Vibrational circular dichroism of a 2,5‐diketopiperazine (DKP) peptide: Evidence for dimer formation in cyclo LL or LD diphenylalanine in the solid state

The diastereomer diketopiperazine (DKP) peptides built on phenylalanine, namely, cyclo diphenylalanine LPhe‐LPhe and LPhe‐DPhe, were studied in the solid phase by vibrational circular dichroism (VCD) coupled to quantum chemical calculations. The unit structure of cyclo LPhe‐LPhe in KBr pellets is a dimer bridged by two strong NH…O hydrogen bonds. The intense bisignate signature in the CO stretch region is interpreted in terms of two contributions arising from the free COs of the dimer and the antisymmetrical combination of the bound COs. In contrast, cyclo LPhe‐DPhe shows no VCD signal in relation to its symmetric nature.     

Ultrafast folding and molecular dynamics of a linear hydrophobic β-hairpin

The first computational study of the folding and dynamics of a hydrophobic β-hairpin containing a central heterochiral diproline segment is reported. Linear hydrophobic sequences containing centrally positioned diproline motifs, heterochiral (DL/LD) and homochiral (LL/DD)), are investigated for their ability to form β-hairpins. Heterochiral diproline motifs (LD/DL) reveal the formation of stable β-hairpins with the backbone adopting β-turn conformation and the formation of backbone hydrogen bonds with antiparallel cross-strand registry, whereas the homochiral diproline (LL/DD) containing sequences tend to adopt PP_II helix conformation. The competition between the β-turn formation and the backbone H-bond ladder of the antiparallel β-strands in heterochiral diproline containing sequences is employed to validate the hypothesis that β-turn formation precedes inter-strand registry in the folding of a β-hairpin (“zipper” mechanism). The observation of noncanonical hydrogen bonds leads to a folded β-hairpin-like conformation and points to the existence of relatively stable transition state intermediates, between the unfolded (extended) and folded (β-hairpin) states. The MD simulations are in excellent agreement with the experimental studies on the model system and constitute the very first computational investigation of the folding and dynamics of a completely hydrophobic synthetic β-hairpin containing heterogeneous residues of mixed chirality.     

Chiral HPLC Separation and Modeling of Four Stereomers of DL‐Leucine‐DL‐Tryptophan Dipeptide on Amylose Chiral Column

Chiral high‐performance liquid chromatography (HPLC) separation and modeling of four stereomers of DL‐leucine‐tryptophan DL‐dipeptide on AmyCoat‐RP column are described. The mobile phase applied was ammonium acetate (10 mM)‐methanol‐acetonitrile (50:5:45, v/v). The flow rate of the mobile phases was 0.8 mL/min with UV detection at 230 nm. The values of retention factors for LL‐, DD‐, DL‐, and LD‐ stereomers were 2.25, 3.60, 5.00, and 6.50, respectively. The values of separation and resolution factors were 1.60, 1.39, and 1.30 and 7.76, 8.05, and 7.19. The limits of detection and quantitation were ranging from 1.0–2.3 and 5.6–14.0 μg/mL. The simulation studies established the elution orders and the mechanism of chiral recognition. It was seen that π–π connections and hydrogen bondings were the main forces for enantiomeric resolution. The reported chiral HPLC method may be applied for the enantiomeric separation of DL‐leucine‐DL‐tryptophan in unknown matrices. Chirality 28:642–648, 2016. © 2016 Wiley Periodicals, Inc.     

The interrelationships between serotonin production and locomotion in different light regimes in southwestern Michigan opilionids, Leiobunum longipes

The comparison was made of the effect of LL and DD with LD 14:10 photoperiods on the 24-h secretion cycle of serotonin secretion and the activity patterns of Leiobunum longipes from Southwestern Michigan. LL and DD altered the normal activity patterns but did not change the pattern of serotonin secretion. The activity pattern in normal photoperiod (LD 14:10) produced a 12-h cosinor pattern, resulting in a 24-h biphasic activity peak model. The activity peaked in both scotophase and photophase . The altered patterns in LL and DD were different. In LL a rhythmic component could not be statistically determined. A high, irregular level of activity was seen, higher than the mean level in LD. In DD a combined 24 and 48 h cosinor pattern best fit the observed data. The major peaks occurred in nature during every other photophase and alternate scotophase time in the constant photoperiod conditions. Serotonin secretion patterns in LD, LL, and DD statistically fitted a 24-h cosinor model. Peak secretion times occurred in mid photophase for LD and LL. A later photophase peak was seen in DD. LL animals showed a mean level of serotonin and secretion pattern which was not statistically different from LD. The hypothesis that LD photoperiods direct a peak of serotonin secretion which initiated the activity pattern could not be accepted.     

Determination of absolute configuration using vibrational circular dichroism spectroscopy: the chiral sulfoxide 1-thiochromanone S-oxide

We reexamined the absolute configuration (AC) of the chiral sulfoxide 1-thiochromanone S-oxide (1) using vibrational circular dichroism (VCD) spectroscopy. The VCD spectrum of 1 was analyzed using density functional theory (DFT). DFT predicts two stable conformations of 1, separated by <1 kcal/mole. Their VCD spectra were calculated using the DFT/GIAO methodology. The VCD spectrum predicted for the equilibrium mixture of the two conformations of (S)-1 is in excellent agreement with the experimental spectrum of (+)-1. The AC of 1 is therefore definitively R(-)/S(+).     

Societal synchronization in groups of rats or quail submitted to various lighting regimens

Groups of rats or of quail that had been previously synchronized in a light (L = 100 lux) dark (D) phase opposition (PO = LD and DL) were placed together in a L12:D12 or D12:L12 alternation or in continuous light (LL) or continuous darkness (DD). Emission of carbon dioxide (VCO2) which was continuously recorded in groups of individuals placed in respiratory chambers under controlled environmental conditions allows an index of their overall respiratory and metabolic exchanges to be found. In PO animals placed in LD or DL, the VCO2 circadian light dark synchronization comes back less quickly in rats than in quail, and the VCO2 variations at the light dark transitions (L-D and D-L) remain unchanged in rats, but are modified in quail. When PO animals are placed for 18 days in LL or DD, respiratory circadian rhythms disappear except in the grouped rats where they reappear after 4-5 days in DD.     

Chiroptical Studies on Supramolecular Chirality of Molecular Aggregates

The attempts of applying chiroptical spectroscopy to supramolecular chirality are reviewed with a focus on vibrational circular dichroism (VCD). Examples were taken from gels, solids, and monolayers formed by low‐molecular mass weight chiral gelators. Particular attention was paid to a group of gelators with perfluoroalkyl chains. The effects of the helical conformation of the perfluoroalkyl chains on the formation of chiral architectures are reported. It is described how the conformation of a chiral gelator was determined by comparing the experimental and theoretical VCD spectra together with a model proposed for the molecular aggregation in fibrils. The results demonstrate the potential utility of the chiroptical method in analyzing organized chiral aggregates. Chirality 27:659–666, 2015. © 2015 Wiley Periodicals, Inc.     

Influence of the light regimen on the circadian rhythm of serum lipids in the laboratory rat

Young male rats of a conventional Wistar breed were adapted for several weeks (3-6) to a 12:12 h light:dark (7 a.m. - 7 p.m., 7 p.m. - 7 a.m.) regimen (LD), to inversion of this standard regimen (DL), to continuous darkness (DD) or to continuous light (LL). After adaptation, groups of animals were killed at 3-hour intervals during the day and the basic lipid fractions were determined in their serum. Under the standard light regimen the cosinor test demonstrated a rhythm in all the indicators (triacylglycerols, total cholesterol, phospholipids) except for non-esterified fatty acids. When the regimen was inverted (DL), the peaks of the circadian non-esterified fatty acid and triacylglycerol curves shifted to the antiphase. The acrophases of "free-running" serum lipid rhythm under the DD regimen of the standard regimen rhythms differed in the case of cholesterol and phospholipids. In the case of triacylglycerols and phospholipids there was disagreement between the DD and LL regimen curves. With reference to the localization of the acrophase under the DD and LD regimens it was assumed that the influence of the light regimen on the synchronization of circadian rhythms is small in the case of serum non-esterified fatty acids and triacylglycerols and that it is greater in the case of serum cholesterol and phospholipids.     

Circadian clock controlling egg hatching in the cricket (Gryllus bimaculatus)

Adult crickets (Gryllus bimaculatus) were maintained under a 12-h light:12-h dark cycle (LD 12:12). After oviposition, their eggs were incubated under different lighting regimens at 23 degrees C, and temporal profiles of egg hatching were examined. When the eggs were incubated in LD 12:12 or in DL 12:12 with a phase difference of 12h from LD 12:12, throughout embryogenesis, 88% to 97% of hatching occurred within 3 h of the dark-light transition on days 17 and 18 of embryogenesis; the phases of the egg-hatching rhythms in the LD 12:12 and DL 12:12 groups differed by about 12 h. In eggs incubated in constant darkness (DD) throughout embryogenesis, a circadian (about 24 h) rhythm of hatching was found, and the phase of the rhythm was similar to that seen in eggs incubated in LD 12:12, but not DL 12:12, throughout embryogenesis. When eggs that had been incubated in DD after oviposition were transferred to DL 12:12 in the middle or later stages of embryogenesis and were returned to DD after three cycles of DL 12:12, the rhythm of hatching synchronized (entrained) to DL 12:12. However, when eggs in the earlier stages of embryogenesis were transferred from DD to DL 12:12 and returned to DD after three cycles, 52% to 94% of hatching did not entrain to DL 12:12. To determine whether photoperiodic conditions to which the parents had been exposed influenced the timing of egg hatching, adult crickets were maintained in DL 12:12, and their eggs were incubated in LD 12:12, DL 12:12, or DD throughout embryogenesis. The egg-hatching rhythm was also found in the eggs incubated under these three lighting regimens. In DD, the phase of the rhythm was similar to that seen in eggs incubated in DL 12:12, not LD 12:12, throughout embryogenesis. The results indicate that in the cricket, the timing of egg hatching is under circadian control and that the circadian rhythm of hatching entrains to 24-h light:dark cycles, but only if the light:dark cycles are imposed midway through embryogenesis. Therefore, by midembryogenesis, a circadian clock has been formed in the cricket, and this is entrainable to light:dark cycles. In addition, the photoperiodic conditions to which the parents (probably the mothers) have been exposed influence the timing of hatching, suggesting that maternal factors may regulate the timing of egg hatching.     

Effect of Different Light Regimes on Pre-Adult Fitness in Drosophila melanogaster Populations Reared in Constant Light for over Six Hundred Generations

Egg to eclosion development time and survivorship were assayed on four laboratory populations of Drosophila melanogaster that had been reared for over 600 generations in continuous light (LL) and constant temperature. The assays were performed in three environments: continuous light (LL), periodically varying light/dark cycles (LD 12:12 hr), and continuous darkness (DD). Development time in LL was significantly less than that in LD, which, in turn, was significantly less than that in DD, whereas survivorship did not differ significantly among the three treatments. The results indicate that individuals from Drosophila populations routinely maintained in LL do not suffer any deleterious effects of LL treatment on pre-adult fitness. Other studies on these populations have shown that free-running period (t) of the eclosion rhythm in DD is greater than that in LD. Our results are, thus, also consistent with the notion that development time may be a function of the free-running period.     

Effect of Different Light Regimes on Pre-Adult Fitness in Drosophila melanogaster Populations Reared in Constant Light for over Six Hundred Generations

Egg to eclosion development time and survivorship were assayed on four laboratory populations of Drosophila melanogaster that had been reared for over 600 generations in continuous light (LL) and constant temperature. The assays were performed in three environments: continuous light (LL), periodically varying light/dark cycles (LD 12:12 hr), and continuous darkness (DD). Development time in LL was significantly less than that in LD, which, in turn, was significantly less than that in DD, whereas survivorship did not differ significantly among the three treatments. The results indicate that individuals from Drosophila populations routinely maintained in LL do not suffer any deleterious effects of LL treatment on pre-adult fitness. Other studies on these populations have shown that free-running period (t) of the eclosion rhythm in DD is greater than that in LD. Our results are, thus, also consistent with the notion that development time may be a function of the free-running period.     

Circadian Rhythm of Blood Ethanol Clearance Rates in Rats: Response to Reversal of the L/D Regimen and to Continuous Darkness and Continuous Illumination

Phase relationships of the circadian rhythms of blood ethanol clearance (metabolic) rates and body temperature were studied in rats successively exposed to 4 illumination regimens: LD (light from 0800-2000 hr), DL (light from 2000-0800 hr), constant darkness (DD) and, lastly, constant light (LL). After a 4-wk standardization to each regimen, body temperatures were taken at 9 × 4-hr intervals to establish baseline circadian profiles. One week later, groups (N = 8) received 1.5 g/kg ethanol (i.p.) at 6 equally spaced timepoints during a 24-hr span, when temperatures were again measured. Ethanol clearance rates were estimated from decreasing blood ethanol levels sampled every 20 min from 60-200 min after dosing, and the resultant elimination curves were subjected to cosinor analysis. These studies show for the first time that the high amplitude circadian rhythm in ethanol metabolism persists under constant conditions of illumination (DD and LL), demonstrating that it may well be a truly internal circadian rhythm and not a response to exogenous cues of the light/dark cycle. During both LD and DL, maximal and minimal ethanol clearance rates fell near the end of the dark and light phases, respectively, and followed circadian peak and trough control temperatures by approximately 6 hr. A fixed internal phase relationship between the core body temperature and the circadian rhythm in ethanol metabolism is demonstrated, thus establishing the rhythm in body temperature as a suitable and convenient internal marker rhythm for studies of the metabolism of low-to-moderate ethanol doses. These studies demonstrate that the phase relationships of blood ethanol clearance rate and body temperature can be manipulated by the illumination regimen selected, an observation of both basic and practical importance.     

Stocking density affects circadian rhythms of locomotor activity in African catfish, Clarias gariepinus

The effect of stocking density on the locomotor activity of African catfish C. gariepinus under different light regimes was investigated. C. gariepinus were stocked under different densities (1, 5, or 10 fish/tank), and their locomotor activity recorded under light-dark (LD), constant light (LL), constant darkness (DD), and LD-reversed (DL) regimens. Under the LD cycle, catfish showed a crepuscular activity pattern, irrespective of stocking density, with most of the daily activity concentrated around the light-onset and light-offset times. When fish were subjected to DD, all 4 tanks with medium (5 fish) and high (10 fish) stocking densities showed circadian rhythmicity, with an average period (?) of 23.3???0.5 and 24.6???0.5?h, respectively. In contrast, only 2 low (1 fish) density tanks showed free-running rhythms. Under LL, activity levels decreased significantly in comparison with levels observed under LD and DD. Moreover, fish of 1, 2, and 3 out of the 4 tanks with low, medium, and high densities, respectively, showed free-running rhythms under these conditions. When the photocycle was reversed (DL), fish of 3, 2, and 4 out of the 4 tanks with low, medium, and high stocking densities, respectively, showed gradual resynchronization to the new phase, and transient cycles of activity were observed. These results suggest that stocking density of fish affected the display of circadian rhythmicity and the intensity of activity levels. Thus, fish kept in higher densities showed more robust rhythmicity and higher levels of daily activity, indicating that social interactions may have an influence on behavioral patterns in the African catfish.     

Cytoplasm Rescues an Arrhythmic Mutant on the Circadian Rhythm of Mating Reactivity in Paramecium bursaria

Cells of an unusual Paramecium bursaria stock (Sj2) expressed rhythmic mating reactivity in a light/dark cycle (LD) and under continuous illumination (LL). When placed in continuous darkness (DD), did not show rhythmicity but rather demonstrated a continuous high mating reactivity. However, mating reactivity was reduced following exposure to a 6-h light pulse interrupting the DD, and then recovered to its former condition. Genetic analysis showed the arrhythmicity in DD to be a dominant character inherited in a Mendelian ratio. On the other hand, a clone (MCIw) that did not show the rhythmicity in either DD or LL was isolated from the parent stock Sj2w following a 5-h treatment with 2 μg/ml nitrosoguanidine (MNNG). The MCIw cells expressed weak rhythmicity in LD, but were insensitive to a 6-h light pulse in DD. The arrhythmicity in LL was inherited cytoplasmically. In addition to this, rhythmicity in LL could be recovered by injection of cytoplasm from the wild-type cell when the recipient cell was homozygous for the wild-type nuclear gene (+/+). The cytoplasmic components or factors are assumed to control the functional circadian system and genetically determine the rhythmicity of mating reactivity.     

Isotope-labeled vibrational circular dichroism studies of calmodulin and its interactions with ligands

In this work we have studied ligand-induced secondary structure changes in the small calcium regulatory protein calmodulin (CaM) using vibrational circular dichroism (VCD) spectroscopy. We find that, due to its chiral sensitivity, VCD spectroscopy has increased ability over IR spectroscopy to detect changes in the structure and flexibility of secondary structure elements upon ligand binding. Moreover, we demonstrate that the uniform isotope labeling of CaM with (13)C shifts its amide I' VCD band by about approximately 43 cm(-1) to lower wavenumbers, which opens up a spectral window to simultaneously visualize a bound target protein. Therefore this study also provides the first example of how isotope labeling enables protein-protein interactions to be studied by VCD with good separation of the signals for both isotope-labeled and unlabeled proteins.     

Circadian oscillatory patterns of oxygen uptake in individual workers of the ant Camponotus rufipes

Abstract. Respiratory rates of individual workers of Camponotus rufipes Fabricius (Hymenoptera: Formicidae) were measured at 25C and LD 12:12 h (lights on 06.00 hours), DL 12:12h (lights on 18.00 hours), LL (850 lux) and DD (red light, 20–30 lux), using the micro-Warburg technique. Worker ants were collected from natural nest during the winter of 1987 in a woodland park in the region of Rio Claro, Sāo Paulo, Brazil. The respiration of ants showed a circadian rhythm with acrophase ranging from 20 h 41 min to O1 h 18 min and from 10 h 32 min to 12 h 22 min at LD and DD, respectively. In constant darkness the rhythmometric variables were similar to those presented by ants kept at LD 12:12 h. Under constant light no circadian rhythm in the respiration rates was found. A reduction in the amplitude was observed, indicating an inhibitory effect of this light regime on the respiration process.     

Circadian rhythm in endogenous nerve activity in the eye of Musca dornestica L.

The frequency of occurrence of endogenous bursts of spikes was monitored by external electrode placed on the surface of housefly eyes in darkness. In LD 16:8 the frequency of these bursts showed an entrained rhythm, with a c. 10-fold change in level from rest to active periods. The rate began to increase in anticipation of dawn. The free-running period in DD was c. 21 h and in LL, 16–17 h. The active/rest ratio was 1.0 in DD and 2.5 in LL, the active phase being 10.4 h in DD and 12.3 h in LL. In these respects the rhythm conforms to Aschoff's rule. In groups of flies, the entrained rhythm was apparently lost 4–6 days after transfer from LD to LL, because the individual flies' rhythms changed from the 24 h entrained state to the LL, free-running state at differing rates, leading to asynchrony. After four cycles the phase angles in a sample of ten flies differed by 120 (8 h). In contrast, when flies were transferred from LD to DD, the phase angle variation did not differ markedly, even after 9 days, from that of entrained flies. The findings are discussed in terms of Truman's (1972) clock types.     

Comparative uptake of nitrate by intact seedlings of C3 (barley) and C4 (corn) plants: Effect of light and exogenously supplied sucrose

The effect of light and exogenously supplied sucrose on NO₃ ⁻ uptake was studied in 9-day-old intact C₃ (barley) and C₄ (corn) seedlings. The seedlings used were uninduced for nitrate uptake system (i.e. had never seen nitrogen during germination and growth) and were exposed to continuous light for 3 days to avoid any diurnal variation and to load the seedlings fully with photosynthates. The uptake assay was conducted either in light or in darkness. Prior to assay, seedlings were treated with darkness or light for 24 h. Accordingly, four sets of seedlings, i.e. pretreated with light and assayed in light (LL); pretreated and assayed in darkness (DD); pretreated with light and assayed in darkness (LD); and pretreated with darkness and assayed in light (DL) were formed. Barley exhibited 55% higher NO₃ ⁻ uptake than corn during light (LL) and 91% higher during darkness (DD). Shifting barley seedlings from light to dark (LD) or dark to light (DL) for uptake assay, did not affect NO₃ ⁻ uptake, i.e. in LD the uptake was similar to LL and in DL it was similar to DD. However, in corn, the light conditions during the assay determined the uptake regardless of the conditions during the period preceding the assay. One percent sucrose in the medium increased NO₃ ⁻ uptake by 31% in barley and 70% in corn during light (LL). The corresponding increase during darkness (DD) was 38% in both barley and corn. Removal of the corn residual endosperm decreased NO₃ ⁻ uptake by 40% during darkness. Etiolated seedlings (those having never seen light) of both barley and corn were able to take up significant amount of NO₃ ⁻ during darkness. Externally supplied sucrose in the assay medium of etiolated seedlings increased the NO₃ ⁻ uptake to about 4 and 2 fold in barley and corn, respectively. The data presented here provide evidence that: 1. In intact seedlings, light per se is not obligatory for NO₃ ⁻ uptake and that the carbohydrate supply may mimic light. 2. Light affected the NO₃ ⁻ uptake differently in barley and corn. This revised version was published online in June 2006 with corrections to the Cover Date.     

Myeloid bodies in the pigment epithelium of a teleost embryo, the viviparous Poecilia reticulata

Appearance of myeloid bodies (MB) in the retinal pigment epithelium (RPE) precedes photoreceptor outer segment development in Poecilia reticulata embryos reared under a 12 hrs LD cycle, in constant darkness (DD) and constant light (LL). When first formed, MB are predominantly continuous with rough endoplasmic reticulum (RER). The same is observed in the peripheral growth zone of the developed eye, whereas in differentiated parts, MB are continuous with the smooth endoplasmic reticulum (SER). At onset of photomechanical movements, wavy MB predominate in light-adapted LD embryos, are exclusively present in LL and are located in the RPE processes. SER abounds. Straight MB predominate in dark-adapted LD embryos, are exclusively present in DD and contain electrondense material between lamellae. Diurnal appearance of electrondense material may be coupled with transfer of retinol, mediated by various transport proteins.     

Effects of constant darkness and constant light on circadian organization and reproductive responses in the ram

The relationship between circadian rhythms in the blood plasma concentrations of melatonin and rhythms in locomotor activity was studied in adult male sheep (Soay rams) exposed to 16-week periods of short days (8 hr of light and 16 hr of darkness; LD 8:16) or long days (LD 16:8) followed by 16-week periods of constant darkness (dim red light; DD) or constant light (LL). Under both LD 8:16 and LD 16:8, there was a clearly defined 24-hr rhythm in plasma concentrations of melatonin, with high levels throughout the dark phase. Periodogram analysis revealed a 24-hr rhythm in locomotor activity under LD 8:16 and LD 16:8. The main bouts of activity occurred during the light phase. A change from LD 8:16 to LD 16:8 resulted in a decrease in the duration of elevated melatonin secretion (melatonin peak) and an increase in the duration of activity corresponding to the changes in the ratio of light to darkness. In all rams, a significant circadian rhythm of activity persisted over the first 2 weeks following transfer from an entraining photoperiod to DD, with a mean period of 23.77 hr. However, the activity rhythms subsequently became disorganized, as did the 24-hr melatonin rhythms. The introduction of a 1-hr light pulse every 24 hr (LD 1:23) for 2 weeks after 8 weeks under DD reinduced a rhythm in both melatonin secretion and activity: the end of the 1-hr light period acted as the dusk signal, producing a normal temporal association of the two rhythms. Under LL, the 24-hr melatonin rhythms were disrupted, though several rams still showed periods of elevated melatonin secretion. Significant activity rhythms were either absent or a weak component occurred with a period of 24 hr. The introduction of a 1-hr dark period every 24 hr for 2 weeks after 8 weeks under LL (LD 23:1) failed to induce or entrain rhythms in either of the parameters. The occurrence of 24-hr activity rhythm in some rams under LL may indicate nonphotoperiodic entrainment signals in our experimental facility. Reproductive responses to the changes in photoperiod were also monitored. After pretreatment with LD 8:16, the rams were sexually active; exposure to LD 16:8, DD, or LL resulted in a decline in all measures of reproductive function. The decline was slower under DD than LD 16:8 or LL.(ABSTRACT TRUNCATED AT 400 WORDS)