Use of nucleofection to efficiently transfect primary rabbit lacrimal gland acinar cells

Lacrimal gland acinar cells are an important cell type to study due to their role in production and release of tear proteins, a function essential for ocular surface integrity and normal visual acuity. However, mechanistic studies are often limited by problems with transfection using either plasmid DNA or siRNA. Although various gene delivery methods are available, many have been unproductive due to consistently low transfection efficiencies. We have developed a method using nucleofection that can result in 50% transfection efficiency and 60% knockdown efficiency for plasmid DNA and siRNA, respectively. These results are vastly improved relative to previous studies, demonstrating that nucleofection offers an efficient transfection technique for primary lacrimal gland acinar cells.     

Reduction of diffusion barriers in isolated rat islets improves survival, but not insulin secretion or transplantation outcome

For people with type 1 diabetes and severe hypoglycemic unawareness, islet transplants offer hope for improving the quality of life. However, islet cell death occurs quickly during or after transplantation, requiring large quantities of islets per transplant. The purpose of this study was to determine whether poor function demonstrated in large islets was a result of diffusion barriers and if removing those barriers could improve function and transplantation outcomes. Islets were isolated from male DA rats and measured for cell viability, islet survival, glucose diffusion and insulin secretion. Modeling of diffusion barriers was completed using dynamic partial differential equations for a sphere. Core cell death occurred in 100% of the large islets (diameter >150 μm), resulting in poor survival within 7 days after isolation. In contrast, small islets (diameter <100 μm) exhibited good survival rates in culture (91%). Glucose diffusion into islets was tracked with 2-NBDG; 4.2 μm/min in small islets and 2.8 μm/min in large islets. 2-NBDG never permeated to the core cells of islets larger than 150 μm diameter. Reducing the diffusion barrier in large islets improved their immediate and long-term viability in culture. However, reduction of the diffusion barrier in large islets failed to improve their inferior in vitro insulin secretion compared to small islets, and did not return glucose control to diabetic animals following transplantation. Thus, diffusion barriers lead to low viability and poor survival for large islets, but are not solely responsible for the inferior insulin secretion or poor transplantation outcomes of large versus small islets.     

Early cytokinin response proteins and phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome profiling

Cytokinins are plant hormones involved in regulation of diverse developmental and physiological processes in plants whose molecular mechanisms of action are being intensely researched. However, most rapid responses to cytokinin signals at the proteomic and phosphoproteomic levels are unknown. Early cytokinin responses were investigated through proteome-wide expression profiling based on image and mass spectrometric analysis of two-dimensionally separated proteins and phosphoproteins. The effects of 15 min treatments of 7-day-old Arabidopsis thaliana seedlings with four main cytokinins representing hydroxyisopentenyl, isopentenyl, aromatic, and urea-derived type cytokinins were compared to help elucidate their common and specific function(s) in regulating plant development. In proteome and phosphoproteome maps, significant differences were reproducibly observed for 53 and 31 protein spots, respectively. In these spots, 96 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), providing a snapshot of early links in cytokinin-regulated signalling circuits and cellular processes, including light signalling and photosynthesis, nitrogen metabolism, the CLAVATA pathway, and protein and gene expression regulation, in accordance with previously described cytokinin functions. Furthermore, they indicate novel links between temperature and cytokinin signalling, and an involvement of calcium ions in cytokinin signalling. Most of the differentially regulated proteins and phosphoproteins are located in chloroplasts, suggesting an as yet uncharacterized direct signalling chain responsible for cytokinin action in chloroplasts. Finally, first insights into the degree of specificity of cytokinin receptors on phosphoproteomic effects were obtained from analyses of cytokinin action in a set of cytokinin receptor double mutants.     

Seasonal influence on reproduction in chimpanzees of gombe national park

Although wild chimpanzees are not seasonal breeders, there are seasonal effects on several aspects of chimpanzee reproduction. I examined the seasonal incidence of anogenital swelling in cyclic, pregnant, and acyclic female chimpanzees in Gombe National Park, May 1975–April 1992, and surveyed important reproductive events to determine whether there is a seasonal effect. I analyzed data by season (wet vs. dry) and seasonal quarter;early dry season = May–July;late dry = August–October;early wet = November–January;late wet = February–April. When data for the 17 years are combined, the percentage of females in each reproductive state remains consistent throughout the year. In a given month, 30–35% of subjects were in the cyclic category, 11–15% were pregnant, and 54–61% were acyclic. Cyclic females showed full swelling more often during the late dry season. Pregnant females exhibited anogenital swelling more often during the late dry and early wet seasons. Acyclic females also exhibited a seasonal effect with more anogenital swelling during the late dry season. There is no seasonal difference in frequency of live births (dry, 20;wet, 23). However, the timing of conception showed a seasonal effect (dry, 32;wet, 16). Consistent with earlier reports, the onset of postpartum cycles is highly seasonal;30 occurred during dry season, 9 during wet season. The occurrence of first full swellings for young females is also concentrated in the late dry season. It appears that the dry season is a time of great change for Gombe chimpanzee reproductive physiology. Previous studies indicated that seasonal changes in food availability play a role in increasing group size during the dry season and social contact between females can enhance cyclicity. Accordingly, I suggest that seasonal changes in diet may play a role, either directly (food content) or indirectly (social contact), to alter reproductive physiology.     

Sphingolipids in inflammation: roles and implications

Sphingolipids, historically described as potential reservoirs for bioactive lipids, presently define a new family of cellular mediators, joining the well-established glycerolipid-derived mediators of signal transduction such as diacylglycerol, phosphatidylinositides, and eicosanoids. Sphingolipid metabolism is clearly involved in the regulation of cell growth, differentiation, and programmed cell death. Indeed, a majority of the greater than four thousand studies conducted on sphingolipids during the past five years were investigations of the role of sphingolipids as cellular bioregulators. Studies spanning more than a decade have shown multiple interactions and intersections of the sphingolipid-mediated pathways and the eicosanoid pathway. This review will discuss the emerging mechanisms by which sphingolipids induce inflammatory responses via the eicosanoid pathway in addition to linking previous literature on sphingolipids and inflammation with newer findings of distinct roles for sphingosine-1-phosphate in regulating cyclooygenase-2 and ceramide-1-phosphate in the regulation of cytosolic phospholipase A2alpha. Finally, the relationship between bioactive sphingolipids and inflammation is discussed.     

Ancestral and derived protein import pathways in the mitochondrion of Reclinomonas americana

The evolution of mitochondria from ancestral bacteria required that new protein transport machinery be established. Recent controversy over the evolution of these new molecular machines hinges on the degree to which ancestral bacterial transporters contributed during the establishment of the new protein import pathway. Reclinomonas americana is a unicellular eukaryote with the most gene-rich mitochondrial genome known, and the large collection of membrane proteins encoded on the mitochondrial genome of R. americana includes a bacterial-type SecY protein transporter. Analysis of expressed sequence tags shows R. americana also has components of a mitochondrial protein translocase or "translocase in the inner mitochondrial membrane complex." Along with several other membrane proteins encoded on the mitochondrial genome Cox11, an assembly factor for cytochrome c oxidase retains sequence features suggesting that it is assembled by the SecY complex in R. americana. Despite this, protein import studies show that the RaCox11 protein is suited for import into mitochondria and functional complementation if the gene is transferred into the nucleus of yeast. Reclinomonas americana provides direct evidence that bacterial protein transport pathways were retained, alongside the evolving mitochondrial protein import machinery, shedding new light on the process of mitochondrial evolution.     

Flexible mate choice when mates are rare and time is short

Female mate choice is much more dynamic than we once thought. Mating decisions depend on both intrinsic and extrinsic factors, and these two may interact with one another. In this study, we investigate how responses to the social mating environment (extrinsic) change as individuals age (intrinsic). We first conducted a field survey to examine the extent of natural variation in mate availability in a population of threespine sticklebacks. We then manipulated the sex ratio in the laboratory to determine the impact of variation in mate availability on sexual signaling, competition, and mating decisions that are made throughout life. Field surveys revealed within season heterogeneity in mate availability across breeding sites, providing evidence for the variation necessary for the evolution of plastic preferences. In our laboratory study, males from both female‐biased and male‐biased treatments invested most in sexual signaling late in life, although they competed most early in life. Females became more responsive to courtship over time, and those experiencing female‐biased, but not male‐biased sex ratios, relaxed their mating decisions late in life. Our results suggest that social experience and age interact to affect sexual signaling and female mating decisions. Flexible behavior could mediate the potentially negative effects of environmental change on population viability, allowing reproductive success even when preferred mates are rare.     

Histochemical localization of FMRFamide, serotonin and catecholamines in embryonic Crepidula fornicata (Gastropoda, Prosobranchia)

 Recent reports indicate that neuronal elements develop in early larval stages of some Gastropoda from the Pulmonata and Opisthobranchia prior to the appearance of any ganglia of the future adult central nervous system (CNS). The present study describes similar early neuronal elements in Crepidula fornicata. A posterior FMRFamide-like immunoreactive (LIR) cell with anteriorly projected fibers was observed in the trochophore stage. Additional FMRFamide-LIR and serotonin-LIR cells and fibers were found in the apical organ in the trochophore and early veliger stages. FMRFamide-LIR and serotonin-LIR projections to the velum and foot were also detected at this time. As the veliger developed, peripheral FMRFamide-LIR and later catecholaminergic cells were located in the foot region. Also during this stage, catecholaminergic cells and processes were observed near the mouth. In addition, this study tentatively identified the first serotonin- and FMRFamide-LIR cells and fibers within the developing ganglia of the adult CNS, which appeared in close proximity to the earlier developing elements. These observations are consistent with the hypothesis that, in addition to its presumed role in the control of larval behaviors, the larval nervous system guides the development of the adult CNS. Larvae from the class Bivalvia and other invertebrate phyla also have neuronal elements marked by the presence of FMRFamide, serotonin, and catecholamines, and, therefore, this study may provide additional insights into phylogenetic relationships of the Gastropoda with other representatives of the Mollusca and different invertebrate phyla. Accepted: 10 February 1999     

Predator experience overrides learned aversion to heterospecifics in stickleback species pairs

Predation risk can alter female mating decisions because the costs of mate searching and selecting attractive mates increase when predators are present. In response to predators, females have been found to plastically adjust mate preference within species, but little is known about how predators alter sexual isolation and hybridization among species. We tested the effects of predator exposure on sexual isolation between benthic and limnetic threespine sticklebacks (Gasterosteus spp.). Female discrimination against heterospecific mates was measured before and after females experienced a simulated attack by a trout predator or a control exposure to a harmless object. In the absence of predators, females showed increased aversion to heterospecifics over time. We found that predator exposure made females less discriminating and precluded this learned aversion to heterospecifics. Benthic and limnetic males differ in coloration, and predator exposure also affected sexual isolation by weakening female preferences for colourful males. Predator effects on sexual selection were also tested but predators had few effects on female choosiness among conspecific mates. Our results suggest that predation risk may disrupt the cognitive processes associated with mate choice and lead to fluctuations in the strength of sexual isolation between species.