The potato tuber,maize endosperm and a chimeric maize-potato ADP-glucose pyrophosphorylase exhibit fundamental differences in Pi inhibition

ADP-glucose pyrophosphorylase (AGPase) is highly regulated by allosteric effectors acting both positively and negatively. Enzymes from various sources differ, however, in the mechanism of allosteric regulation. Here, we determined how the effector, inorganic phosphate (Pi), functions in the presence and absence of saturating amounts of the activator, 3-phosphoglyceric acid (3-PGA). This regulation was examined in the maize endosperm enzyme, the oxidized and reduced forms of the potato tuber enzyme as well as a small subunit chimeric AGPase (MP), which contains both maize endosperm and potato tuber sequences paired with a wild-type maize large subunit. These data, combined with our previous kinetic studies of these enzymes led to a model of Pi inhibition for the various enzymes. The Pi inhibition data suggest that while the maize enzyme contains a single effector site that binds both 3-PGA and Pi, the other enzymes exhibit more complex behavior and most likely have at least two separate interacting binding sites for Pi. The possible physiological implications of the differences in Pi inhibition distinguishing the maize endosperm and potato tuber AGPases are discussed.     

Secondary amines containing one aromatic nitro group: Preparation,nitrosation, sustained nitric oxide release,and the synergistic effects of released nitric oxide and an arginase inhibitor on vascular smooth muscle cell proliferation

Atherosclerosis, a leading cause of death worldwide, is associated with the excessive proliferation of vascular smooth muscle cells. Nitrogen monoxide, more commonly known as nitric oxide, inhibits this uncontrolled proliferation. Herein we report the preparation of two families of nitric oxide donors; beginning with the syntheses of secondary amine precursors, obtained through the reaction between 2 equiv of various monoamines with 2,4 or 2,6-difluoronitrobenzene. The purified secondary amines were nitrosated then subjected to a Griess reagent test to examine the slow and sustained nitric oxide release rate for each compound in both the absence and presence of reduced glutathione. The release rate profiles of these two isomeric families of NO-donors were strongly dependent on the number of side chain methylene units and the relative orientations of the nitro groups with respect to the N-nitroso moieties. The nitrosated compounds were then added to human aortic smooth muscle cell cultures, individually and in tandem with S-2-amino-6-boronic acid (ABH), a potent arginase inhibitor. Cell viability studies indicated a lack of toxicity of the amine precursors, in addition to anti-proliferative effects exhibited by the nitrosated compounds, which were enhanced in the presence of ABH.     

Characterization of culturable bacterial endophytes of switchgrass (Panicum virgatum L.) and their capacity to influence plant growth

Switchgrass (Panicum virgatum L.) is a perennial warm season grass that is native to the plains of North America and is widely grown as a forage, bioenergy or groundcover crop. Despite its importance, a bottleneck in switchgrass production is poor seedling vigor, which as a perennial crop represents an important time for management. Herein, data identify a suite of culturable bacterial microflora extracted from switchgrass, and show their capability to influence host plant growth and development. A total of 307 bacterial isolates were cultured and isolated from surface sterilized switchgrass biomass and sequence identified into 76 strains (subspecies classification), 36 species and 5 phyla. Approximately 58% of bacterial strains, when reintroduced into surface‐sterilized switchgrass seeds, were documented to increase lamina length (cm from base to tip after 60 days growth) relative to uninoculated controls. Ecologically, Phylum Firmicutes was the most abundant bacterial classification and encompassed 75% of all isolates. Although the culturable bacterial community studies herein represent an unknown and assumedly minor proportion of the total microbiome, by focusing on culturable bacteria, we delineate functional feedback between the presence of isolated bacteria and switchgrass seedling growth.     

ATP Consumption by Sarcoplasmic Reticulum Ca2+ Pumps Accounts for 40-50% of Resting Metabolic Rate in Mouse Fast and Slow Twitch Skeletal Muscle

The main purpose of this study was to directly quantify the relative contribution of Ca²⁺ cycling to resting metabolic rate in mouse fast (extensor digitorum longus, EDL) and slow (soleus) twitch skeletal muscle. Resting oxygen consumption of isolated muscles (VO₂, µL/g wet weight/s) measured polarographically at 30^°C was ~20% higher (P<0.05) in soleus (0.326 ± 0.022) than in EDL (0.261 ± 0.020). In order to quantify the specific contribution of Ca²⁺ cycling to resting metabolic rate, the concentration of MgCl₂ in the bath was increased to 10 mM to block Ca²⁺ release through the ryanodine receptor, thus eliminating a major source of Ca²⁺ leak from the sarcoplasmic reticulum (SR), and thereby indirectly inhibiting the activity of the sarco(endo) plasmic reticulum Ca²⁺-ATPases (SERCAs). The relative (%) reduction in muscle VO₂ in response to 10 mM MgCl₂ was similar between soleus (48.0±3.7) and EDL (42.4±3.2). Using a different approach, we attempted to directly inhibit SERCA ATPase activity in stretched EDL and soleus muscles (1.42x optimum length) using the specific SERCA inhibitor cyclopiazonic acid (CPA, up to 160 µM), but were unsuccessful in removing the energetic cost of Ca²⁺ cycling in resting isolated muscles. The results of the MgCl₂ experiments indicate that ATP consumption by SERCAs is responsible for 40–50% of resting metabolic rate in both mouse fast- and slow-twitch muscles at 30^°C, or 12–15% of whole body resting VO₂. Thus, SERCA pumps in skeletal muscle could represent an important control point for energy balance regulation and a potential target for metabolic alterations to oppose obesity.     

The Induction of microRNA-16 in Colon Cancer Cells by Protein Arginine Deiminase Inhibition Causes a p53-Dependent Cell Cycle Arrest

Protein Arginine Deiminases (PADs) catalyze the post-translational conversion of peptidyl-Arginine to peptidyl-Citrulline in a calcium-dependent, irreversible reaction. Evidence is emerging that PADs play a role in carcinogenesis. To determine the cancer-associated functional implications of PADs, we designed a small molecule PAD inhibitor (called Chor-amidine or Cl-amidine), and tested the impact of this drug on the cell cycle. Data derived from experiments in colon cancer cells indicate that Cl-amidine causes a G1 arrest, and that this was p53-dependent. In a separate set of experiments, we found that Cl-amidine caused a significant increase in microRNA-16 (miRNA-16), and that this increase was also p53-dependent. Because miRNA-16 is a putative tumor suppressor miRNA, and others have found that miRNA-16 suppresses proliferation, we hypothesized that the p53-dependent G1 arrest associated with PAD inhibition was, in turn, dependent on miRNA-16 expression. Results are consistent with this hypothesis. As well, we found the G1 arrest is at least in part due to the ability of Cl-amidine-mediated expression of miRNA-16 to suppress its'' G1-associated targets: cyclins D1, D2, D3, E1, and cdk6. Our study sheds light into the mechanisms by which PAD inhibition can protect against or treat colon cancer.     

Early network activity propagates bidirectionally between hippocampus and cortex

Spontaneous activity in the developing brain helps refine neuronal connections before the arrival of sensory‐driven neuronal activity. In mouse neocortex during the first postnatal week, waves of spontaneous activity originating from pacemaker regions in the septal nucleus and piriform cortex propagate through the neocortex. Using high‐speed Ca²⁺ imaging to resolve the spatiotemporal dynamics of wave propagation in parasagittal mouse brain slices, we show that the hippocampus can act as an additional source of neocortical waves. Some waves that originate in the hippocampus remain restricted to that structure, while others pause at the hippocampus‐neocortex boundary and then propagate into the neocortex. Blocking GABAergic neurotransmission decreases the likelihood of wave propagation into neocortex, whereas blocking glutamatergic neurotransmission eliminates spontaneous and evoked hippocampal waves. A subset of hippocampal and cortical waves trigger Ca²⁺ waves in astrocytic networks after a brief delay. Hippocampal waves accompanied by Ca²⁺ elevation in astrocytes are more likely to propagate into the neocortex. Finally, we show that two structures in our preparation that initiate waves—the hippocampus and the piriform cortex—can be electrically stimulated to initiate propagating waves at lower thresholds than the neocortex, indicating that the intrinsic circuit properties of those regions are responsible for their pacemaker function. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 661–672, 2016     

Nontuberculous Mycobacterial Disease in Children – Epidemiology,Diagnosis & Management at a Tertiary Center

Background

There are limited data on the epidemiology, diagnosis and optimal management of nontuberculous mycobacterial (NTM) disease in children.

Methods

Retrospective cohort study of NTM cases over a 10-year-period at a tertiary referral hospital in Australia.

Results

A total of 140 children with NTM disease, including 107 with lymphadenitis and 25 with skin and soft tissue infections (SSTIs), were identified. The estimated incidence of NTM disease was 0.6–1.6 cases / 100,000 children / year; no increasing trend was observed over the study period. Temporal analyses revealed a seasonal incidence cycle around 12 months, with peaks in late winter/spring and troughs in autumn. Mycobacterium-avium-complex accounted for most cases (77.8%), followed by Mycobacterium ulcerans (14.4%) and Mycobacterium marinum (3.3%). Polymerase chain reaction testing had higher sensitivity than culture and microscopy for acid-fast bacilli (92.0%, 67.2% and 35.7%, respectively). The majority of lymphadenitis cases underwent surgical excision (97.2%); multiple recurrences in this group were less common in cases treated with clarithromycin and rifampicin compared with clarithromycin alone or no anti-mycobacterial drugs (0% versus 7.1%; OR:0.73). SSTI recurrences were also less common in cases treated with two anti-mycobacterial drugs compared with one or none (10.5% versus 33.3%; OR:0.23).

Conclusions

There was seasonal variation in the incidence of NTM disease, analogous to recently published observations in tuberculosis, which have been linked to seasonal variation in vitamin D. Our finding that anti-mycobacterial combination therapy was associated with a reduced risk of recurrences in patients with NTM lymphadenitis or SSTI requires further confirmation in prospective trials.