Differential effects of acute and regular physical exercise on cognition and affect.

The effects of regular exercise versus a single bout of exercise on cognition, anxiety, and mood were systematically examined in healthy, sedentary young adults who were genotyped to determine brain-derived neurotrophic factor (BDNF) allelic status (i.e., Val-Val or Val66Met polymorphism). Participants were evaluated on novel object recognition (NOR) memory and a battery of mental health surveys before and after engaging in either (a) a 4-week exercise program, with exercise on the final test day, (b) a 4-week exercise program, without exercise on the final test day, (c) a single bout of exercise on the final test day, or (d) remaining sedentary between test days. Exercise enhanced object recognition memory and produced a beneficial decrease in perceived stress, but only in participants who exercised for 4 weeks including the final day of testing. In contrast, a single bout of exercise did not affect recognition memory and resulted in increased perceived stress levels. An additional novel finding was that the improvements on the NOR task were observed exclusively in participants who were homozygous for the BDNF Val allele, indicating that altered activity-dependent release of BDNF in Met allele carriers may attenuate the cognitive benefits of exercise. Importantly, exercise-induced changes in cognition were not correlated with changes in mood/anxiety, suggesting that separate neural systems mediate these effects. These data in humans mirror recent data from our group in rodents. Taken together, these current findings provide new insights into the behavioral and neural mechanisms that mediate the effects of physical exercise on memory and mental health in humans.

Human amygdala sensitivity to the pupil size of others.

Stimulation of the amygdala produces pupil dilation in animal and human subjects. The present study examined whether the amygdala is sensitive to variations in the pupil size of others. Male subjects underwent event-related functional magnetic resonance imaging while passively viewing unfamiliar female faces whose pupils were either unaltered (natural variations in large and small pupils) or altered to be larger or smaller than their original size. Results revealed that the right amygdala and left amygdala/substantia innominata were sensitive to the pupil size of others, exhibiting increased activity for faces with relatively large pupils. Upon debrief, no subject reported being aware that the pupils had been manipulated. These results suggest a function for the amygdala in the detection of changes in pupil size, an index of arousal and/or interest on the part of a conspecific, even in the absence of explicit knowledge.

Regulation of daily locomotor activity and sleep by hypothalamic EGF receptor signaling.

The circadian clock in the suprachiasmatic nucleus (SCN) is thought to drive daily rhythms of behavior by secreting factors that act locally within the hypothalamus. In a systematic screen, we identified transforming growth factor-alpha (TGF-alpha) as a likely SCN inhibitor of locomotion. TGF-alpha is expressed rhythmically in the SCN, and when infused into the third ventricle it reversibly inhibited locomotor activity and disrupted circadian sleep-wake cycles. These actions are mediated by epidermal growth factor (EGF) receptors on neurons in the hypothalamic subparaventricular zone. Mice with a hypomorphic EGF receptor mutation exhibited excessive daytime locomotor activity and failed to suppress activity when exposed to light. These results implicate EGF receptor signaling in the daily control of locomotor activity, and identify a neural circuit in the hypothalamus that likely mediates the regulation of behavior both by the SCN and the retina.

Gene integration and expression and extracellular secretion of Erwinia chrysanthemi endoglucanase CelY (celY) and CelZ (celZ) in ethanologenic Klebsiella oxytoca P2.

The development of methods to reduce costs associated with the solubilization of cellulose is essential for the utilization of lignocellulose as a renewable feedstock for fuels and chemicals. One promising approach is the genetic engineering of ethanol-producing microorganisms that also produce cellulase enzymes during fermentation. By starting with an ethanologenic derivative (strain P2) of Klebsiella oxytoca M5A1 with the native ability to metabolize cellobiose, the need for supplemental beta-glucosidase was previously eliminated. In the current study, this approach has been extended by adding genes encoding endoglucanase activities. Genes celY and celZ from Erwinia chrysanthemi have been functionally integrated into the chromosome of P2 using surrogate promoters from Zymomonas mobilis for expression. Both were secreted into the extracellular milieu, producing more than 20,000 endoglucanase units (carboxymethyl cellulase activity) per liter of fermentation broth. During the fermentation of crystalline cellulose with low levels of commercial cellulases of fungal origin, these new strains produced up to 22% more ethanol than unmodified P2. Most of the beneficial contribution was attributed to CelY rather than to CelZ. These results suggest that fungal enzymes with substrate profiles resembling CelY (preference for long-chain polymers and lack of activity on soluble cello-oligosaccharides of two to five glucosyl residues) may be limiting in commercial cellulase preparations.

Retinopathy and attenuated circadian entrainment in Crx-deficient mice.

Crx, an Otx-like homeobox gene, is expressed specifically in the photoreceptors of the retina and the pinealocytes of the pineal gland. Crx has been proposed to have a role in the regulation of photoreceptor-specific genes in the eye and of pineal-specific genes in the pineal gland. Mutations in human CRX are associated with the retinal diseases, cone-rod dystrophy-2 (adCRD2; refs 3, 4, 5), retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), which all lead to loss of vision. We generated mice carrying a targeted disruption of Crx. Crx-/- mice do not elaborate photoreceptor outer segments and lacked rod and cone activity as assayed by electroretinogram (ERG). Expression of several photoreceptor- and pineal-specific genes was reduced in Crx mutants. Circadian entrainment was also affected in Crx-/- mice.

Biosynthetic burden and plasmid burden limit expression of chromosomally integrated heterologous genes (pdc, adhB) in Escherichia coli.

Previous studies have shown an unexpectedly high nutrient requirement for efficient ethanol production by ethanologenic recombinants of Escherichia coli B such as LY01 which contain chromosomally integrated Zymomonas mobilis genes (pdc,adhB) encoding the ethanol pathway. The basis for this requirement has been identified as a media-dependent effect on the expression of the Z. mobilis genes rather than a nutritional limitation. Ethanol production was substantially increased without additional nutrients simply by increasing the level of pyruvate decarboxylase activity. This was accomplished by adding a multicopy plasmid containing pdc alone (but not adhB alone) to strain LY01, and by adding multicopy plasmids which express pdc and adhB from strong promoters. New strong promoters were isolated from random fragments of Z. mobilis DNA and characterized but were not used to construct integrated biocatalysts. These promoters contained regions resembling recognition sites for 3 different E. coli sigma factors: sigma(70), sigma(38), and sigma(28). The most effective plasmid-based promoters for fermentation were recognized by multiple sigma factors, expressed both pdc and adhB at high levels, and produced ethanol efficiently while allowing up to 80% reduction in complex nutrients as compared to LY01. The ability to utilize multiple sigma factors may be advantageous to maintain the high levels of PDC and ADH needed for efficient ethanol production throughout batch fermentation. From this work, we propose that the activation of biosynthetic genes in nutrient-poor media creates a biosynthetic burden that reduces the expression of chromosomal pdc and adhB by competing for transcriptional and translational machinery. This reduced expression can be viewed as analogous to the effect of plasmids (plasmid burden) on the expression of native chromosomal genes.

Free-running circadian period does not shorten with age in female Syrian hamsters.

It has been reported that the free-running period of circadian rhythms shortens with age in mammals, including humans, and this shortening has been suggested to be the underlying cause of early morning awakening and difficulty maintaining sleep in older people. A recent study found that the free-running period of male hamsters does not change with age. The present study extends those findings to female hamsters. We studied the locomotor activity rhythm of 22 female hamsters kept in constant conditions from early adulthood until their death, and compared their data to those from male hamsters. We found no shortening of free-running period with age in the female hamsters, and no difference in free-running period between females and males. In contrast, mean activity level and amount of time per cycle spent running declined with age in females and males. These findings demonstrate that the free-running period in hamsters does not systematically shorten with age, and suggest that alternative explanations for the observed age-related advance of sleep-wake times in humans should be explored.

Enhancement of expression and apparent secretion of Erwinia chrysanthemi endoglucanase (encoded by celZ) in Escherichia coli B.

Escherichia coli B has been engineered as a biocatalyst for the conversion of lignocellulose into ethanol. Previous research has demonstrated that derivatives of E. coli B can produce high levels of Erwinia chrysanthemi endoglucanase (encoded by celZ) as a periplasmic product and that this enzyme can function with commercial fungal cellulase to increase ethanol production. In this study, we have demonstrated two methods that improve celZ expression in E. coli B. Initially, with a low-copy-number vector, two E. coli glycolytic gene promoters (gap and eno) were tested and found to be less effective than the original celZ promoter. By screening 18,000 random fragments of Zymomonas mobilis DNA, a surrogate promoter was identified which increased celZ expression up to sixfold. With this promoter, large polar inclusion bodies were clearly evident in the periplasmic space. Sequencing revealed that the most active surrogate promoter is derived from five Sau3A1 fragments, one of which was previously sequenced in Z. mobilis. Visual inspection indicated that this DNA fragment contains at least five putative promoter regions, two of which were confirmed by primer extension analysis. Addition of the out genes from E. chrysanthemi EC16 caused a further increase in the production of active enzyme and facilitated secretion or release of over half of the activity into the extracellular environment. With the most active construct, of a total of 13,000 IU of active enzyme per liter of culture, 7,800 IU was in the supernatant. The total active endoglucanase was estimated to represent 4 to 6% of cellular protein.

Transient entrainment of a circadian pacemaker during development by dopaminergic activation in Syrian hamsters.

Maternal cues entrain a circadian pacemaker in fetal Syrian hamsters. These cues may act through dopaminergic activation of the fetal suprachiasmatic nucleus (SCN); injection of the dopamine D1 agonist SKF38393 to pregnant hamsters entrains activity rhythms of their pups and induces expression of c-fos in the fetal SCN. The aim of this study was to examine the ability of SKF38393 to entrain neonatal Syrian hamsters and to determine the age at which this effect is lost. SKF38393 injections given to two groups of pups at opposite times of day on postnatal days (PN) 1-5 entrained the pups' activity rhythms to average phases that differed by 9.25 h. SKF38393 failed to establish different average phases when given on PN 6-10. Injection of SKF38393 on PN 1, but not PN 6, induced expression of Fos. These results demonstrate that dopaminergic activation is a potent entraining stimulus in neonatal hamsters and that its entraining effects, as well as its ability to induce Fos, are lost by PN 6. The phase established by dopaminergic activation was approximately opposite to that previously shown to be established by melatonin injections. Dopaminergic activation and melatonin may mimic separate but complementary maternal entraining signals which represent day and night.

Stability of circadian timing with age in Syrian hamsters.

The causes of age-related disruptions in the timing of human sleep and wakefulness are not known but may include changes in both the homeostatic and circadian regulation of sleep. In Syrian hamsters the free running period of the circadian activity/rest rhythm has been reported to shorten with age. Although this has been observed under a variety of experimental conditions, the changes have been small and their consistency uncertain. In the present study, the wheel running activity/rest rhythm was continuously measured in male Syrian hamsters (Mesocricetus auratus) in dim constant light (<1 lx) from 8 wk of age until death. Fifteen hamsters survived to at least 90 wk (28%). The average free running period of these hamsters did not change with age. In 18 hamsters that died between 50 and 88 wk, free running period also did not change before death. In contrast to free running period, other measures related to activity level changed significantly with age and before death. Despite changes in the expression of the activity/rest rhythm, the free running period of the hamster circadian pacemaker remained remarkably stable with age.

Melatonin entrains the restored circadian activity rhythms of syrian hamsters bearing fetal suprachiasmatic nucleus grafts.

A circadian pacemaker consists of at least three essential features: the ability to generate circadian oscillations, an output signal, and the ability to be entrained by external signals. In rodents, ablation of the suprachiasmatic nucleus (SCN) results in the loss of circadian rhythms in activity. Rhythmicity can be restored by transplanting fetal SCN into the brain of the lesioned animal, demonstrating the first two of the essential pacemaker features within the grafts. External signals, such as the light/dark cycle, have not, however, been shown to entrain the restored rhythms. Melatonin injections are an effective entraining stimulus in fetal and neonatal Syrian hamsters of the same developmental ages used to provide donor tissue for transplantation. Therefore, melatonin was used to test the hypothesis that SCN grafts contain an entrainable pacemaker. Daily injections of melatonin were given to SCN-lesioned hosts beginning on the day after transplantation of fetal SCN. Two groups that received melatonin at different times of day 12 hr apart each showed significantly clustered phases but with average phases that differed by 8.67 hr. Thus melatonin was able to entrain the restored circadian activity rhythms. In contrast to these initial injections, injections given 6 weeks after transplantation were unable to entrain or phase shift the rhythms. The results demonstrate that SCN grafts contain an entrainable circadian pacemaker. In addition, the results also indicate that the fetal SCN is directly sensitive to melatonin and, as with intact hamsters, sensitivity to melatonin is lost during SCN development.

Role of the CLOCK protein in the mammalian circadian mechanism.

The mouse Clock gene encodes a bHLH-PAS protein that regulates circadian rhythms and is related to transcription factors that act as heterodimers. Potential partners of CLOCK were isolated in a two-hybrid screen, and one, BMAL1, was coexpressed with CLOCK and PER1 at known circadian clock sites in brain and retina. CLOCK-BMAL1 heterodimers activated transcription from E-box elements, a type of transcription factor-binding site, found adjacent to the mouse per1 gene and from an identical E-box known to be important for per gene expression in Drosophila. Mutant CLOCK from the dominant-negative Clock allele and BMAL1 formed heterodimers that bound DNA but failed to activate transcription. Thus, CLOCK-BMAL1 heterodimers appear to drive the positive component of per transcriptional oscillations, which are thought to underlie circadian rhythmicity.

A screen for genes induced in the suprachiasmatic nucleus by light.

The mechanism by which mammalian circadian clocks are entrained to light-dark cycles is unknown. The clock that drives behavioral rhythms is located in the suprachiasmatic nucleus (SCN) of the brain, and entrainment is thought to require induction of genes in the SCN by light. A complementary DNA subtraction method based on genomic representational difference analysis was developed to identify such genes without making assumptions about their nature. Four clones corresponded to genes induced specifically in the SCN by light, all of which showed gating of induction by the circadian clock. Among these genes are c-fos and nur77, two of the five early-response genes known to be induced in the SCN by light, and egr-3, a zinc finger transcription factor not previously identified in the SCN. In contrast to known examples, egr-3 induction by light is restricted to the ventral SCN, a structure implicated in entrainment.

Melatonin: role in development.

Melatonin is the mammalian fetus's window to periodicity of the outside world. Through melatonin, the fetus "knows" what time of year it is and, in all likelihood, also knows the time of day. The best known function of melatonin during development is to communicate information about photoperiod and thereby adaptively regulate reproductive development. A second likely function of melatonin during development, which may be related to but more widespread than the first, is to entrain the developing circadian pacemaker. Prenatal maternal entrainment occurs in all of the eutherian mammals in which it has been examined, and in Syrian hamsters exogenous melatonin during development causes entrainment. The broader distribution and greater abundance of melatonin receptors during development, relative to mature animals, suggests that developmental effects of melatonin are greater and more diverse. The human fetal suprachiasmatic nucleus expresses melatonin binding sites and is therefore likely to be affected by both endogenous and exogenous melatonin with consequences for the prenatal and postnatal expression and entrainment of circadian rhythms. Caution is warranted, not only concerning the use of exogenous melatonin during pregnancy and lactation but also concerning behavior that might disrupt the mother's endogenous melatonin rhythm.

Single prenatal injections of melatonin or the D1-dopamine receptor agonist SKF 38393 to pregnant hamsters sets the offsprings' circadian rhythms to phases 180 degrees apart.

Pregnant Syrian hamsters with lesions of the suprachiasmatic nucleus (SCN) received single injections of melatonin or the D1-dopamine receptor agonist, SKF 38393 on day 15 of gestation (1 day before birth). Pups were weaned on postnatal day 20 and their freerunning activity rhythms recorded for 3-4 weeks. The pups' phases on the day of weaning were significantly clustered in both of the treatment groups, but the average phases differed by approximately 180 degrees. The results demonstrate that a single prenatal stimulus is sufficient to set the phases of the hamsters' rhythms and that the phase established depends on the stimulus. Both c-fos mRNA and Fos protein were expressed in the fetal SCN after SKF 38393 injection but neither were expressed after melatonin injection. Simulations showed that a single stimulus could produce the observed synchrony from a population of uniformally distributed phases if the phase shifts were three to four times the magnitude of the adult hamster light phase response curve (PRC). A light pulse PRC mimicked the effect of an SKF 38393 injection and a dark-pulse PRC mimicked the effects of a melatonin injection. Together these results suggest that dopamine and melatonin either are, or mimic, maternal entraining signals that represent day and night.

Cloning of cellobiose phosphoenolpyruvate-dependent phosphotransferase genes: functional expression in recombinant Escherichia coli and identification of a putative binding region for disaccharides.

Genomic libraries from nine cellobiose-metabolizing bacteria were screened for cellobiose utilization. Positive clones were recovered from six libraries, all of which encode phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) proteins. Clones from Bacillus subtilis, Butyrivibrio fibrisolvens, and Klebsiella oxytoca allowed the growth of recombinant Escherichia coli in cellobiose-M9 minimal medium. The K. oxytoca clone, pLOI1906, exhibited an unusually broad substrate range (cellobiose, arbutin, salicin, and methylumbelliferyl derivatives of glucose, cellobiose, mannose, and xylose) and was sequenced. The insert in this plasmid encoded the carboxy-terminal region of a putative regulatory protein, cellobiose permease (single polypeptide), and phospho-beta-glucosidase, which appear to form an operon (casRAB). Subclones allowed both casA and casB to be expressed independently, as evidenced by in vitro complementation. An analysis of the translated sequences from the EIIC domains of cellobiose, aryl-beta-glucoside, and other disaccharide permeases allowed the identification of a 50-amino-acid conserved region. A disaccharide consensus sequence is proposed for the most conserved segment (13 amino acids), which may represent part of the EIIC active site for binding and phosphorylation.

The effect of transplanting one or two suprachiasmatic nuclei on the period of the restored rhythm.

A fundamental property of circadian rhythms is the free-running period expressed by organisms when isolated from environmental periodicity. The physiological determinants of the free-running period, including variation among and within individuals and among species, are not known. The circadian rhythms of mammals are regulated by a circadian pacemaker within the suprachiasmatic nucleus (SCN) of the hypothalamus. To examine possible determinants of the free-running period, one or two SCNs were transplanted into hamsters that had their own SCNs ablated. Wheel-running behavior was measured to estimate the free-running period of restored rhythmicity. Hosts received grafts containing either the left or right SCN from a single fetus or both SCNs from a single fetus. In some cases, both the left and right SCNs from a single fetus restored rhythmicity in different hosts, demonstrating that each of the right and left SCN alone is a competent circadian pacemaker. The average free-running period of the restored rhythms was significantly longer in hamsters that received both of the SCNs from a single fetus. The sizes of grafts were estimated using immunoreactivity for vasoactive intestinal polypeptide as a marker of SCN tissue. Grafts never grew to be larger than an intact SCN, and a graft only 6.5% the size of the combined left and right intact SCNs restored rhythmicity. The average volume of grafted SCN in hamsters that received two SCNs was larger than that in hamsters that received a single SCN. The results demonstrate that SCN graft volume and/or the number of SCNs that comprise the graft influence the free-running period.

The effect of melatonin on cleavage rate of C57BL/6 and CBA/Ca preimplantation embryos cultured in vitro.

There is growing interest in using melatonin as a therapeutic agent for the treatment of a variety of medical conditions, including cancer, heart disease, glaucoma, stress, jet lag, and sleep disorders. In addition, melatonin is being evaluated in a clinical trial to test its efficacy as an oral contraceptive. In order to test any possible adverse effects of melatonin on preimplantation embryos, we used the mouse as a model system. Two strains of mice, a Ped fast, melatonin-deficient strain, C57BL/6, and a Ped slow strain previously found to have detectable melatonin levels at nighttime, CBA/Ca, were studied. Two cell embryos were incubated with melatonin concentrations from 10(-5) M to 10(-13) M for 48 or 72 hours and the number of cells per embryo assessed quantitatively at the end of the incubation period. We used sufficiently high levels of melatonin to mimic the pharmacological concentration used in the oral contraceptive. It was found that there was no effect of melatonin on embryos from either mouse strain at any of the concentrations tested. Our results suggest that if conception occurs while melatonin is being administered to treat a range of conditions, it would not adversely affect the embryo.

Entrainment of Syrian hamster circadian activity rhythms by neonatal melatonin injections.

The circadian rhythms of fetal and neonatal rodents are entrained by their mother. This entrainment is dependent upon the maternal suprachiasmatic nucleus (SCN), but the mechanism of entrainment is unknown. Administration of the pineal hormone melatonin to pregnant, SCN-lesioned female Syrian hamsters entrains the activity rhythms of their hamster pups. The aim of this study was to determine whether melatonin injected directly in neonatal Syrian hamsters is able to entrain circadian rhythms and, if so, for how long this effect persists during development. Injection of melatonin in two groups of hamster pups at opposite phases on postnatal days 1-5 entrained the onset of activity rhythms on the day of weaning to two phases 10.67 h apart. Melatonin injection did not entrain activity rhythms to opposite phases on either postnatal days 6-10 or 21-25. Vehicle injection did not entrain animals to opposite phases at any of the ages studied. These results demonstrate that melatonin is able to act directly on the neonate to cause entrainment and that this effect disappears after postnatal day 6.

Suprachiasmatic nucleus grafts restore circadian function in aged hamsters.

The regulation of circadian rhythms changes with age. In humans, changes in the timing of sleep and wakefulness are especially common. In Syrian hamsters Mesocricetus auratus the free running period of the activity/rest rhythm shortens with age. The present study tested the hypothesis that critical age-related changes occur within the hypothalamic suprachiasmatic nucleus (SCN), known to contain a circadian pacemaker. Fetal SCN were transplanted into the brains of younger (20 weeks) and older (81 weeks) hamsters which had had their own SCNs ablated. The restoration of rhythmicity and the free running period of the rhythmicity were determined from continuous records of wheel-running activity. Transplantation restored rhythmicity in hosts of both ages. In older hamsters, the mean free running period after transplantation was longer than that measured before SCN ablation, but a similar lengthening of period was not observed after transplantation to younger hamsters. In addition, the mean period after transplantation was the same for both younger and older hosts even when there was a difference between the groups before SCN ablation. When the grafts were allowed to age, the mean free running period of the restored rhythms became shorter, indicating that the grafts can also undergo age-related changes. The results indicate that age-related changes specifically in the SCN are responsible for an age-related change in free running period.