Jagua blue derived from Genipa americana L. fruit: A natural alternative to commonly used blue food colorants?

Due to consumers' increasing health awareness, food industry aims at replacing synthetic dyes by natural counterparts. The substitution of blue synthetic dyes is particularly challenging since current natural alternatives such as phycocyanin (Spirulina) suffer from poor stability. Jagua blue (produced from Genipa americana L. fruit) might represent a potential novel blue pigment source. However, only little is known about its color properties, and application in food systems. Therefore, the blue color and the stability of Jagua blue were assessed for the first time and compared to commonly used colorants, namely, Spirulina, brilliant blue FCF (Blue no. 1), and indigo carmine (Blue no. 2). The reaction rate of Jagua blue was independent of its concentration, confirming thermal degradation to follow first-order kinetics. Between pH 3.6 and 5.0, the color hue of Jagua blue solutions was similar to that of Blue no. 2. However, Jagua blue revealed markedly higher storage stabilities (t1/2=86-105days) than Blue no. 2 (t1/2≤9days) and was less susceptible to acidic pH of 3.6 (t1/2=86days) than Spirulina (t1/2=70days). High negative b* values (blueness) of colored gelatin gels were only obtained for Jagua blue and Spirulina, and the former exhibited higher light stabilities (t1/2=15days) than Spirulina gels (t1/2=4days). Our findings indicate Jagua blue to be a most promising alternative to synthetic dyes, providing relevant information regarding potential food applications.

Point mutations in the avian sarcoma/leukosis virus 3' untranslated region result in a packaging defect.

The 3' untranslated region (3' UTR) between the 3' end of env and the long terminal repeat is well conserved among avian retroviruses and is essential for efficient replication. Deletion of the dr1 element within the 3' UTR has been reported to have various effects, including reduced levels of unspliced RNA in the cytoplasm, decreased stability of unspliced RNA, decreased particle production, and decreased genomic RNA packaging. To probe the role of specific sequences within dr1 in virus replication, site-directed mutagenesis was utilized to perturb parts of the predicted secondary structure of dr1. Seven of thirteen mutations had no significant effect; the others resulted in an approximately 10- to 20-fold reduction in replication. These mutants were further characterized and found to impair cytoplasmic accumulation of unspliced RNA only slightly. Furthermore, no decreases were observed in the stability of the unspliced RNA or in the production of virus particles. Genomic RNA packaging, however, was reduced by about 10-fold. Similar amounts of particles were produced by cells containing the mutant and wild-type DNA, and all particles contained similar levels of reverse transcriptase activity. The results suggest that the region of the dr1 disrupted by the mutations plays a role in genomic RNA packaging, although that packaging may not be the only role for dr1.

Masking and parametric effects of high-frequency light-dark cycles.

Light seems to be the only zeitgeber signal that reaches the pacemaker via specific pathways. For all nonphotic entraining agents, there might be a common mechanism that operates by feedback processes via "arousal." When entrained, the circadian system keeps a distinct phase relationship to the zeitgeber. This is achieved by daily corrections of frequency and/or phase. The phase response curves (PRCs) obtained with brief light- or dark-pulses are satisfactory in explaining such non-parametric entrainment. However, many organisms are exposed daily to one major step-up and one step-down in light intensity; hence, attention should be payed to the usefulness of step-PRCs. Zeitgebers not only entrain circadian rhythms by controlling the phase and period of the pacemaker, but also mask the overt rhythm. Many experimental data demonstrate a dependence on phase of the masking effects. It is due to this dependence on phase that organisms that become arrhythmic in constant conditions can show freerunning rhythms under the influence of permanent high-frequency light-dark (LD) cycles (the "demasking" effect). Under natural conditions, most animals are exposed to continuously fluctuating light intensities. As demonstrated by experiments with high-frequency LD cycles, either self-selected or externally controlled, the circadian system integrates over the intensities experienced, and changes its frequency accordingly. It seems likely that non-parametric entrainment plays a predominant role, but contributions of parametric effects are probably underrated.

Human perception of short and long time intervals: its correlation with body temperature and the duration of wake time.

Time estimation was studied in seven human subjects during prolonged sojourn is isolation from time cues. They wore rectal temperature probes throughout the experiments, and during wakefulness recorded each time they thought one hour had passed. At the end of each of these subjective hours they produced a subjective 5 or 10 sec interval. The produced intervals on the 1-h task were not related to body temperature but were correlated with and proportional to the duration of waketime in all subjects. The produced 5 and 10 sec intervals were in all subjects negatively correlated with rectal temperature, but were not associated with wake time. Brief and long time intervals are subjectively experienced via different mechanisms.

Circadian parameters as individual characteristics.

Human circadian rhythms that are free running in temporal isolation can show an extreme variability of the sleep-wake cycle without the subject being aware of it. This is due mainly to a systematic adjustment of certain circadian time structures to the varying length of the "day": the perception of long time intervals in the range of 1 h and the intervals between meals have a strong positive correlation with the duration of wake time alpha, in contrast to the amount of hourly locomotor activity that is negatively correlated with alpha. So far, the dependence of locomotor activity, of the "1-h" intervals, and of the intermeal intervals on alpha is well documented for the data sets of single subjects. The present article demonstrates that the correlations found intraindividually also apply if data from several subjects are compared. If plotted as a function of the mean individual alpha, the individual means of "1-h" intervals and of the intermeal intervals show the same positive correlation and the means of locomotor activity show the same negative correlation with alpha as known from single individuals. It is to be concluded that each individual has its characteristic place on the regression lines that describe the dependence of the three circadian parameters on the duration of wake time.

Human time perception in temporal isolation: effects of illumination intensity.

Living in isolation from time cues under relatively high and low light intensities for a total (on average) of 24 days, 18 subjects estimated the passage of time by "producing" short (10 to 120 seconds) and long (1h) intervals throughout the experiments. The 1h productions were independent of light intensity and highly positively correlated with the duration of wake times. The short-interval productions were markedly increased under high light intensity. In a subsample of 6 subjects, the interaction between effects of body temperature and light condition on 10-second production was analyzed. Productions were negatively correlated with body temperature. In both dim and bright light, productions decreased by a factor of 0.7 per degree C. In bright light, production was increased by a factor of 1.2 relative to dim light. This effect was not mediated by body temperature, which itself was on average slightly increased in bright light. Since subjective time is slowed by bright light, objective time seems to pass faster in bright light.

[Physostigmine in treatment of cerebellar ataxia]. / Physostigmin in der Behandlung von Kleinhirnataxien.

Cerebellar ataxias are still a challenging problem for neurologists, and to this day there exists no medical, physiotherapeutic, psychot-erapeutic or surgical therapy which constantly leads to a reduction of ataxic symptoms. In the pathophysiology of cerebellar ataxia a cholinergic defect has often been described. In a double-blind, cross-over study with 14 patients with cerebellar ataxia and an open follow-up, long-term study with 21 patients, the clinical effects of physostigmine capsules in doses up to 10 mg per day were studied. Moreover, a transdermal application (physostigmine patch) was developed, achieving constant physostigmine plasma levels for 24 h. Of 14 patients treated with physostigmine during the double-blind, cross-over study, nine could correctly distinguish between verum and placebo. They all showed small but constant improvements, clearly experienced by the patients. With the physostigmine patch, 12 of 14 patients improved. Thirteen of 14 patients decided to take part in open follow-up studies with physostigmine, and most of them preferred the physostigmine patch as long-term medication. To date, 21 patients with cerebellar ataxia have been treated with physostigmine. As far as we can judge at the end of a treatment period of at least 2 years, the progression of the disease could be stopped in 17 of 21 patients. These patients reported small but constant effects, and none wants to live without the physostigmine patch.

Electrophysiological effects of aconitine in rat hippocampal slices.

The electrophysiological effects of aconitine were investigated in the rat hippocampal slice and compared with those of veratridine. Both alkaloids are known to bind at site 2 of sodium channels and to block its inactivation. Extracellular recordings revealed that aconitine and veratridine exert inhibitory effects on neuronal excitability. Aconitine slowly and reversibly decreased the population spike recorded in the CA1 pyramidal cell layer. The reduction of the spike amplitude was similar whether orthodromically or antidromically activated. The aconitine-induced inhibition did not differ from that of veratridine. However, following washout of aconitine, the amplitude of the antidromic spike was increased compared to the control amplitude. The veratridine-induced inhibition was only partially reversible. This inhibition was also observed during suppression of synaptic transmission by a low Ca2+/high Mg2+-medium, indicating an inhibition of axonal conductance. The results show that in the absence of synaptic transmission the antidromic (alvear) spike is more sensitive to the inhibitory action of aconitine than the presynaptic fiber spike elicited by stimulation of the Schaffer collaterals. Furthermore, it is shown that aconitine acts in an activity-dependent manner, in that the latency of onset of the inhibition is prolonged when the stimulation frequency is decreased. Field excitatory postsynaptic potentials were also suppressed by aconitine, whereas excitatory postsynaptic currents recorded by the patch clamp technique were not influenced by aconitine when cells were held at -60 mV.

[Changing gears in the human circadian mechanism]. / Uber Gangschaltungen im circadianen Getriebe des Menschen.

Subjects who live in isolation and without information on time of day, develop, in all their functions, free-running circadian rhythms whose common periods usually scatter around 25 h. That means that the circadian system, though desynchronized from the 24-h day, remains internally synchronized. However, if the sleep-wake cycle gets longer than 28 h, or shorter than 22 h, the rhythms of autonomic functions uncouple from the sleep-wake cycle, resulting in a state of internal desynchronization. In internally synchronized as well as in desynchronized systems, the intervals between meals change in strong proportionality to the duration of wake time, nevertheless, neither the caloric intake per meal nor body weight are altered. Hence, there must be a saving of energy with increasing wake time. This is accomplished by a reduction of muscular work, as becomes evident from the negative correlation between locomotor activity per hour and wake time. On the occurrence of internal desynchronization, the regression lines describing the correlations mentioned above are shifted against each other in such a way that the consumption of energy adapts to the needs economically. This mechanism is comparable to the effects of a variable geer.

Relative quantification of angiotensin-converting enzyme mRNA in human smooth muscle cells, monocytes, and lymphocytes by the polymerase chain reaction.

A method was developed for relative quantification of angiotensin-converting enzyme (ACE) mRNA in as few as 100 cells. After reverse transcription of total RNA to cDNA, multiplexed polymerase chain reaction with two sets of primers amplified ACE cDNA and that of an internal standard glyceraldehyde phosphate dehydrogenase (GAPDH) simultaneously. By adjusting primer pair concentrations, both ACE and GAPDH were amplified with constant efficiencies. Macrophage-like U937 histiocytic lymphoma cells expressed ACE mRNA constitutively. Freshly isolated human monocytes did not express ACE mRNA initially, but after 4 days in culture had 8% of the amount found in U937 cells. After phorbol ester stimulation, monocytes transcribed ACE at levels comparable to U937 cells. Human smooth muscle cells had ninefold more ACE mRNA than 4-day monocytes, but 30% less than U937 cells. In contrast, a mixed population of lymphocytes was devoid of ACE mRNA.

Angiotensin converting enzyme (kininase II) mRNA production and enzymatic activity in human peripheral blood monocytes are induced by GM-CSF but not by other cytokines.

Peripheral blood monocytes (PBM) do not possess angiotensin converting enzyme (ACE) activity in the inactive state. However, measurable PBM ACE activity is found in patients with certain inflammatory diseases. We have examined the effect of cytokines likely to be present during granulomatous inflammation on the regulation of ACE mRNA in PBM. The presence of ACE mRNA in human PBM cultured in vitro with various cytokines for up to 6 days was analyzed using polymerase chain reaction. PBM not exposed to cytokines did not express ACE mRNA, while incubation of PBM with recombinant human GM-CSF resulted in high levels of ACE mRNA expression after 72 h of cell culture, which persisted through day six. Increased ACE mRNA expression occurred concomitantly with phenotypic changes in cell size and shape consistent with cell activation. A 5-fold increase in ACE enzymatic activity also occurred. Incubation of PBM with all other cytokines tested failed to induce ACE mRNA expression. Alveolar macrophages expressed ACE mRNA immediately following their isolation, but mRNA expression decreased markedly during a 24-h period of incubation and was only partially reversed with exogenous GM-CSF. We conclude that GM-CSF enhances ACE mRNA levels in human PBM, but not in alveolar macrophages.

The timing of defecation within the sleep-wake cycle of humans during temporal isolation.

Data were collected from 14 human subjects who lived singly in an isolation unit without temporal cues. The subjects used buttons to signal the times when they woke up, took a meal, defecated, and retired. Under these conditions, the "free-running" circadian rhythms (e.g., the sleep-wake cycles and the rhythm of body temperature) remained internally synchronized in 7 subjects (mean circadian period = 24.47 hr); in the remaining 7 subjects the sleep-wake cycle lengthened beyond 28 hr, desynchronizing from the rhythm of body temperature (internal desynchronization; mean sleep-wake cycle = 33.45 hr). In all subjects, the interval from wake-up to defecation increased with the duration of wake time (alpha); on average, the interval varied proportionally with alpha. Furthermore, the interval from the last main meal (dinner) to defecation the following day was proportional to the sleep-wake cycle--either that which included dinner but preceded the defecation, or that which followed the dinner but included the defecation. It is concluded that a lengthening of the sleep-wake cycle (and of the wake time) results in a slowing down of the processes of digestion and evacuation of the bowels, in parallel with an apparent reduction of total energy expenditure.

Naps as integral parts of the wake time within the human sleep-wake cycle.

Thirteen subjects lived singly in an isolation unit without temporal cues for an average time span of 32 days. They signaled the times when they woke up, took a meal, and retired, noting in their diaries what kind of sleep (nap or main sleep) they were going to have. In five subjects the free-running circadian rhythms remained internally synchronized. The other eight subjects became internally desynchronized, with different periods in the sleep-wake cycle and in the rhythm of body temperature, or developed a "circa-bi-dian" rhythmicity (i.e., a state of the circadian system in which the temperature rhythm regains synchrony with the sleep-wake cycle in a 2:1 ratio). The duration of naps was positively correlated with the duration of wake time (after subtraction of the nap), as well as with the duration of wakefulness preceding the nap. In contrast, the duration of main sleep was negatively correlated with the preceding wake time. It is concluded that naps are integral parts of the wake time, and that they follow the rules known from intermeal intervals and the perception of long time intervals such as 1 hr.

On the relationship between motor activity and the sleep-wake cycle in humans during temporal isolation.

In the free-running circadian rhythms of 14 human subjects (4 females, 10 males) who lived singly in an isolation unit without temporal clues, locomotor activity was recorded by means of contact plates installed below the carpet in the main room. During sleep, movements in bed were picked up by spring contacts attached to the mattress. In all subjects, the hourly means of locomotion during wakefulness (alpha) were negatively correlated with the duration of alpha to such an extent that the total amount of locomotion per cycle remained constant when alpha varied from 14 to 23 hr. The hourly values of movements in bed were independent of the duration of sleep (rho), so that the total number of movements was almost proportional to rho. The "homeostatic control" of locomotion during wake time is considered as a means to conserve energy when the duration of wakefulness increases.

Estimates on the duration of sleep and wakefulness made in isolation.

Fifteen subjects who lived singly in an isolation unit without temporal cues were asked to note every day after awakening how many hours they thought they had slept, and in the evening before retiring how many hours they had been awake. These estimates of the duration of sleep and wakefulness were compared with the intervals between two signals given by the subjects by pressing a button at the time of waking up and when turning off their bedside reading lamp. The results can be summarized as follows: (a) the daily estimated durations of sleep and wakefulness were positively correlated with the actual durations in all but one subject; (b) sleep and wake times were better estimated in the presence of a light-dark cycle even if the subjects were not entrained by the zeitgeber; (c) for both episodes, there was a consistent trend from an overestimation of relatively short to an underestimation of long durations; (d) with equal durations in the two episodes, sleep was estimated to be shorter than wake time; (e) the most accurate estimates centered around 10.5 h of sleep and 13.5 h of wake time; (f) the sleep and wake times added up to 24 h in subjects who did not deliberately "compensate" for relatively long sleep estimates with a short wake estimate, with the full cycle being adjusted to 24 h.

Hufeland's interest in plant movements.

Christoph Wilhelm Hufeland (1762-1836) was one of the eminent physicians at the time of Goethe. When only 21 years old, he followed his father as a medical practitioner in Weimar. In 1793 he became Professor of Medicine at the University of Jena, from where he moved, in 1801, to Berlin as the physician in ordinary to king Friedrich Wilhelm III, council of state, and Professor at the leading hospital, the Charité. Hufeland pioneered in what today would be called public hygiene. Many of his lectures and publications were addressed to the educated laymen. In his most read book, the 'Makrobiotik', he emphasizes the importance of the 24-h periodicity as a basic unit of biological chronometry. In view of this, Hufeland has become a kind of 'patron saint' to modern chronobiologists.

Activity in anticipation and in succession of a daily meal.

When exposed, in otherwise constant conditions, to a schedule with one single meal per day, rodents anticipate the time of food availability by an increase in locomotor activity while the main circadian rhythm continues to free-run with a period different from 24 h. The anticipatory activity (AA) is considered a component which is uncoupled from the light-entrainable circadian system and controlled by a food-entrainable oscillator. In this report it is shown that, in addition to AA, sometimes a burst or band of activity appears which succeeds the feeding time (SA). AA and SA seem to belong to one another, both being controlled by the same food-entrainable oscillator. The band of activity constituted by the combination of both AA and SA, though temporarily suppressed during and immediately after the meal, follows, as a whole, the rules of entrainment as known from circadian systems.