Evaluation of the cell growth of mycobacteria using Mycobacterium smegmatis mc2 155 as a representative species

The study of the in vitro cell growth of mycobacteria still remains a fastidious, difficult, and time-consuming procedure. In addition, assessing mycobacterial growth in the laboratory is often complicated by cell aggregation and slow growth-rate. We now report that the use of a stainless steel spring in the culture led to an absence of large cell clumps, to a decrease of dead cells in the exponential phase and to growth of a more homogeneous population of large cells. We also report that flow cytometry is a rapid, simple and reliable approach to monitor mycobacterial cell growth and viability. Here, we monitored Mycobacterium smegmatis cellular growth by optical density, dry cell mass, and colony forming units; in addition, viability, cell size and granularity profiles were analyzed by flow cytometry, and cell morphology by electron microscopy. Cultures monitored by flow cytometry may lead to a better understanding of the physiology of mycobacteria. Moreover, this methodology may aid in characterizing the cell growth of other fastidious species of microorganisms.     

Neuropeptide Y5 receptor antagonism does not induce clinically meaningful weight loss in overweight and obese adults

Neuropeptide Y (NPY) is a potent orexigenic neuropeptide, and antagonism of NPY Y1 and NPY Y5 receptors (NPYxR) is considered a potentially important anti-obesity drug target. We tested the hypothesis that blockade of the NPY5R will lead to weight loss in humans using MK-0557, a potent, highly selective, orally active NPY5R antagonist. The initial series of experiments reported herein, including a multiple-dose positron-emission tomography study and a 12 week proof-of concept/dose-ranging study, suggested an optimal MK-0557 dose of 1 mg/day. The hypothesis was then tested in a 52 week, multicenter, randomized, double-blind, placebo-controlled trial involving 1661 overweight and obese patients. Although statistically significant at 52 weeks, the magnitude of induced weight loss was not clinically meaningful. These observations provide the first clinical insight into the human NPY-energy homeostatic pathway and suggest that solely targeting the NPY5R in future drug development programs is unlikely to produce therapeutic efficacy.     

A multi-proxy study of deeper-water carbonates (Upper Jurassic, southern Germany): combining sedimentology, chemostratigraphy and palynofacies

Deeper shelf carbonates are often composed of relatively monotonous successions with few diagnostic sedimentological characteristics. The Upper Jurassic of southern Germany provides a classical example for deeper ramp carbonate environments, dominated by limestone/marl sequences including conspicuous sponge/microbial bioherms. Sedimentological analysis was integrated with stable isotope (O, C) and palynofacies analysis in an attempt to reconstruct the dominant depositional controls (sea level, climate, nutrients) as well as to delineate genetic sequences and their stacking patterns. Small-scale (3–10 m thick), medium-scale (5–25 m thick) and large-scale (45–60 m thick) sequences could be recognised, which all share similar patterns and trends. Oxygen isotopes from bulk rock carbonate samples were interpreted as records of temperature trends which were related to climatically induced sea level fluctuations. A positive oxygen isotope trend (i.e., cooling and associated relative sea-level fall) in combination with increasing absolute palynoclast abundances (increasing proximality) are inferred to mark regressive hemi-sequences. Negative trends in oxygen isotopes (i.e., warming and associated relative sea-level rise) and a decrease in absolute palynoclast abundances (increasing distality) are interpreted to indicate transgressive hemi-sequences. In contrast to the small-scale sequences, the medium-scale sequences could be correlated on a basin-wide scale by means of stable isotope trends and gamma-ray logs. Borehole scans were found to be useful for the recognition of major facies associations and sequence types when core data are not available.     

Tree species, root decomposition and subsurface denitrification potential in riparian wetlands

Patches of organic matter have been found to be important `hotspots' of denitrification in both surface and subsurface soils, but the factors controlling the formation and maintenance of these patches are not well established. We compared the concentration of patches of organic matter and root biomass in the subsurface (saturated zone) beneath poorly drained riparian wetland soils at four sites in Rhode Island, USA - two dominated by red maple (Acer rubrum) and two dominated by white pine (Pinus strobus). Denitrification enzyme activity (DEA) and carbon (C) content of patch material were compared between sites and between patches with different visual characteristics. Root decomposition was measured in an 8-week ex-situ incubation experiment that compared the effects of water content, root species, and soil matrix origin on CO₂ evolution. We observed significantly greater concentrations of patches at 55 cm at one red maple site than all other sites. DEA and percent C in patches was generally higher in patches than matrix soil and did not vary between sites or by patch type. White pine roots decomposed at a faster rate than red maple roots under unsaturated conditions. Our results suggest that faster root decomposition could result in lower concentrations of patches of organic material in subsurface soils at sites dominated by white pine. Tree species composition and root decomposition may play a significant role in the formation of patches and the creation and maintenance of groundwater denitrification hotspots in the subsurface of riparian wetlands. Abbreviations: DEA – denitrification enzyme activity; DOC – dissolved organic carbon; PD – poorly drained; RM-1 – red maple-1 site; RM-2 – red maple-2 site; WP-1 – white pine-1 site; WP-2 – white pine-2 site.     

Differential effects of nicotine and aging on splenocyte proliferation and the production of Th1- versus Th2-type cytokines

Nicotine has a multitude of biological actions in the central and peripheral nervous systems where nicotinic acetylcholine receptors are found. Nicotinic acetylcholine receptors have also been identified on immune cells, but the effects of nicotine on immune responses are not well characterized. These studies tested the hypotheses that nicotine has an effect on both T-lymphocyte proliferation and the production of cytokines by activated T cells, processes that are necessary for effective T-cell-mediated immune responses. In addition, the effects of nicotine on these immune responses in aging animals and the effects of nicotine exposure prior to immunostimulation were investigated. Murine splenocytes were exposed to nicotine and stimulated with concanavalin A (ConA). The highest concentration of nicotine (128 microg/ml) significantly depressed proliferation of T cells both when nicotine and ConA were added concurrently and when nicotine was added 3 hr prior to ConA. Nicotine, added concurrently with ConA at concentrations between 0. 25 and 64 microg/ml, significantly inhibited the production of IL-10 by splenocytes from young adult mice, whereas the inhibition of production of IL-10 by splenocytes from old mice was significantly inhibited, but the response was more variable, depending on the nicotine concentration. In contrast, the production of IFN-gamma by splenocytes from either young adult or old mice was not affected when nicotine (0.016-64 microg/ml) was added concurrently with ConA. Pre-exposure to 1 microg/ml of nicotine for 3 hr significantly enhanced the production of IFN-gamma by splenocytes from young adult mice, whereas pre-exposure to 0.016 microg/ml of nicotine tended to but did not significantly enhance IFN-gamma production. Nicotine is now being used as an over-the-counter drug by people who differ in age and general immunocompetence. Therefore, the effects of nicotine on immune responses, independent from the effects of the other chemicals found in tobacco, need to be investigated.     

The driving force for life's emergence: kinetic and thermodynamic considerations

The principles that govern the emergence of life from non-life remain a subject of intense debate. The evolutionary paradigm built up over the last 50 years, that argues that the evolutionary driving force is the Second Law of Thermodynamics, continues to be promoted by some, while severely criticized by others. If the thermodynamic drive toward ever-increasing entropy is not what drives the evolutionary process, then what does? In this paper, we analyse this long-standing question by building on Eigen's "replication first" model for life's emergence, and propose an alternative theoretical framework for understanding life's evolutionary driving force. Its essence is that life is a kinetic phenomenon that derives from the kinetic consequences of autocatalysis operating on specific biopolymeric systems, and this is demonstrably true at all stages of life's evolution--from primal to advanced life forms. Life's unique characteristics--its complexity, energy-gathering metabolic systems, teleonomic character, as well as its abundance and diversity, derive directly from the proposition that from a chemical perspective the replication reaction is an extreme expression of kinetic control, one in which thermodynamic requirements have evolved to play a supporting, rather than a directing, role. The analysis leads us to propose a new sub-division within chemistry--replicative chemistry. A striking consequence of this kinetic approach is that Darwin's principle of natural selection: that living things replicate, and therefore evolve, may be phrased more generally: that certain replicating things can evolve, and may therefore become living. This more general formulation appears to provide a simple conceptual link between animate and inanimate matter.     

Differentiation and redifferentiation of a transfer cell: Development of septal nectaries ofAloe andGasteria

Summary The epithelial cells of the septal nectaries ofGasteria andAloe have a second wall layer consisting of numerous irregular wall protuberances along the outer wall. They, thus, are typical transfer cells. The wall protuberances develop a short time before the nectar secretion begins. At this time and during the secretory phase the amount of microtubules is at a minimum. After the secretion phase the protuberances disappear gradually while often callose is formed between them. Simultaneously a third wall layer is deposited which covers the remnants of the second layer. During the first developmental stages the cells contain amyloplasts with changing amounts of starch. In the redifferentiation phase they become chloroplasts with a crystalline prolamellar body and small stacks of thylakoids.