Stimulation of Ca(2+)-dependent exocytosis of the sperm acrosome by cAMP acting downstream of phospholipase A2

Spermatozoa undergo exocytosis in response to agonists that induce Ca2+ influx and, in turn, activation of phosphoinositidase C, phospholipase C, phospholipase A2, and cAMP formation. Since the role of cAMP downstream of Ca2+ influx is unknown, this study investigated whether cAMP modulates phospholipase C or phospholipase A2 using a ram sperm model stimulated with A23187 and Ca2+. Exposure to dibutyryl-cAMP, phosphodiesterase inhibitors or forskolin resulted in enhancement of exocytosis. However, the effect was not due to stimulation of phospholipase C or phospholipase A2: in spermatozoa prelabelled with [3H]palmitic acid or [14C]arachidonic acid, these reagents did not enhance [3H]diacylglycerol formation or [14C]arachidonic acid release. Spermatozoa were treated with the phospholipase A2 inhibitor aristolochic acid, and dibutyryl-cAMP to test whether cAMP acts downstream of phospholipase A2. Under these conditions, exocytosis did not occur in response to A23187 and Ca2+. However, inclusion of dibutyryl-cAMP and the phospholipase A2 metabolite lysophosphatidylcholine did result in exocytosis (at an extent similar to that seen when cells were treated with A23187/Ca2+ and without the inhibitor). Inclusion of lysophosphatidylcholine alone, without dibutyryl-cAMP, enhanced exocytosis to a lesser extent, demonstrating that cAMP requires a phospholipase A2 metabolite to stimulate the final stages of exocytosis. These results indicate that cAMP may act downstream of phospholipase A2, exerting a regulatory role in the exocytosis triggered by physiological agonists.     

Development of a specific fluorescent phage endolysin for in situ detection of Clostridium species associated with cheese spoilage

Late blowing defect (LBD) is a major cause of spoilage in cheeses, caused by the growth of Clostridium spp. in the cheese matrix. We investigated the application of CTP1L, a bacteriophage endolysin active against Clostridium tyrobutyricum, and its enzymatically active and cell wall‐binding domains (EAD and CBD) attached to green fluorescent protein (GFP) to detect dairy‐related Clostridium species by fluorescence microscopy. GFP‐CTP1L and GFP‐CBD demonstrated specificity for Clostridium spp. by labelling 15 and 17 of 20 Clostridium strains, respectively, but neither bound to other members of the cheese microbiota. However, GFP‐EAD did not label any Clostridium strain tested. Unexpectedly, GFP‐CTP1L and GFP‐CBD were also able to bind to clostridial spores. In addition, GFP‐CBD allowed us to visualize the vegetative cells of C. tyrobutyricum directly in the matrix of a LBD cheese. Site‐directed mutants of GFP‐CTP1L and GFP‐CBD were made to examine the amino acids involved in binding and oligomer formation. Oligomerization was not essential for binding, but specific mutations in the CBD which affected oligomer formation also affected binding and lytic activity. We conclude that GFP‐CTP1L and GFP‐CBD could be good biomarkers for rapid detection of Clostridium spores in milk, so measures can be taken for the prevention of LBD in cheese, and also provide effective tools to study the development of Clostridium populations during cheese ripening.     

Diurnal Urinary 6‐Sulfatoxymelatonin Levels among Healthy Danish Nurses during Work and Leisure Time

The present study aims to examine the influence of evening and night shift work, compared to day shift work, on melatonin secretion in nurses in a field setting. Effects were examined during a workday and during a day off. Both fixed schedules and mixed or rotating schedules were studied. In total, 170 nurses were studied: 89 nurses worked fixed schedules, 27 nurses worked the day shift, 12 nurses worked the evening shift, 50 nurses worked the night shift, and 82 nurses worked mixed schedules, with data collected during a day (n=17), evening (n=14), or night shift (n=50). All spot urine samples were collected during 24 h from the participants on a work day and on a day off and were analyzed for 6‐sulphatoxymelatonin. On the day of urine sampling, participants filled in the Karolinska Sleep Diary. Additional information was collected through a telephone interview. Data were analyzed using a mixed procedure with autoregressive covariance structure. The present study showed that shift work affected the concentrations of 6‐sulphatoxymelatonin in the short term by lower excretion in urine from nurses working the night compared to day shift on a workday and on a day off as well. No significant differences were observed between a workday and a day off when doing day and evening shifts, irrespective of mixed and fixed schedules. Sleep length was reduced workdays (from 6.1–6.8 h) among all nurses, compared to days off (from 7.8–8.7 h).     

Building‐Site Camps and Extended Work Hours: A Two‐Week Monitoring of Self‐Reported Physical Exertion,Fatigue, and Daytime Sleepiness

Large‐scale construction work often requires people to work longer daily hours and more than the ordinary five days in a row. In order to minimize transportation times and optimize the use of personnel, workers are sometimes asked to live in temporary building‐site camps in the proximity of the work site. However, little is known about the biological and psychological effects of this experience. The objective of the present study was to investigate whether exposure to long work hours and extended workweeks while living in building‐site camps in between work shifts was associated with a build‐up of increased complaints of poor sleep, daytime sleepiness, physical exertion, and fatigue across a two‐week work cycle. Two groups of construction workers were examined. The camp group of 13 participants (mean age: 42±11 S.D. yrs) lived in building‐site camps and worked extended hours (between 07:00 and 18:00 h) and extended workweeks (six days in a row, one day off, five days in a row, nine days off). The home group of 16 participants (mean age 40±9 yrs) worked ordinary hours between 07:00 and 15:00 h and returned home after each workday. Self‐ratings of daytime sleepiness (Karolinska Sleepiness Scale), physical exertion (Borg CR‐10), and mood were obtained six or seven times daily during two workweeks. Fatigue ratings were obtained once daily in the evening, and ratings of sleep disturbances were obtained once daily in the morning with the Karolinska Sleep Diary. Data were evaluated in a repeated measures design. The results showed that both groups reported a similar level of daytime sleepiness, physical exertion, and mood across workdays and time points within a workday (all three‐way interactions had p>0.898). Although the home group reported earlier wake‐up times, the pattern of sleep disturbance ratings across the workdays did not differ between the groups. Both groups reported few sleep disturbances and good mood. However, the camp group reported higher physical exertion already at the start of work and showed a more gentle increase in ratings during the work shift and a smaller decline between the end of work and bedtime. The camp group also reported higher fatigue scores than the home group. However, none of the groups showed signs of increasing ratings in the progress of the two workweeks. For both groups, the ratings of daytime sleepiness formed a U‐shaped pattern, with the highest scores at awakening and at bedtime. Yet, the camp group reported higher daytime sleepiness than the home group at lunch break and at the second break in the afternoon. In conclusion, there were no signs of fatigue build‐up or accumulation of daytime sleepiness, physical exertion, or sleep disturbances in either group. Despite the fact that the camp group showed some signs of having trouble in recuperating in between work shifts, as indicated by the higher physical exertion ratings at the start of work, higher fatigue scores, and higher daytime sleepiness, the results constitute no real foundation for altering the camp group's current work schedule and living arrangements.     

Using quantum dots to visualize clathrin associations

Our current understanding of clathrin-mediated endocytosis proposes that the process is initiated at a specialized anatomical structure called a coated pit. Electron microscopy has been required for elucidation of the morphology of coated pits and the vesicles produced therein, and the presence of a bristle coat has been taken as suggestive of clathrin surrounding these vesicles. More recently, immunocytochemical methods have confirmed that endocytic vesicles are surrounded by clathrin and its adaptor proteins, but there is a need to identify precisely and to follow the fate of the cellular organelles seen by fluorescence microscopy. We used quantum immune-electron microscopy to localize clathrin in a human adrenal cortical cell line (SW-13). Clathrin was shown to associate with a variety of vesicle types including the classic clathrin-coated vesicles and pits used in receptor internalization, pentilaminar annular gap junction vesicles, and multivesicular bodies. The images obtained with quantum dot technology allow accurate and specific localization of clathrin and the clathrin adaptor protein, AP-2, with cellular organelles and suggest that some of the structures classified as typical coated vesicles by immunocytochemical light microscopic techniques actually may be membrane bound pits.     

Evidence of structural balance in spatial ecological networks

Despite recent advances in applying networks to study ecological systems, most of the network datasets are built attending only to a single type of interaction between nodes, which can be an oversimplification. In the present work, we built ecological networks that had positive and negative links for multiple plant communities based on the local spatial association between species. Then, we evaluated whether those networks were in balance, a hypothesis commonly formulated for real signed graphs but never tested in systems other than social networks. Specifically, we quantified the global and the local structural balance in the networks. We found that plant community networks were more balanced than expected by chance, and that this pattern was due to a large number of balanced triads to the detriment of unbalanced ones. Furthermore, this pattern was consistent among all of the types of the plant communities examined, which suggests that configurations that promote structural balance might be common in ecological signed networks. We also found that almost all networks had some unbalanced components, which might be responsible for the adaptation of the system. Mechanisms behind these structure and possible applications for community ecology are discussed. Our results encourage testing structural balance in other ecological networks to confirm if it is a widespread architecture of natural systems.     

Geographic variation in cranial morphology of narwhals (Monodon monoceros) from Greenland and the eastern Canadian Arctic

We analyzed variation in nine non-metric and eight metric variables in the skulls of 132 narwhals (Monodon monoceros) from five localities in Greenland (Inglefield Bredning, Melville Bay, Uummannaq, Disko Bay, and Scoresby Sound) and one in the eastern Canadian Arctic (Eclipse Sound). Metric variables were used to compare the combined Disko Bay and Uummannaq samples with the samples from Inglefield Bredning and Scoresby Sound using three different multivariate techniques for each sex. None of the results were significant. Seven of the non-metric variables were independent of age and sex and were used in comparing samples from the six localities. No differences were found among the four localities in West Greenland, but differences were found in two of the non-metric variables between the combined West Greenland sample and the one from Scoresby Sound. A major shortcoming of the analysis based on metric data was the small sample size from several of the areas, which resulted in low statistical power. Genetic as well as environmental factors could explain the differences detected here between narwhals living along the west and the east coasts of Greenland.     

Application of a novel method for age estimation of a baleen whale and a porpoise

Eyeballs from 121 fin whales (Balaenoptera physalus) and 83 harbor porpoises (Phocoena phocoena) were used for age estimation using the aspartic acid racemization (AAR) technique. The racemization rate (k_Asp) for fin whales was established from 15 fetuses (age 0) and 15 adult whales where age was estimated by reading growth layer groups (GLGs) in the earplugs. The (k_Asp) for harbor porpoises was derived from 15 porpoises (two calves and 13 > 1 yr old) age‐estimated by counting GLGs in the teeth and two calves classified to age based on length. The (k_Asp) values were estimated by regression of GLGs against D/L ratios. For the fin whales an (k_Asp) of 1.15 × 10^?3/yr (SE ± 0.00005) and a D/L ratio at birth [(D/L)₀] of 0.028 (SE ± 0.0012) were estimated, which is in agreement with rates for other mysticeti. For the harbor porpoises a (k_Asp) of 3.10 × 10^?3/yr (SE ± 0.0004) and a (D/L)₀ value of 0.023 (SE ± 0.0018) were estimated, which is considerably higher than found for other cetaceans. Correlation between chosen age estimates from AAR and GLG counts indicated that AAR might be an alternative method for estimating age in marine mammals.     

Effects of UVB radiation on marine phytoplankton communities

The impact of enhanced and reduced UVB radiation (UVBR) on pelagic ecosystems was studied during two mesocosm experiments in May and June/July 1994. The ambient UVBR exposure was either reduced with mylar foil or artificially enhanced with UVB fluorescent tubes. Developments in the phytoplankton communities were followed during 11 and 8 day periods using several structural and functional parameters. In the May experiment, enhanced UVBR significantly stimulated carbon dioxide fixation, activity of alkaline phosphatase and content of fatty acids. In the June-July experiment, the effects induced by changed UVBR were smaller with some indications of decreased algal biomass with enhanced UVBR. Several of the measured parameters indicated that the two experiments represented different stages in the plankton community development. In the May experiment, the community was in a development stage, moving from nutrient-replete to nutrient-depleted conditions, while the community in June/July was depleted of nutrients from the start. The stimulating effects of UVBR in May are suggested to be the secondary effects of a photochemically induced breakdown of dissolved organic matter, resulting in an increase in available nutrients.