Separation and properties of prestalk and prespore cells of Dictyostelium discoideum

We describe a method of separating prestalk and prespore cells of Dictyostelium discoideum slugs using a self-generating Percoll gradient. This method gives quantitative recovery of cells and good purity. Separated prestalk and prespore cells possess different levels of the enzymes UDP galactose :polysaccharide transferase, cAMP phosphodiesterase and glycogen phosphorylase. We have used this method, as well as mechanical dissection of slugs, to examine the fate of separated prestalk and prespore cells in Dictyostelium strains that are able to give rise to mature stalk and spore cells in cell monolayers. The results from such experiments provide direct evidence that prestalk and prespore cells from the migrating slug stage are programmed to differentiate into stalk and spore cells respectively.     

Salt-water coupling in leaky epithelia

Summary The theory of quasi-isotonic transport by cellular osmosis (the standing-gradient theory) has been challenged on the grounds that the osmotic permeabilities of the mucosal and interspace membranes are too low; if they were as high as the theory requires then the osmotic permeability of the whole epithelium would be 2–3 orders of magnitude higher than observed. This objection has basically been accepted for it is now claimed that these enormous permeabilities do exist, but are masked by unstirred-layer effects; I show that this is incorrect because unstirredlayer corrections are small and that the situation has not changed since 1975.The view that the route of fluid transport is junctional is replacing the cellular theory, and trans-junctional water flows seem to account for major fractions of the flow in various epithelia. I argue on grounds of general theory that these are unlikely to be osmotic flows because the junctional pores cannot satisfy both the osmotic and diffusive properties required of them, but the basic osmotic theory is also rather vague here.Non-osmotic theories, if junctional flow is accepted, must be either electro-kinetic or peristaltic.     

Behavioral responses to intracerebroventricularly administered neurohypophyseal peptides in mice

Lysine vasopressin (LVP), arginine vasopressin, oxytocin, and arginine vasotocin administered intraventricularly (icv) to mice all provoked a dose-dependent behavioral response in the range 0.1 – 1.0 μg. This response included a pronounced hyperactivity, extensive foraging, increased grooming, and at higher doses, stereotyped scratching, squeaking, and occasional barrel rolling. The four hormones were all approximately equipotent. Desglycinamide lysine vasopressin and [desaminocys₁, D-Arg₈] vasopressin produced some of the characteristic behaviors, but were much less potent. While pretreatment of the animals with reserpine (5 mg/kg ip), haloperidol (0.5 mg/kg ip), or physostigmine (0.5 mg/kg ip) sedated the animals and attenuated the locomotion and grooming, these drugs did not substantially alter the characteristic behavioral responses to LVP. Pretreatment with α-methyl-p-tyrosine (400 mg/kg ip), p-chlorophenylalanine (320 mg/kg ip), 6-hydroxydopamine (100 μg icv), ergotamine (0.5 μg icv), ethoxolamide (52 ng icv), diphenhydramine (20 μg icv), prostaglondin F_2α (2 μg icv), or naloxone (1 mg/kg ip) did not alter the LVP-induced behaviors. None of these drugs or -amphetamine (0.5 to 20 mg/kg ip) or nicotine (0.1 or 1 μg icv) mimicked the behavioral effects of the hormones.     

Optimising Immunogenicity with Viral Vectors: Mixing MVA and HAdV-5 Expressing the Mycobacterial Antigen Ag85A in a Single Injection

The Bacillus Calmette - Guerin (BCG) vaccine provides a critical but limited defense against Mycobacterium tuberculosis (M.tb). More than 60 years after the widespread introduction of BCG, there is an urgent need for a better vaccine. A large body of pre-clinical research continues to support ongoing clinical trials to assess whether viral vectors expressing M.tb antigens that are shared by BCG and M.tb, can be used alongside BCG to enhance protection. A major focus involves using multiple unique viral vectors to limit anti-vector immunity and thereby enhance responses to the insert antigen delivered. The successful introduction of viral vector vaccines to target M.tb and other pathogens will be reliant on reducing the costs when using multiple vectors and inhibiting the development of unwanted anti-vector responses that interfere with the response to insert antigen. This study examines methods to reduce the logistical costs of vaccination by mixing different viral vectors that share the same insert antigen in one vaccine; and whether combining different viral vectors reduces anti-vector immunity to improve immunogenicity to the insert antigen. Here we show that a homologous prime-boost regimen with a mixture of MVA (Modified Vaccinia virus Ankara) and Ad5 (human adenovirus type 5) vectors both expressing Ag85A in a single vaccine preparation is able to reduce anti-vector immunity, compared with a homologous prime-boost regimen with either vector alone. However, the level of immunogenicity induced by the homologous mixture remained comparable to that induced with single viral vectors and was less immunogenic than a heterologous Ad5 prime-MVA-boost regimen. These findings advance the understanding of how anti-vector immunity maybe reduced in viral vector vaccination regimens. Furthermore, an insight is provided to the impact on vaccine immunogenicity from altering vaccination methods to reduce the logistical demands of using separate vaccine preparations in the field.     

Elimination of African Onchocerciasis: Modeling the Impact of Increasing the Frequency of Ivermectin Mass Treatment

The African Programme for Onchocerciasis Control (APOC) is currently shifting its focus from morbidity control to elimination of infection. To enhance the likelihood of elimination and speed up its achievement, programs may consider to increase the frequency of ivermectin mass treatment from annual to 6-monthly or even higher. In a computer simulation study, we examined the potential impact of increasing the mass treatment frequency for different settings. With the ONCHOSIM model, we simulated 92,610 scenarios pertaining to different assumptions about transmission conditions, history of mass treatment, the future mass treatment strategy, and ivermectin efficacy. Simulation results were used to determine the minimum remaining program duration and number of treatment rounds required to achieve 99% probability of elimination. Doubling the frequency of treatment from yearly to 6-monthly or 3-monthly was predicted to reduce remaining program duration by about 40% or 60%, respectively. These reductions come at a cost of additional treatment rounds, especially in case of 3-monthly mass treatment. Also, aforementioned reductions are highly dependent on maintained coverage, and could be completely nullified if coverage of mass treatment were to fall in the future. In low coverage settings, increasing treatment coverage is almost just as effective as increasing treatment frequency. We conclude that 6-monthly mass treatment may only be worth the effort in situations where annual treatment is expected to take a long time to achieve elimination in spite of good treatment coverage, e.g. because of unfavorable transmission conditions or because mass treatment started recently.     

Queuosine Biosynthesis Is Required for Sinorhizobium meliloti-Induced Cytoskeletal Modifications on HeLa Cells and Symbiosis with Medicago truncatula

Rhizobia are symbiotic soil bacteria able to intracellularly colonize legume nodule cells and form nitrogen-fixing symbiosomes therein. How the plant cell cytoskeleton reorganizes in response to rhizobium colonization has remained poorly understood especially because of the lack of an in vitro infection assay. Here, we report on the use of the heterologous HeLa cell model to experimentally tackle this question. We observed that the model rhizobium Sinorhizobium meliloti, and other rhizobia as well, were able to trigger a major reorganization of actin cytoskeleton of cultured HeLa cells in vitro. Cell deformation was associated with an inhibition of the three major small RhoGTPases Cdc42, RhoA and Rac1. Bacterial entry, cytoskeleton rearrangements and modulation of RhoGTPase activity required an intact S. meliloti biosynthetic pathway for queuosine, a hypermodifed nucleoside regulating protein translation through tRNA, and possibly mRNA, modification. We showed that an intact bacterial queuosine biosynthetic pathway was also required for effective nitrogen-fixing symbiosis of S. meliloti with its host plant Medicago truncatula, thus indicating that one or several key symbiotic functions of S. meliloti are under queuosine control. We discuss whether the symbiotic defect of que mutants may originate, at least in part, from an altered capacity to modify plant cell actin cytoskeleton.