Effects of oxygen and chloramphenicol on Frankia nitrogenase activity

The kinetics of asymbiotic nitrogenase activity in three strains of the actinomycete Frankia were studied. Decay rates for enzyme activity were determined by adding chloramphenicol to active acetylene-reducing cells and measuring the time required for all activity to cease. Synthesis rates were measured by bubbling oxygen through actively-reducing cells (which totally destroyed all activity) and then measuring the time required for activity to return to normal. Decay rates (t _1/2) for these three strains were approximately 30 to 40 min. Synthesis rates were slower and initial nitrogenase activities were recorded about 110 min (DDB 011610) or 210 min (DDB 020210 and WgCc1.17) after return to air-equilibrated cultures. Frankia strain WgCc1.17 showed a greater sensitivity to oxygen and nitrogenase activity was totally lost when cells were bubbled only with atmospheric concentrations of oxygen. The results presented here indicate that nitrogenase activity turnover time is relatively rapid, on the order of minutes rather than hours or days. However, regulation of nitrogenase activity will differ from one strain to another and asmmbiotic characterization will be useful for understanding nitrogenase regulation in the bacterial-plant symbiosis.Contribution no. 879 from the Battelle-Kettering Laboratory     

One-year survey of a single Micronesian reef reveals extraordinarily rich diversity of <Emphasis Type="Italic">Symbiodinium</Emphasis> types in soritid foraminifera

Recent molecular studies of symbiotic dinoflagellates (genus Symbiodinium) from a wide array of invertebrate hosts have revealed exceptional fine-scale symbiont diversity whose distribution among hosts, regions and environments exhibits significant biogeographic, ecological and evolutionary patterns. Here, similar molecular approaches using the internal transcribed spacer-2 (ITS-2) region were applied to investigate cryptic diversity in Symbiodinium inhabiting soritid foraminifera. Approximately 1,000 soritid specimens were collected and examined during a 12-month period over a 40 m depth gradient from a single reef in Guam, Micronesia. Out of 61 ITS-2 types distinguished, 46 were novel. Most types found are specific for soritid hosts, except for three types (C1, C15 and C19) that are common in metazoan hosts. The distribution of these symbionts was compared with the phylotype of their foraminiferal hosts, based on soritid small subunit ribosomal DNA sequences, and three new phylotypes of soritid hosts were identified based on these sequences. Phylogenetic analyses of 645 host-symbiont pairings revealed that most Symbiodinium types associated specifically with a particular foraminiferal host genus or species, and that the genetic diversity of these symbiont types was positively correlated with the genetic diversity found within each of the three host genera. Compared to previous molecular studies of Symbiodinium from other locations worldwide, the diversity reported here is exceptional and suggests that Micronesian coral reefs are home to a remarkably large Symbiodinium assemblage.     

Travelling gradients in interacting morphogen systems

Morphogen gradients are well known to play several important roles in development; however the mechanisms underlying the formation and maintenance of these gradients are often not well understood. In this work, we investigate whether the presence of a secondary morphogen can increase the robustness of the primary morphogen gradient to perturbation, thereby providing a more stable mechanism for development. We base our model around the interactions of Fibroblast Growth Factor 8 and retinoic acid, which have been shown to act as morphogens in many developmental systems. In particular, we investigate the formation of opposing gradients of these morphogens along the antero-posterior axis of vertebrate embryos, thereby controlling temporal and spatial aspects of axis segmentation and neuronal differentiation.     

Oxidative stress and antioxidant capacity of a terrestrially hibernating hatchling turtle

Hatchlings of the painted turtle, Chrysemys picta, hibernate terrestrially and can survive subfreezing temperatures by supercooling or by tolerating the freezing of their tissues. Whether supercooled or frozen, an ischemic hypoxia develops because tissue perfusion is limited by low temperature and/or freezing. Oxidative stress can occur if hatchlings lack sufficient antioxidant defenses to minimize or prevent damage by reactive oxygen species. We examined the antioxidant capacity and indices of oxidative damage in hatchling C. picta following survivable, 48 h bouts of supercooling (−6°C), freezing (−2.5°C), or hypoxia (4°C). Samples of plasma, brain, and liver were collected after a 24 h period of recovery (4°C) and assayed for Trolox-equivalent antioxidant capacity (TEAC), thiobarbituric acid reactive substances (TBARS), and carbonyl proteins. Antioxidant capacity did not vary among treatments in any of the tissues studied. We found a significant increase in TBARS in plasma, but not in the brain or liver, of frozen/thawed hatchlings as compared to untreated controls. No changes were found in the concentration of TBARS or carbonyl proteins in supercooled or hypoxia-exposed hatchlings. Our results suggest that hatchling C. picta have a well-developed antioxidant defense system that minimizes oxidative damage during hibernation.     

Interannual variability of cyanobacterial blooms in lake erie

After a 20-year absence, severe cyanobacterial blooms have returned to Lake Erie in the last decade, in spite of negligible change in the annual load of total phosphorus (TP). Medium-spectral Resolution Imaging Spectrometer (MERIS) imagery was used to quantify intensity of the cyanobacterial bloom for each year from 2002 to 2011. The blooms peaked in August or later, yet correlate to discharge (Q) and TP loads only for March through June. The influence of the spring TP load appears to have started in the late 1990 s, after Dreissenid mussels colonized the lake, as hindcasts prior to 1998 are inconsistent with the observed blooms. The total spring Q or TP load appears sufficient to predict bloom magnitude, permitting a seasonal forecast prior to the start of the bloom.     

Modulation of drug resistance in homoharringtonine-resistant C-1300 neuroblastoma cells with cyclosporine A and dipyridamole.

The development of resistance accounts for therapy failure in the majority of advanced cases of neuroblastoma in children. A new transplantable murine C-1300 neuroblastoma cell line was developed in vitro, by repeated exposure of a sensitive cell line to increasing, but sublethal, doses of Homoharringtonine (HHT). The ED50 of the highly resistant cells for HHT, using a standard agar colony assay, is 480 ng/ml, compared with 13 ng/ml for the sensitive parental line. The resistant cells have cross-resistance to a number of other agents, including adriamycin, vinca alkaloids, melphalan, and CCNU. Western blot analysis revealed progressive increases in P-glycoprotein, parallel to the graded development of resistance with a 29-fold elevation in the highest resistant cells. High-performance liquid chromatography (HPLC) indicated that resistant cells have a significantly lower uptake of HHT than parental sensitive cells. cyclosporine A (CsA) and dipyridamole (DPM) could modulate the acquired resistance and completely restore the cytotoxic effects of HHT and adriamycin as determined by the clonogenic assay. The reversal of resistance by CsA and DPM was dose dependent. With the relative low toxicity of dipyridamole and CsA in doses required for modulation of resistance, these agents may be candidates for clinical utilization in chemotherapy of resistant neuroblastoma.     

Cannabinoid Receptor Type 1 Protects against Age- Related Osteoporosis by Regulating Osteoblast and Adipocyte Differentiation in Marrow Stromal Cells

Age-related osteoporosis is characterized by reduced bone formation and accumulation of fat in the bone marrow compartment. Here, we report that the type 1 cannabinoid receptor (CB1) regulates this process. Mice with CB1 deficiency (CB1^−/−) had increased peak bone mass due to reduced bone resorption, but developed age-related osteoporosis with reduced bone formation and accumulation of adipocytes in the bone marrow space. Marrow stromal cells from CB1^−/− mice had an enhanced capacity for adipocyte differentiation, a reduced capacity for osteoblast differentiation, and increased expression of phosphorylated CREB (pCREB) and PPARγ. Pharmacological blockade of CB1 receptors stimulated adipocyte differentiation, inhibited osteoblast differentiation, and increased cAMP and pCREB in osteoblast and adipocyte precursors. The CB1 receptor is therefore unique in that it regulates peak bone mass through an effect on osteoclast activity, but protects against age-related bone loss by regulating adipocyte and osteoblast differentiation of bone marrow stromal cells.     

Regional specificity of functional sensory connections in developing spinal cord cultures varies with the incidence of spontaneous bioelectric activity

Summary Cultured spinal cord explants in which little spontaneous bioelectric activity was present showed, when monitored using sensory ganglion-evoked monosynaptic action potentials, diffuse innervation by ingrowing afferent fibers at 3–4 weeks in vitro. In contrast, highly active cultures of the same age showed a strong tendency for functional sensory connections to be made within the dorsal half of the cord. Regional specificity was present in mature cultures (4–5 weeks in vitro), however, even when their spontaneous activity level was low. The results support earlier results using tetrodotoxin, and make it appear likely that centrally generated neuronal discharges can influence the topography of afferent terminals within the developing spinal cord.