Time of the sidereal year affects responsiveness to the phase-resetting effects of photoperiod in the ewe

Two groups of ovary-intact ewes were placed in separate photochambers on the day of the vernal equinox (VE). One group was exposed to a 16 h light:8 h dark (16L:8D) photoperiod and the other to 8L:16D. On the day of the summer solstice (SS) and at 90-91-day intervals thereafter [autumnal equinox (AE), winter solstice (WS), VE and SS], each group was changed to the opposite photoperiod. The latent period between each change and either onset or cessation of cycles, as determined by measuring blood progesterone concentrations, was recorded. The latent period between change to 8L:16D and onset of cycles was shortest after the exposure at AE and longest after exposure at WS (P less than 0.001). The latent period after AE was shorter (P less than 0.001) than after VE. The correlations were small between ambient temperature and interval to onset of cycles. The latent period to cessation of cycles in response to 16L:8D was shorter after SS exposure than after WS exposure (P less than 0.01), but other differences were not significant. There was a strong (r = -0.94, P less than 0.05) negative correlation between interval to cessation of cycles and ambient temperature. Cessation of cycles in response to 16L:8D occurred more rapidly (P less than 0.001) than onset in response to 8L:16D. These results show that responsiveness to the inductive effects of photoperiod varies significantly with time of the sidereal year.     

Synthesis of high mobility group proteins in regenerating rat liver.

Incorporation of [3H]lysine into the non-histone chromosomal proteins HMG1, HMG2, and HMG17 and into each of the five major classes of histones was measured in rat liver at various times after partial hepatectomy. Histone synthesis was closely coupled temporally to that of DNA, although a small amount of histone was shown to be produced before DNA replication began. In contrast, the incorporation curves for the high mobility group (HMG) proteins showed little correlation with that for DNA. At 4 h after partial hepatectomy, protein synthesis had virtually ceased. Thereafter, the rates of synthesis of the HMG proteins rose steadily so that by 12 h, well before the onset of DNA replication they had reached about two-thirds of the maximum rates attained during the first cell division cycle. Histones had only reached about one-sixth of their maximum rates at this time. The lack of coupling betweeen the synthesis of the HMG proteins and DNA was confirmed by experiments with inhibitors of DNA replication. Reduction of DNA synthesis to less than 10% of the uninhibited rate had little or no effect on incorporation into the HMG proteins, whereas, under similar conditions, the rate of synthesis of histones was reduced by more than 50%.     

Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics*

Microglia, the resident immune cells of the brain, have been shown to display a complex spectrum of roles that span from neurotrophic to neurotoxic depending on their activation status. Microglia can be classified into four stages of activation, M1, which most closely matches the classical (pro-inflammatory) activation stage, and the alternative activation stages M2a, M2b, and M2c. The alternative activation stages have not yet been comprehensively analyzed through unbiased, global-scale protein expression profiling. In this study, BV2 mouse immortalized microglial cells were stimulated with agonists specific for each of the four stages and total protein expression for 4644 protein groups was quantified using SILAC-based proteomic analysis. After validating induction of the various stages through a targeted cytokine assay and Western blotting of activation states, the data revealed novel insights into the similarities and differences between the various states. The data identify several protein groups whose expression in the anti-inflammatory, pro-healing activation states are altered presumably to curtail inflammatory activation through differential protein expression, in the M2a state including CD74, LYN, SQST1, TLR2, and CD14. The differential expression of these proteins promotes healing, limits phagocytosis, and limits activation of reactive nitrogen species through toll-like receptor cascades. The M2c state appears to center around the down-regulation of a key member in the formation of actin-rich phagosomes, SLP-76. In addition, the proteomic data identified a novel activation marker, DAB2, which is involved in clathrin-mediated endocytosis and is significantly different between M2a and either M1 or M2b states. Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells.Microglia, along with astrocytes, form the backbone of the immune response in the brain. Microglia, in particular, comprise 10–15% of the brain, varying by region and predominating in areas of the midbrain such as the hippocampus and substantia nigra (1). Separated from the systemic immune system by the blood-brain barrier, the brain''s immune response relies on the ability of microglia to act as a multifaceted immune cell; microglia are able to sense pathogens, toxins, injury, and cytokine levels, as well as respond in a neurotrophic or neurotoxic manner similar to the macrophage in the systemic immune system (2).Microglia can respond to insult and injury in a neurotoxic manner (3, 4) where activated microglia are able to induce pro-inflammatory cytokines to recruit other microglia and astrocytes in response to bacterial infection and produce a wide and varied array of factors including reactive oxygen species (ROS)¹, and reactive nitrogen species (RNS), cytokines and lipid mediators as well as remove cellular debris as a post-infection response through phagocytosis (5). As such, microglia protect themselves from their own toxic products through a series of antioxidant proteins regulated through the actions of nuclear factor, erythroid 2-like 2 protein (NFE2L2) (6). Microglia have been implicated in a growing number of CNS-associated diseases; classically activated microglia have been found in brain regions afflicted with Parkinson''s disease, Alzheimer''s disease, and AIDS-related dementia (7–9). Microglial activation has also been reported to play a role in brain injury because of chronic alcohol exposure (10–13).Raivich et al. described microglia response and phases as a linear set of stages that microglia pass through in response to injury, pathogens, or antibodies from the systemic immune system that have crossed the blood-brain barrier (14). The first stage is a quiescent resting state, followed by an alert stage characterized by increased expression of integrin-binding proteins, or cell adhesion molecules, such as CD11b. The homing stage of activation that follows is characterized by increased cell mobility and adhesion as microglia target sites of injury or invasion. The fourth stage is a phagocytic stage that is often termed the classical microglia response, characterized by production of neurotoxic factors such as ROS through a cell membrane-bound NADPH oxidase complex and RNS through the action of inducible nitric oxide synthase, iNOS, as well as phagocytosis of cellular debris. The final stage, known as the bystander activation stage, potentiates the microglia response by activating additional microglia through the production and release of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and interleukin-6 (IL-6).Our understanding of the role of microglia has broadened in recent years to include neurotrophic as well as neurotoxic features (15, 16). The presence of activated microglia does not always correlate to an inflammatory state in the local brain region, implying a noninflammatory or possibly neurotrophic role for these microglia. Microglia that display multiple activation states have been observed in the brains of Alzheimer''s patients (17). It has been suggested that microglia that enter an inflammatory neurotoxic state first change into a neurotrophic healing response prior to returning to their quiescent resting phase (1). As such, a new schema to describe microglia phenotype was required. M1 phase, which can be triggered in vivo and in vitro by lipopolysaccharide (LPS) and inflammatory cytokines, has been established to describe classically activated microglial cells that are similar to those found in the fourth and fifth stages of Raivich''s microglial hierarchy. Microglia do not return to a resting state without first receiving anti-inflammatory triggers that are released by other microglia. These additional stages have been classified as alternative activation and have multiple healing responses. Microglia can be induced into the first alternative activation stage, M2a, through treatment with interleukin-4 (IL-4), and/or interleukin-13 (IL-13). M2a is a healing phase typified by tissue repair and growth stimulation through the actions of various extracellular matrix factors. Most importantly, M2a microglia act as an anti-inflammatory counterpart to M1 phase microglia by competing for arginine, a nitrogen pool for the production of RNS during M1 phase; M2a phase microglia compete for this pool through the production of arginase-1 (ARG1) which converts arginine into ornithine (18). M2b phase is a mixed activation state that responds to viral infection and activated antibodies characterized by the production of the pro-inflammatory cytokines, TNFα and IL-6, in addition to reduction of IL-12 and increased production of IL-10 (19). M2b phase microglia can be reproduced, in vitro, by treating with IL-1β and LPS concurrently or activated IgA complexes, which bind to Fcγ receptors. M2c phase microglia can be induced through IL-10 exposure in vivo and in vitro, and the emergence of M2c microglia shuts down microglial immune response.In order to study microglia in a laboratory setting, enriched ex vivo microglia, primary microglia, or immortalized cell lines are required. BV2 immortalized mouse microglia have been described as producing 41% of the cytokines and chemokines produced by ex vivo cells as compared with 96% coverage by primary microglia. However, Wilcock et al. showed that BV2 cells were successful at producing the classical activators for all four microglia activation stages as measured by real-time polymerase chain reaction (17). In addition, proteomic analysis of pathway level changes may be able to smooth over the lack of full expression through high levels of accurate protein quantification.Because of their importance in immune response and possible role in multiple disease states, a thorough investigation of the differential proteomic expression in the various microglial activation states is required. Using SILAC-labeled immortalized BV2 microglial cells treated with activators of the various activation stages, a proteome profile that includes the major canonical microglial pathways across all four activation states, providing crucial information as to where in these pathways of various states diverge, was established. In addition, using the differential protein expression data, a novel marker of microglia activation, DAB2, was identified and confirmed in primary mouse microglia through Western blot analysis. The abundance of this protein, as well as other differentially expressed proteins identified in this study, may prove as novel indicators in differentiating and categorizing activated microglia in the brain.     

Using partial genomic fosmid libraries for sequencing complete organellar genomes

Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However; for some organisms, it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. A minimum tiling path of clones comprising the entire genome sequence of each plastid was selected, and these clones were shotgun-sequenced and assembled into complete genomes. Although this method worked well for both heterotrophic and autotrophic plants, nuclear genome size had a dramatic effect on the proportion of screened clones containing plastid DNA and, consequently, the overall number of clones that must be screened to ensure full plastid genome coverage. This technique makes it possible to determine complete plastid genome sequences for organisms that defy other available organellar genome sequencing methods, especially those for which limited amounts of tissue are available.     

Melatonin implantation during spring and summer does not affect the seasonal rhythm of feeding in anadromous Arctic charr (Salvelinus alpinus)

Plasma melatonin levels in the high-latitude teleost Arctic charr (Salvelinus alpinus) are constantly low during summer when feeding activity is high, and high during the dark winter when they eat little and loose weight. The question arises if melatonin is involved in the phase-setting of annual rhythms of feeding and growth and if low summer melatonin production is permissive for high summer growth in this species. The present study was therefore set out to compare the seasonal appetite and growth rhythms in Arctic charr with constantly high plasma melatonin levels from February throughout the Arctic summer (melatonin implanted, average mid-day plasma melatonin levels 1,106 ± 147 pg/ml) with those of fish with natural plasma melatonin levels (vehicle implanted and untreated fish with average mid-day plasma melatonin levels of 94 ± 13 and 58 ± 6 pg/ml, respectively). Feed intake, body mass or body length, as well as the timing of the seasonal growth rhythm, were not affected by the high summer plasma melatonin level. Further, Arctic charr fasted for 3 months had a 24 h plasma profile of melatonin which was consistently higher throughout the scotophase compared to fed charr. Although the daily melatonin production seems to be affected by the energy status of the fish, melatonin does not seem to be directly involved in regulation of the seasonal feeding and growth rhythm in the high-latitude, anadromous Arctic charr.     

Ovarian stimulation of squirrel monkeys (Saimiri boliviensis boliviensis) using pregnant mare serum gonadotropin

The application of assisted reproductive technologies (ART) to nonhuman primates has created opportunities for improving reproductive management in breeding colonies, and for creation of new animal models by genetic modification. One impediment to the application of ART in Saimiri spp. has been the lack of an effective gonadotropin preparation for ovarian stimulation. Pregnant mare serum gonadotropin (PMSG) is inexpensive and readily available, but its repeated use in rhesus monkeys has been associated with induction of a refractory state. We have compared PMSG to recombinant human follicle stimulating hormone (rhFSH) for controlled ovarian stimulation in Bolivian squirrel monkeys. Groups of mature squirrel monkeys received rhFSH (75 IU daily) or PMSG (250 IU twice daily) by subcutaneous injection for 4 d during the breeding season (November to January) or nonbreeding season (March to September). Serum estradiol (E2) was measured daily. Follicular growth was monitored by abdominal ultrasound. During the breeding season, PMSG induced a higher E2 response than did rhFSH, with mean E2 levels being significantly higher within 3 d of stimulation. Superior follicular development in PMSG animals was confirmed by abdominal ultrasonography. During the nonbreeding season, PMSG elicited a similar increase in serum E2 levels despite the fact that basal serum E2 is typically low during the nonbreeding season. Repeated use of PMSG (< or = 3 cycles of administration) produced no attenuation of the E2 response. We conclude that PMSG is highly effective for repeated cycles of controlled ovulation stimulation in the squirrel monkey.