Cytoplasmic filaments and cellular wound healing in amoeba proteus

The flexibility and self-healing properties of animal cell surface membranes are well known. These properties have been best exploited in various micrurgical studies on living cells (2, 3), especially in amoebae (7, 20). During nuclear transplantation in amoebae, the hole in the membrane through which a nucleus passes can have a diameter of 20-30 μm, and yet such holes are quickly sealed, although some cytoplasm usually escapes during the transfer. While enucleating amoebae in previous studies, we found that if a very small portion of a nucleus was pushed through the membrane and exposed to the external medium, the amoeba expelled such a nucleus on its own accord. When this happened, a new membrane appeared to form around the embedded portion of the nucleus and no visible loss of cytoplasm occurred during nuclear extrusion. In the present study, we examined amoebae that were at different stages of expelling partially exposed nuclei, to follow the sequence of events during the apparent new membrane formation. Unexpectedly, we found that a new membrane is not formed around the nucleus from inside but a hole is sealed primarily by a constriction of the existing membrane, and that cytoplasmic filaments are responsible for the prevention of the loss of cytoplasm.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

An approach to the use of stable isotopes for DNA sequencing

The sequencing of DNA by current procedures involves the use of radioisotopic or fluorescent labels. We propose that stable isotopes can be used as such labels and that the large number of stable isotopes available would allow multiplexing so that many DNA segments could be sequenced simultaneously. We have developed methods to use 57Fe2O3 to synthesize ferrocene and to attach the ferrocene to the 5' end of oligonucleotides. The 57Fe-labeled M13 universal primer functioned normally in a Sanger sequencing procedure. When a 57Fe-labeled oligonucleotide had migrated on a polyacrylamide gel it was readily located on the dried gel by scanning with resonance ionization spectroscopy (RIS) coupled with mass spectrometry. Using a 57Fe-labeled primer in a PCR reaction a 2000-bp DNA was produced that was detected by RIS on nylon membrane after agarose electrophoresis. The rapid analysis features of RIS coupled with the multispectral multiplexing possibilities of stable isotopes should significantly increase the rate of determination of DNA sequences.     

Characterization of dog prenodal peripheral lymph lipoproteins. Evidence for the peripheral formation of lipoprotein-unassociated apoA-I with slow pre-beta electrophoretic mobility

Dog plasma and prenodal peripheral lymph apoA-I distribution was examined by nondenaturing gradient gel electrophoresis-immunoblot analysis. In control dogs, plasma apoA-I could be localized to two distinct populations of particles with modal diameters of 8.4 nm and 10.4 nm. The smaller sized population accounted for over 50% of plasma apoA-I. Peripheral lymph apoA-I distribution was significantly different. The percentage of apoA-I localized to the 10.4 nm population was reduced by 40% and the modal diameter of the smaller HDL apoA-I population was significantly decreased by 0.1 nm. Additionally, peripheral lymph apoA-I could be localized to particles smaller than albumin (lipoprotein-unassociated apoA-I). The presence of lipoprotein-unassociated apoA-I particles was confirmed by gel filtration chromatography. Immunoblots of column fractions subjected to agarose electrophoresis revealed that these particles had slow pre-beta electrophoretic mobility. In dogs fed an atherogenic diet, lipoprotein-unassociated apoA-I particles with slow pre-beta electrophoretic mobility could be found in both plasma and peripheral lymph. With increasing degree of hypercholesterolemia, the relative amount of plasma lipoprotein-unassociated apoA-I tended to increase. In peripheral lymph, an increasing degree of hypercholesterolemia was associated with a decrease in the relative amount of lipoprotein-unassociated apoA-I. Instead, a population of large apoA-I particles (11-25 nm) became increasingly prominent.     

Progesterone Receptor Membrane Component 1 Is a Functional Part of the Glucagon-like Peptide-1 (GLP-1) Receptor Complex in Pancreatic β Cells

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates glucose homeostasis. Because of their direct stimulation of insulin secretion from pancreatic β cells, GLP-1 receptor (GLP-1R) agonists are now important therapeutic options for the treatment of type 2 diabetes. To better understand the mechanisms that control the insulinotropic actions of GLP-1, affinity purification and mass spectrometry (AP-MS) were employed to uncover potential proteins that functionally interact with the GLP-1R. AP-MS performed on Chinese hamster ovary cells or MIN6 β cells, both expressing the human GLP-1R, revealed 99 proteins potentially associated with the GLP-1R. Three novel GLP-1R interactors (PGRMC1, Rab5b, and Rab5c) were further validated through co-immunoprecipitation/immunoblotting, fluorescence resonance energy transfer, and immunofluorescence. Functional studies revealed that overexpression of PGRMC1, a novel cell surface receptor that associated with liganded GLP-1R, enhanced GLP-1-induced insulin secretion (GIIS) with the most robust effect. Knockdown of PGRMC1 in β cells decreased GIIS, indicative of positive interaction with GLP-1R. To gain insight mechanistically, we demonstrated that the cell surface PGRMC1 ligand P4-BSA increased GIIS, whereas its antagonist AG-205 decreased GIIS. It was then found that PGRMC1 increased GLP-1-induced cAMP accumulation. PGRMC1 activation and GIIS induced by P4-BSA could be blocked by inhibition of adenylyl cyclase/EPAC signaling or the EGF receptor–PI3K signal transduction pathway. These data reveal a dual mechanism for PGRMC1-increased GIIS mediated through cAMP and EGF receptor signaling. In conclusion, we identified several novel GLP-1R interacting proteins. PGRMC1 expressed on the cell surface of β cells was shown to interact with the activated GLP-1R to enhance the insulinotropic actions of GLP-1.Glucagon-like peptide-1 (GLP-1)¹ is a gastrointestinal hormone secreted by intestinal L cells upon food intake that is best known for its role in controlling glucose homeostasis. Acting through its cognate glucagon-like peptide-1 receptor (GLP-1R), GLP-1 has several important physiological and pharmacological functions. GLP-1 is best known for enhancing glucose-stimulated insulin secretion (GSIS) from the pancreatic β cells. Importantly, the insulinotropic properties of GLP-1 are maintained in patients with type 2 diabetes (1), which is characterized by insufficient insulin secretion from pancreatic β cells and an inability to maintain glucose homeostasis. Therefore, therapeutic strategies targeting GLP-1R have been developed to treat type 2 diabetes (2, 3). In addition to augmenting insulin secretion, GLP-1 has been known to improve glucose sensing, proinsulin biosynthesis, survival, and proliferation of β cells (3, 4) in a variety of experimental models. GLP-1 also has several extrapancreatic effects, including actions on the central nervous system to inhibit food intake (5), the stomach to decrease gastric emptying and gastric acid secretion (6), and the lungs to stimulate secretion of macromolecules from airways (7). Additionally, GLP-1 has an effect on the heart and possibly the kidney to modulate blood pressure and heart rate (8, 9).The GLP-1R is a member of the B1 family of G protein–coupled receptors (secretin receptor family). In mammals, GLP-1R is expressed in multiple tissues, including pancreatic β cells and δ cells (10), hypothalamus, lung, stomach, heart, kidney (11), and thyroid (12), which in part explains its diverse actions. Upon ligand binding, the GLP-1R is capable of coupling to diverse cell signal transduction pathways, but it is best known for its actions on G protein Gs α and adenylate cyclase activity to increase intracellular cAMP. It is known that other proteins can affect GLP-1R activity in addition to G proteins, including β-arrestin and caveolin, which affect receptor internalization and trafficking. β-Arrestin 1 is also required for proper GLP-1-stimulated cAMP production (13–15). More recently, it was shown that another B1 family member, gastric inhibitory polypeptide receptor heterodimerizes with GLP-1R, decreasing GLP-1-induced β-arrestin recruitment and mobilization (16). Very recently, our group identified several novel potential GLP-1R interactors using a membrane-based split-ubiquitin yeast two-hybrid (MYTH) assay (17). Three β cell–expressing membrane-bound interactors, solute carrier family 15 member 4 (SLC15A4), amyloid β A4 precursor-like protein 1 (APLP1), and adaptor-related protein complex 2 subunit mu (AP2M1), were further selected for individual knockdown in mouse insulinoma (MIN6) β cells using small interfering RNAs (siRNAs). GLP-1-induced insulin secretion was significantly enhanced when these genes were silenced, suggesting that these interactor proteins attenuate GLP-1R activity. These findings demonstrated that GLP-1R protein interactions are complex and the interactors can have measurable effects on receptor trafficking and downstream signaling. Such interactions may in part explain the diverse tissue-specific effects of GLP-1 and offer avenues for controlling GLP-1 actions in a tissue-selective manner.Although the MYTH system is well established (18) and has been applied to study G protein–coupled receptor interactomes (17), it is limited on two fronts. Firstly, it must be performed in yeast which is not an ideal representation of the mammalian system. Secondly, it is technically difficult to activate the receptor in MYTH, thus, effects of ligand stimulation on the receptor interactome cannot be assessed. Recently, affinity purification–mass spectrometry (AP-MS) has become a powerful tool for discovering and examining novel protein–protein interactions, including those between membrane-bound proteins in mammalian cells (19–21). In the current study, we applied AP-MS to discover novel GLP-1R interactors and employed a human GLP-1R harboring a FLAG^® epitope. GLP-1R-Flag was expressed in either Chinese hamster ovary (CHO) cells or MIN6 β cells, and interactors were studied in the presence or absence of GLP-1.     

The population biology of mitigation: impacts of habitat creation on an endangered plant species

Conservation practitioners widely agree that optimal conservation strategies will maximize the amount of genetic variation preserved in target taxa, but there is ongoing debate about how that variation should be distributed through restoration and mitigation activities. Here, we evaluate the impacts of ~10 years of mitigation on the population genetic structure of Limnanthes vinculans, a state- and federally-listed endangered plant species restricted to ephemeral vernal pool wetlands in the Santa Rosa Plain of California. Using microsatellite loci to estimate patterns of neutral molecular variation, we found that created pools support similar levels of variation in L. vinculans as natural pools. Habitat creation and seed translocation have not disrupted the largest-scale patterns of population structure across the species range, but a concentration of mitigation activity towards the range center has reduced the extent of isolation-by-distance operating in this region and shifted the location of at least one genetic boundary. Patterns of genetic variation among populations in remnant vernal pools reveal that gene flow has historically occurred beyond the scale of individual pools at the center of the species range, while small genetic populations have differentiated around the range margins. On average, L. vinculans in created pools exhibit less cover and more restricted local distributions than those in remnant pools, but these patterns were driven by two particularly productive natural sites rather than consistent differences between natural and created sites. We conclude that mitigation activities have changed the historical patterns of gene flow within the species range to a moderate degree, that these changes will likely impact remnant pools through gene flow, and that current created sites provide less heterogeneous habitat for L. vinculans than do natural pools. Studies that track individual plants will be needed to determine if the changes in gene flow due to mitigation will have positive or negative impacts on the demographic and microevolutionary trajectories of L. vinculans. More generally, this study provides a retrospective analysis of the outcome of managing an endangered plant species through intensive mitigation, and yields several insights to inform future conservation strategies.     

A sensitive, inexpensive method for determining minute quantities of lipase activity

We describe a sensitive and reproducible lipase assay based on the binding of 63Ni to fatty acid. This method can detect down to 1 nmol of fatty acid per milliliter of solution. It has been adapted for measuring low concentrations of lipoprotein lipase and hepatic triacylglycerol lipase. Furthermore, in the presence of tritiated triolein, the method is insensitive to radiolabel interference, even when the fatty acid is labeled.     

Which population level environmental factors are associated with asthma,rhinoconjunctivitis and eczema? Review of the ecological analyses of ISAAC Phase One

The International Study of Asthma and Allergies in Childhood (ISAAC) Phase One showed large worldwide variations in the prevalence of symptoms of asthma, rhinoconjunctivitis and eczema, up to 10 to 20 fold between countries. Ecological analyses were undertaken with ISAAC Phase One data to explore factors that may have contributed to these variations, and are summarised and reviewed here.In ISAAC Phase One the prevalence of symptoms in the past 12 months of asthma, rhinoconjunctivitis and eczema were estimated from studies in 463,801 children aged 13 - 14 years in 155 centres in 56 countries, and in 257,800 children aged 6-7 years in 91 centres in 38 countries. Ecological analyses were undertaken between symptom prevalence and the following: Gross National Product per capita (GNP), food intake, immunisation rates, tuberculosis notifications, climatic factors, tobacco consumption, pollen, antibiotic sales, paracetamol sales, and outdoor air pollution.Symptom prevalence of all three conditions was positively associated with GNP, trans fatty acids, paracetamol, and women smoking, and inversely associated with food of plant origin, pollen, immunisations, tuberculosis notifications, air pollution, and men smoking. The magnitude of these associations was small, but consistent in direction between conditions. There were mixed associations of climate and antibiotic sales with symptom prevalence.The potential causality of these associations warrant further investigation. Factors which prevent the development of these conditions, or where there is an absence of a positive correlation at a population level may be as important from the policy viewpoint as a focus on the positive risk factors. Interventions based on small associations may have the potential for a large public health benefit.