mitochondria. Higher concentrations of anthracycline were needed to quench the fluorescence of tryptophan. The results suggest that these drugs interact with both phospholipids and proteins and that the cardiotoxicity of adriamycin is unlikely to be related to the amount of drug bound to heart mitochondria.

Continuous wave Doppler ultrasound in evaluation of cerebral blood flow in neonates.

The cerebral circulation of 25 normal term infants was investigated using continuous wave Doppler ultrasound. Serial blood flow velocity signals were obtained from the common carotid and anterior cerebral arteries during the first week of life. The records were processed using a frequency spectrum analyser to provide cerebral blood velocity waveforms. The pulsatility index (PI), A/B ratio, and rise and fall slope of the waveforms were calculated. The results indicated that cerebrovascular resistance was raised appreciably on day 1 of life compared with later in the first week. In 18 of 25 infants (72%) there was no continuous carotid blood flow in the first hours of life. We suggest that the human cerebral circulation adapts to the process of birth in a similar fashion to that of animal models.

Variations in intralipid tolerance in newborn infants.

Intravenous Intralipid tolerance tests (IVLTT) were done in 26 newborn infants of 26-40 wk gestational ages. The clearance constants (k2) ranged from 1.2-12.7 (%/min) after bolus injections given within 4.5 h (n = 12) or daily (n = 13). Significant variation (17-31%) occurred, similar to adults, and was unrelated to the time or dose given. Eleven infants received continuous Intralipid infusions for 10-24 h at a rate calculated to maintain a plasma Intralipid plateau concentration of 100 mg/dl. Nine infants did not exceed this optimal plasma level, although four could have tolerated more Intralipid. Two infants exceeded the ideal plasma concentration (greater than 100 mg/dl). All infants achieved and maintained plateaus within 5 h. Neither day-to-day variations nor the bolus dose used to establish clearance characteristics, accounted for the discrepancies in plateaus achieved. These studies identify some limitations of the IVLTT as a predictor of Intralipid utilization during continuous infusion, and the need for early monitoring of plasma Intralipid concentrations to optimize the therapeutic dose given to newborn infants.

Evaluation of cerebral blood flow in the normal neonate by Doppler spectrum analysis.

Continuous wave Doppler ultrasound has seldom been applied to the evaluation of the cerebral circulation of the newborn infant. Twenty-five term infants were studied and records taken from the common carotid and anterior cerebral arteries during the first week of life using a bidirectional Doppler instrument. The best Doppler signals obtained were audiofrequency analysed and sonagrams recorded. Analysis of the carotid sonagrams revealed a mean Pulsatility Index (PI) on the first day of life of 0.98 compared to 0.88 on Day 2 (p less than 0.001). An unexpected finding was the absence of continuous carotid blood flow in the majority of infants in the first hours of life. The PI on subsequent days did not differ significantly from Day 2. The PI of the anterior cerebral arteries on Day 1 was significantly elevated (p less than 0.001) compared to Day 2 and subsequent days. The significance of these findings and their importance in the further evaluation of pathological cerebral states in the neonate, especially birth asphyxia and intracranial haemorrhage are discussed.