Size-related variation in protein abundance in the brain and abdominal tissue of bumble bee workers.

Female bumble bee workers of the same species often show a profound body size variation that is linked to a division of labour. Large individuals are more likely to forage whereas small individuals are more likely to perform in-nest activities. A higher sensory sensitivity, stronger circadian rhythms as well as better learning and memory performances appear to better equip large individuals for outdoor activities compared to their smaller siblings. The molecular mechanisms underlying worker functional polymorphism remain unclear. Proteins are major determinants of an individual's morphology and behaviour. We hypothesized that the abundance of proteins such as metabolic enzymes as well as proteins involved in neuronal functions would differ with body size and provide insights into the mechanisms underlying size-dependent physiological specialization in bumble bee workers. We conducted protein quantification measurements based on liquid chromatography coupled with tandem mass spectrometry on tissue samples derived from small and large Bombus impatiens and Bombus terrestris workers. Proteins found to differ significantly in abundance between small and large workers belong to the categories of structure, energy metabolism and stress response. These findings provide the first proteomic insight into mechanisms associated with size-based division of labour in social insects.

Raalin, a transcript enriched in the honey bee brain, is a remnant of genomic rearrangement in Hymenoptera.

We identified a predicted compact cysteine-rich sequence in the honey bee genome that we called 'Raalin'. Raalin transcripts are enriched in the brain of adult honey bee workers and drones, with only minimum expression in other tissues or in pre-adult stages. Open-reading frame (ORF) homologues of Raalin were identified in the transcriptomes of fruit flies, mosquitoes and moths. The Raalin-like gene from Drosophila melanogaster encodes for a short secreted protein that is maximally expressed in the adult brain with negligible expression in other tissues or pre-imaginal stages. Raalin-like sequences have also been found in the recently sequenced genomes of six ant species, but not in the jewel wasp Nasonia vitripennis. As in the honey bee, the Raalin-like sequences of ants do not have an ORF. A comparison of the genome region containing Raalin in the genomes of bees, ants and the wasp provides evolutionary support for an extensive genome rearrangement in this sequence. Our analyses identify a new family of ancient cysteine-rich short sequences in insects in which insertions and genome rearrangements may have disrupted this locus in the branch leading to the Hymenoptera. The regulated expression of this transcript suggests that it has a brain-specific function.

The influence of body mass index, age and gender on current illness: a cross-sectional study.

CONTEXT: Obesity poses a significant health risk, but health risk is not equivalent to actual health status. Further, age and gender might alter the effect of body weight on physical health. OBJECTIVE: To determine the relationship between body mass index (BMI), age, gender and current health status. DESIGN: Data from the 1988-1994, 2003-2004 and 2005-2006 National Health & Nutrition Examination Surveys were weighted to represent the US population. BMI, age, gender and current medication use were analyzed in a sample-adjusted 9071 women and 8880 men. MAIN OUTCOME MEASURES: The percentage of participants taking medication and the total number of medications taken. RESULTS: In both the 1988-1994 and 2003-2006 data sets, with few exceptions, medication loads did not increase significantly in overweight compared with normal-weight people. Medication loads increased significantly in obese compared with normal-weight people aged 40+, but only marginally at 25-39 years. Medication loads were higher in women than men, but significantly less so in people aged 55-70. CONCLUSIONS: First, medication loads, a measure of current health status, were increased in obese compared with the normal-weight people, but the effect was mainly at ages over 40 years. In addition, BMI category contributed less to medication loads at ages 25-39 than in older groups. Second, there was little difference in current health status in normal-weight versus overweight people at all ages. Finally, higher medication loads in women than men are more apparent in younger than older people. Although obesity does not substantially affect current health in young people, it is likely that the increased medication loads in obese compared with normal-weight older people originates at least in part from an increased BMI starting at a younger age. Thus, age, gender and onset of high BMI all require consideration when using BMI to assess current health status.

Antiandrogen implanted in brain stimulates male reproductive system.

Chronic intrahypothalanic implantation of cyproterone, an antiandrogen, in male rats resulted in specific stimulation of testes, seminal vesicles, and prostates. Implantation of cholesterol-filled or empty tubes in the median eminence in controls was ineffective. We conclude that decreases in the amount of testosterone reaching specific receptors in the median eminence or in nearby regions activate a mechanism that produces increased gonadotropin secretion.

Monitoring circadian rhythms of individual honey bees in a social environment reveals social influences on postembryonic ontogeny of activity rhythms.

Social factors constitute an important component of the environment of many animals and have a profound influence on their physiology and behavior. Studies of social influences on circadian rhythms have been hampered by a methodological trade-off: automatic data acquisition systems obtain high-quality data but are effective only for individually isolated animals and therefore compromise by requiring a context that may not be sociobiologically relevant. Human observers can monitor animal activity in complex social environments but are limited in the resolution and quality of data that can be gathered. The authors developed and validated a method for prolonged, automatic, high-quality monitoring of focal honey bees in a relatively complex social environment and with minimal illumination. The method can be adapted for studies on other animals. The authors show that the system provides a reliable estimation of the actual path of a focal bee, only rarely misses its location for > 1 min, and removes most nonspecific signals from the background. Using this system, the authors provide the first evidence of social influence on the ontogeny of activity rhythms. Young bees that were housed with old foragers show ~24-h rhythms in locomotor activity at a younger age and with stronger rhythms than bees housed with a similar number of young bees. By contrast, the maturation of the hypopharyngeal glands was slower in bees housed with foragers, similar to findings in previous studies. The morphology and function of the hypopharyngeal glands vary along with age-based division of labor. Therefore, these findings indicate that social inhibition of task-related maturation was effective in the experimental setup. This study suggests that although the ontogeny of circadian rhythms is typically correlated with the age-based division of labor, their social regulation is different.

Neuronal circadian clock protein oscillations are similar in behaviourally rhythmic forager honeybees and in arrhythmic nurses.

Internal clocks driving rhythms of about a day (circadian) are ubiquitous in animals, allowing them to anticipate environmental changes. Genetic or environmental disturbances to circadian clocks or the rhythms they produce are commonly associated with illness, compromised performance or reduced survival. Nevertheless, some animals including Arctic mammals, open sea fish and social insects such as honeybees are active around-the-clock with no apparent ill effects. The mechanisms allowing this remarkable natural plasticity are unknown. We generated and validated a new and specific antibody against the clock protein PERIOD of the honeybee Apis mellifera (amPER) and used it to characterize the circadian network in the honeybee brain. We found many similarities to Drosophila melanogaster and other insects, suggesting common anatomical organization principles in the insect clock that have not been appreciated before. Time course analyses revealed strong daily oscillations in amPER levels in foragers, which show circadian rhythms, and also in nurses that do not, although the latter have attenuated oscillations in brain mRNA clock gene levels. The oscillations in nurses show that activity can be uncoupled from the circadian network and support the hypothesis that a ticking circadian clock is essential even in around-the-clock active animals in a constant physical environment.

Behavioral rhythmicity, age, division of labor and period expression in the honey bee brain.

Young adult honey bees work inside the beehive "nursing" brood around the clock with no circadian rhythms; older bees forage for nectar and pollen outside with strong circadian rhythms. Previous research has shown that the development of an endogenous rhythm of activity is also seen in the laboratory in a constant environment. Newly emerging bees maintained in isolation are typically arrhythmic during the first few days of adult life and develop strong circadian rhythms by about a few days of age. In addition, average daily levels of period (per) mRNA in the brain are higher in foragers or forager-age bees (> 21 days of age) relative to young nest bees (approximately 7 days of age). The authors used social manipulations to uncouple behavioral rhythmicity, age, and task to determine the relationship between these factors and per. There was no obligate link between average daily levels of per brain mRNA and either behavioral rhythmicity or age. There also were no differences in per brain mRNA levels between nurse bees and foragers in social environments that promote precocious or reversed behavioral development. Nurses and other hive-age bees can have high or low levels of per mRNA levels in the brain, depending on the social environment, while foragers and foraging-age bees always have high levels. These findings suggest a link between honey bee foraging behavior and per up-regulation. Results also suggest task-related differences in the amplitude of per mRNA oscillation in the brain, with foragers having larger diurnal fluctuation in per than nurses, regardless of age. Taken together, these results suggest that social factors may exert potent influences on the regulation of clock genes.

Prepubertal testosterone treatment of neonatally gonadectomized male rats: defeminization and masculinization of behavioral and endocrine function in adulthood.

Testosterone (T) administered well after the neonatal "critical" period to females at a dose approximating male levels permanently defeminizes reproductive function (see companion publication). To obtain comparable data for the male, neonatally gonadectomized (NeoGx) males received T filled or empty Silastic capsules during days 15-30 of age and were studied in adulthood. Compared to controls, the T treatment resulted in reduced lordosis and proceptive behaviors, increased mounting and intromission behaviors without differences in penile reflexes or size, and reduced plasma FSH and LH surges. Twenty of twenty-three sham-NeoGx males, but only one NeoGx male, showed ejaculatory behavior despite equivalence in penile reflexes and size after detaching a frenulum when present on the penis. These results show that T can still act on neural substrates well beyond the neonatal period to defeminize and masculinize endocrine and behavioral function in the male rat. A comparison with effects in females indicates a sex difference, the male appearing to be more sensitive to these actions of T.

Prepubertal testosterone treatment of female rats: defeminization of behavioral and endocrine function in adulthood.

This study assessed the capacity of testosterone (T) administered well after the neonatal "critical" period to permanently sexually differentiate reproductive function. Females received T filled or empty Silastic capsules during days 15-30 of age and vaginal cyclicity, ovarian weight and appearance, lordosis and proceptive behaviors, mounting behavior, and the gonadotropin response to estrogen and progesterone were measured in adulthood. T-treated females (plasma levels of 0.66 ng T/ml) showed constant vaginal estrus from the day of vaginal opening and small, polyfollicular ovaries. Proceptive behaviors were dramatically reduced whether or not the ovaries were present after day 15 of age, but lordosis behavior was not affected. Exposure to T for 5-6 h was ineffective. Compared to controls, T-treated females had dramatically reduced plasma FSH and LH surges. No effects were observed on mounting behavior, phallus size, or body weights. These results suggest that androgen at approximately male levels can act on neural substrates well beyond the neonatal period to permanently defeminize endocrine and behavioral function in the female rat.

Early N-acetylaspartate depletion is a marker of neuronal dysfunction in rats and primates chronically treated with the mitochondrial toxin 3-nitropropionic acid.

N-acetylaspartate (NAA) quantification by 1H-magnetic resonance spectroscopy has been commonly used to assess in vivo neuronal loss in neurodegenerative disorders. Here. the authors used ex vivo and in vivo 1H-magnetic resonance spectroscopy in rat and primate models of progressive striatal degeneration induced by the mitochondrial toxin 3-nitropropionate (3NP) to determine whether early NAA depletions could also be associated with neuronal dysfunction. In rats that were treated for 3 days with 3NP and had motor symptoms, the authors found a significant decrease in NAA concentrations, specifically restricted to the striatum. No cell loss or dying cells were found at this stage in these animals. After 5 days of 3NP treatment, a further decrease in striatal NAA concentrations was observed in association with the occurrence of dying neurons in the dorsolateral striatum. In 3NP-treated primates, a similar striatal-selective and early decrease in NAA concentrations was observed after only a few weeks of neurotoxic treatment, without any sign of ongoing cell death. This early decrease in striatal NAA was partially reversed after 4 weeks of 3NP withdrawal. These results demonstrate that early NAA depletions reflect a reversible state of neuronal dysfunction preceding cell degeneration and suggest that in vivo quantification of NAA 1H-magnetic resonance spectroscopy may become a valuable tool for assessing early neuronal dysfunction and the effects of potential neuroprotective therapies in neurodegenerative disorders.

Sequence-dependence of the conformational changes induced by the 5-methyl cytosine in synthetic RNA oligomers.

The RNA hexamer containing a 5-methyl cytosine (m5C) r(CGUAm5CG) was studied by 1H and 31P NMR at 500 MHz and 121 MHz, respectively. In contrast to r(CGm5CGCG) which exhibits an atypic duplex structure [(1987) J. Am. Chem. Soc. 109, 2539-2541], r(CGUAm5CG) adopts a classical A-type conformation. This result demonstrates that the influence of the m5C on the conformation of RNA hexamers is sequence-dependent.

Estrogen/progesterone treatment in adulthood affects the size of several components of the medial preoptic area in the male rat.

The results of preliminary studies suggested that steroid and/or propylthiouracil (PTU) treatment of adult gonadectomized (Gxd) male rats significantly reduced the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA). Therefore, we designed a study to examine this effect in detail. Groups of adult rats were sham Gxd (intact) or Gxd, then treated with multiple injections of oil (males and females), or estrogen and progesterone (males). Gonadectomized estrogen/progesterone-treated males had a significantly smaller SDN-POA volume, smaller volume of the medial division of the medial preoptic nucleus (MPNm), smaller volume of the anteroventral MPNm (MPNav), and larger volume of the anteroventral periventricular nucleus (AVPv). The volume of the central division of the medial preoptic nucleus (MPNc) or of the suprachiasmatic nucleus was not affected. There were no differences between Gxd estrogen/progesterone-treated males vs the group that received PTU as well, indicating that the PTU treatment was unnecessary. The reduced volume of the SDN-POA was due to a reduced volume of the MPNav and of the portion of the SDN-POA located within the MPNm-exclusive of the MPNav and MPNc. In conclusion, estrogen/progesterone treatment in adulthood caused significant changes in the volume of several medial preoptic structures in two separate groups of Gxd males. Because the steroids produced no significant effects in intact males, testicular hormones appear to "protect" these structures from the effects of the estrogen/progesterone treatment.

Cytoarchitectonic analysis of the SDN-POA of the intact and gonadectomized rat.

The densely staining group of cells referred to as the sexually dimorphic nucleus of the preoptic area (SDN-POA) is greater in volume in the male than in the female rat. Because we and others have reported absolute volumes that have been consistent within individual studies but that vary considerably, we characterized the SDN-POA by describing its morphology with respect to the cytoarchitectonic divisions of the medial preoptic nucleus (MPN) in intact and gonadectomized rats. We report three major findings: the SDN-POA is heterogeneous and is composed of cells belonging to three distinct cytoarchitectonic divisions; the cytoarchitecture of the MPN and its medial and lateral divisions (MPNm and MPNl, respectively) in male rats appear to be influenced by the hormonal status in adulthood; and a small anteroventral division of the MPN (MPNav) is present in males but virtually absent in females. Specifically, the SDN-POA is located within the MPNm, but consists of subcomponents located within the central division of the MPN (MPNc), the MPNav, and part of the MPNm-exclusive of the MPNc and MPNav. The percentage of the total SDN-POA located within the MPNc and MPNav. The percentage of the total SDN-POA located within the MPNc and MPNav was greater in males, and that in the MPNm-exclusive of the MPNc and MPNav was greater in females, indicating that the SDN-POA has a different cytoarchitectonic composition in the two sexes. Gonadectomy produced no significant differences in SDN-POA volume, but the MPN, MPNl, and MPNm were significantly reduced in gonadectomized versus intact males, suggesting an activational effect of testicular hormones on these structures.

Regulation of queen-worker conflict in bumble-bee (Bombus terrestris) colonies

In annual colonies of bumble-bees overt queen-worker conflict is limited to a distinct 'competition phase' (CPh). In unmanipulated Bombus terrestris colonies, the queen's switch to male production (the 'switch point', SP) accounted for only-22% of the variation in the onset of the CPh. In some colonies, the CPh even began before the SP. The CPh was more strongly correlated with the transition in queen production (r=0.79). Replacing the queen eggs with male eggs or doubling the number of workers in young colonies resulted in a significantly earlier onset of the CPh and a significantly earlier transition to queen production. Replacing queen eggs with female eggs did not have this effect. These manipulations did not affect the timing of the queen's switch from female to male production. These findings show that the mechanism underlying the queen-worker conflict in insect societies is more complex than previously appreciated. The onset of queen-worker conflict cannot be attributed simply to a single factor such as the queen's switch to male production or a decrease in queen inhibition. Rather, multiple cues are important.

Diagnosis of muscular glycogenosis by in vivo natural abundance 13C NMR spectroscopy.

Natural abundance 13C NMR (nuclear magnetic resonance) spectroscopy was used to distinguish patients suffering from muscle glycogenosis type V (McArdle's disease) from normal subjects by measuring their muscle glycogen content at rest. Proton-decoupled 13C spectra were obtained in 10-15 min from calf muscles at rest. The ratio of the glycogen/creatine signal areas was 12.9 +/- 1.7 in four McArdle's disease patients and 2.0 +/- 0.7 in seven normal subjects. This technique thus allows the non-invasive diagnosis of muscle glycogenosis.

period expression in the honey bee brain is developmentally regulated and not affected by light, flight experience, or colony type.

Changes in circadian rhythms of behavior are related to age-based division of labor in honey bee colonies. The expression of the clock gene period (per) in the bee brain is associated with age-related changes in circadian rhythms of behavior, but previous efforts to firmly associate per brain expression with division of labor or age have produced variable results. We explored whether this variability was due to differences in light and flight experience, which vary with division of labor, or differences in colony environment, which are known to affect honey bee behavioral development. Our results support the hypothesis that per mRNA expression in the bee brain is developmentally regulated. One-day-old bees had the lowest levels of expression and rarely showed evidence of diurnal fluctuation, while foragers and forager-age bees (> 21 days of age) always had high levels of brain per and strong and consistent diurnal patterns. Results from laboratory and field experiments do not support the hypothesis that light, flight experience, and colony type influence per expression. Our results suggest that the rate of developmental elevation in per expression is influenced by factors other than the ones studied in our experiments, and that young bees are more sensitive to these factors than foragers.

Preconditioning with potassium channel openers. A new concept for enhancing cardioplegic protection?

Ischemic preconditioning defines an adaptive endogenous mechanism in which a brief episode of reversible ischemia renders the heart more resistant to a subsequent period of sustained ischemia. Because the cardioprotective effects of ischemic preconditioning might be mediated by an activation of adenosine triphosphate-sensitive potassium channels, this study was designed to assess whether these effects could be duplicated by the preischemic administration of a potassium channel opener. Fifty isolated isovolumic buffer-perfused rat hearts underwent 45 minutes of normothermic potassium arrest followed by 1 hour of reperfusion. They were divided into five equal groups that differed with regard to the preconditioning regimen: Group 1 hearts were left untreated and served a controls; in group 2, preconditioning was achieved with 5 minutes of total global ischemia followed by 5 minutes of buffer reperfusion before cardioplegic arrest; in group 3, the preconditioning stimulus consisted of a 5-minute infusion of the potassium channel opener nicorandil (10 mumol/L) followed by 5 minutes of drug-free buffer perfusion before arrest; group 4 hearts underwent a similar protocol except that the infusion of nicorandil was preceded by that of the potassium channel blocker glibenclamide (10 mumol/L); group 5 hearts were ischemically preconditioned like those of group 2 except that the no-flow preconditioning period was also preceded by a 5-minute infusion of glibenclamide (50 mumol/L). The results demonstrate that ischemic preconditioning significantly improved contractility and reduced contracture during reperfusion, as compared with results in control hearts. These protective effects were duplicated by pretreatment with nicorandil but were abolished when the drug was antagonized by a prior infusion of glibenclamide. Likewise, the glibenclamide-induced blockade of potassium channels largely blunted the beneficial effects of ischemic preconditioning. These data suggest that opening of adenosine triphosphate-sensitive potassium channels substantially contributes to preconditioning-induced cardiac protection in a surgically relevant model of global ischemia and, consequently, that the use of potassium channel openers like nicorandil could be an effective means of enhancing cardioplegic protection.

Improved recovery of heart transplants with a specific kit of preservation solutions.

In the course of cardiac transplantation, donor hearts undergo a four-step sequence of events (arrest, cold storage, global ischemia during implantation, and reperfusion) during which myocardial damage can occur. We tested the hypothesis that the functional recovery of these hearts could be improved by exposure to two interdependently formulated preservation solutions throughout this four-step sequence. Solution I was used as a perfusion and storage medium during the first three steps, and solution II served as a modified reperfusate. The two solutions share the following principles of formulation: prevention of cell swelling (high concentrations of mannitol, a myocardium-specific impermeant) calcium overload (ionic manipulations), and oxidative damage (reduced glutathione) and enhancement of anaerobic energy production (glutamate). The two solutions differ with respect to the calcium content and buffering capacity. One hundred rat hearts perfused with isolated isovolumic buffer were subjected to cardioplegic arrest; cold (2 degrees C) storage for 5 hours, global ischemia at 15 degrees C for 1 hour, and normothermic reperfusion for 1 additional hour. In a first series of experiments (70 hearts), our kit of solutions was compared with six clinical preservation regimens that involved cardiac arrest with St. Thomas' Hospital or University of Wisconsin solutions followed by storage of the hearts in saline, Euro-Collins, St. Thomas' Hospital, or University of Wisconsin solutions. In a second series of experiments (30 hearts), the effects of the kit were more specifically investigated in relation to two types of additive--oncotic agents (dextran) and thiol-based antioxidants (reduced glutathione and N-acetyl-L-cysteine). According to comparisons of maximal rate of ventricular pressure increase and left ventricular compliance after reperfusion, the best myocardial protection was afforded by our kit of solutions. The addition of dextran during storage did not provide additional protection. Conversely, the omission of reduced glutathione was clearly detrimental; the replacement of reduced glutathione with N-acetyl-L-cysteine failed to improve recovery beyond that provided by antioxidant-free solutions, thereby suggesting the importance, in this model, of an anti-free radical compound that, like reduced glutathione, is operative extracellularly. We conclude that the preservation of heart transplants can be improved with the sequential use of two closely interrelated solutions, the formulations of which integrate the basic principles of organ preservation with those of myocardium-specific metabolism.

Aortoseptal approach for optimal resection of diffuse subvalvular aortic stenosis.

The results of conventional operative resection of diffuse subaortic stenosis (tunnel subaortic stenosis and diffuse idiopathic hypertrophic subaortic stenosis) have been less than satisfactory. A new approach using the concept of aortoventriculoplasty was designed to allow adequate surgical resection of a diffuse subvalvular aortic stenosis. The aorta, the right ventricle, and the septum are incised in the same way as during aortoventriculoplasty , with the aortic anulus being divided carefully across the commissure between the left and right aortic cusps. The septotomy is extended beyond the limits of the stenosis, and fibrous and/or muscular tissue is removed from each edge of the septal incision. After adequate widening of the subvalvular area, the various incisions are closed and the aortic valve is reconstructed. This aortoseptal approach was studied experimentally in the dog and then carried out on two patients, both of whom had excellent hemodynamic and functional results. The aortoseptal approach may be the procedure of choice in the treatment of diffuse stenoses limited to the subvalvular area, whereas other procedures ( aortoventriculoplasty , and apico-aortic valved conduit) should be used when the valvular and/or supravalvular levels are involved.

Surgical management of acute dissections involving the ascending aorta. Early and late results in 38 patients.

Thirty-eight patients (32 men and six women, mean age 48.1 years) were operated upon for acute dissection involving the ascending aorta. The surgical procedure included multiple peripheral arterial cannulations, resection of the initial intimal tear if found (35 cases), and obliteration of the false channel by double cuffing with Teflon of the two layers of the dissecting process proximally and distally. When present (29 cases), aortic regurgitation was usually (21 cases) managed by conservative remodeling of the aortic anulus; 34 prosthetic replacements of the ascending aorta and four replacements of the arch were achieved. The operative mortality was 7.9% (3138) and the overall hospital mortality was 23.7% (9138). Nonfatal complications occurred in 11 patients (29%). There were three late deaths (10.3%). Mean follow-up was 3.4 years (2 months to 8 years, 8 months). Twenty-three (88.5%) of the 26 patients were asymptomatic. Contrast tomodensitometry was performed in 14 patients; in type II (two patients), the aorta was normal; in type I (12 patients), residual abnormalities were noted: patency of the false channel (10 cases), aneurysmal dilatation (seven cases), and reduction of the true lumen by the false channel (four cases). These results emphasize the need for scrupulous long-term follow-up in surgically treated aortic dissections.