Effects of hydroxyurea on Crithidia fasciculata.

Hydroxyurea (HU) inhibits increase in cell number in cultures of Crithidia fasciculata. Complete inhibition is produced by 8 mM and higher concentrations. If HU is not removed, population growth resumes in 45-50 h; if HU is removed, partially synchronous growth occurs through 2 cycles. During HU inhibition, the rate of DNA synthesis is reduced to 1% of that in exponentially growing cultures; protein and RNA syntheses continue at slightly reduced rates. Mean cell size and protein and RNA contents per cell increase; rate of oxygen consumption per mg cell protein remains constant. The behavior of a culture upon addition of HU and upon its removal agrees with predictions based on the hypothesis that the only direct effect of HU is to block DNA synthesis. The synchrony produced by HU is judged satisfactory for investigations of kinetoplast and nuclear replication but not for biochemical characterization of other aspects of the cell cycle.     

Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls

Walls from frozen-thawed cucumber (Cucumis sativus L.) hypocotyls extend for many hours when placed in tension under acidic conditions. This study examined whether such creep is a purely physical process dependent on wall viscoelasticity alone or whether enzymatic activities are needed to maintain wall extension. Chemical denaturants inhibited wall creep, some acting reversibly and others irreversibly. Brief (15 s) boiling in water irreversibly inhibited creep, as did pre-incubation with proteases. Creep exhibited a high Q₁₀ (3.8) between 20° and 30°C, with slow inactivation at higher temperatures, whereas the viscous flow of pectin solutions exhibited a much lower Q₁₀ (1.35). On the basis of its temperature sensitivity, involvement of pectic gel-sol transitions was judged to be of little importance in creep. Pre-incubation of walls in neutral pH irreversibly inactivated their ability to creep, with a half-time of about 40 min. At 1 mM, Cu²⁺, Hg²⁺ and Al³⁺ were strongly inhibitory whereas most other cations, including Ca²⁺, had little effect. Sulfhydryl-reducing agents strongly stimulated creep, apparently by stabilizing wall enzyme(s). The physical effects of these treatments on polymer interactions were examined by Instron and stress-relaxation analyses. Some treatments, such as pH and Cu²⁺, had significant effects on wall viscoelasticity, but others had little or no apparent effect, thus implicating an enzymatic creep mechanism. The results indicate that creep depends on relatively rugged enzymes that are firmly attached to or entangled in the wall. The sensitivity of creep to SH-reducing agents indicates that thiol reduction of wall enzymes might provide a control mechanism for endogenous cell growth.Abbreviations DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetraacetic acid - Hepes N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid     

Single visit versus multiple visit root canal

Single visit endodontics offers many advantages over multi visit treatment. Therefore, it is of interest to assess the preference of single visit over multiple visit root canals. We used 86,000 patient records and selected 9017 records matching the inclusion criteria for the analysis using statistical tools (Chi square test at p value <0.05). Data shows that people between 26 to 45 years are often affected with dental caries. Available data is biased towards multi visits rather than single visit regardless number of canals.     

Activity and movement patterns of Octopus dofleini

Four individual Octopus dofleini were followed over a fifteen day period in their underwater habitat by sonic tracking. Octopuses had small home ranges (250 m²maximum) which were stable over the two weeks of observation. These ranges overlapped those of other octopuses extensively, and animals did not keep a constant distance from one another, which suggests they were solitary and non‐social. Activity was slightly higher at night, and was not correlated with changes in other environmental variables. The O. dofleini made frequent short hunting trips (mode 0.5 hours), tending to have more and longer excursions at night. Octopuses are predators who appear to have flexible activity and, in the short term, restricted home range areas.     

Influence of glutamate and GABA transport on brain excitatory/inhibitory balance

An optimally functional brain requires both excitatory and inhibitory inputs that are regulated and balanced. A perturbation in the excitatory/inhibitory balance—as is the case in some neurological disorders/diseases (e.g. traumatic brain injury Alzheimer’s disease, stroke, epilepsy and substance abuse) and disorders of development (e.g. schizophrenia, Rhett syndrome and autism spectrum disorder)—leads to dysfunctional signaling, which can result in impaired cognitive and motor function, if not frank neuronal injury. At the cellular level, transmission of glutamate and GABA, the principle excitatory and inhibitory neurotransmitters in the central nervous system control excitatory/inhibitory balance. Herein, we review the synthesis, release, and signaling of GABA and glutamate followed by a focused discussion on the importance of their transport systems to the maintenance of excitatory/inhibitory balance.     

mGluRs Modulate Strength and Timing of Excitatory Transmission in Hippocampal Area CA3

Excitatory transmission within hippocampal area CA3 stems from three major glutamatergic pathways: the perforant path formed by axons of layer II stellate cells in the entorhinal cortex, the mossy fiber axons originating from the dentate gyrus granule cells, and the recurrent axon collaterals of CA3 pyramidal cells. The synaptic communication of each of these pathways is modulated by metabotropic glutamate receptors that fine-tune the signal by affecting both the timing and strength of the connection. Within area CA3 of the hippocampus, group I mGluRs (mGluR1 and mGluR5) are expressed postsynaptically, whereas group II (mGluR2 and mGluR3) and III mGluRs (mGluR4, mGluR7, and mGluR8) are expressed presynaptically. Receptors from each group have been demonstrated to be required for different forms of pre- and postsynaptic long-term plasticity and also have been implicated in regulating short-term plasticity. A recent observation has demonstrated that a presynaptically expressed mGluR can affect the timing of action potentials elicited in the postsynaptic target. Interestingly, mGluRs can be distributed in a target-specific manner, such that synaptic input from one presynaptic neuron can be modulated by different receptors at each of its postsynaptic targets. Consequently, mGluRs provide a mechanism for synaptic specialization of glutamatergic transmission in the hippocampus. This review will highlight the variability in mGluR modulation of excitatory transmission within area CA3 with an emphasis on how these receptors contribute to the strength and timing of network activity within pyramidal cells and interneurons.     

Concurrent versus Sequential Sorafenib Therapy in Combination with Radiation for Hepatocellular Carcinoma

Sorafenib (SOR) is the only systemic agent known to improve survival for hepatocellular carcinoma (HCC). However, SOR prolongs survival by less than 3 months and does not alter symptomatic progression. To improve outcomes, several phase I-II trials are currently examining SOR with radiation (RT) for HCC utilizing heterogeneous concurrent and sequential treatment regimens. Our study provides preclinical data characterizing the effects of concurrent versus sequential RT-SOR on HCC cells both in vitro and in vivo. Concurrent and sequential RT-SOR regimens were tested for efficacy among 4 HCC cell lines in vitro by assessment of clonogenic survival, apoptosis, cell cycle distribution, and γ-H2AX foci formation. Results were confirmed in vivo by evaluating tumor growth delay and performing immunofluorescence staining in a hind-flank xenograft model. In vitro, concurrent RT-SOR produced radioprotection in 3 of 4 cell lines, whereas sequential RT-SOR produced decreased colony formation among all 4. Sequential RT-SOR increased apoptosis compared to RT alone, while concurrent RT-SOR did not. Sorafenib induced reassortment into less radiosensitive phases of the cell cycle through G₁-S delay and cell cycle slowing. More double-strand breaks (DSBs) persisted 24 h post-irradiation for RT alone versus concurrent RT-SOR. In vivo, sequential RT-SOR produced the greatest tumor growth delay, while concurrent RT-SOR was similar to RT alone. More persistent DSBs were observed in xenografts treated with sequential RT-SOR or RT alone versus concurrent RT-SOR. Sequential RT-SOR additionally produced a greater reduction in xenograft tumor vascularity and mitotic index than either concurrent RT-SOR or RT alone. In conclusion, sequential RT-SOR demonstrates greater efficacy against HCC than concurrent RT-SOR both in vitro and in vivo. These results may have implications for clinical decision-making and prospective trial design.     

Chronic HCV Infection Affects the NK Cell Phenotype in the Blood More than in the Liver

Although epidemiological and functional studies have implicated NK cells in protection and early clearance of HCV, the mechanism by which they may contribute to viral control is poorly understood, particularly at the site of infection, the liver. We hypothesized that a unique immunophenotypic/functional NK cell signature exists in the liver that may provide insights into the contribution of NK cells to viral control. Intrahepatic and blood NK cells were profiled from chronically infected HCV-positive and HCV-negative individuals. Baseline expression of activating and inhibitory receptors was assessed, as well as functional responses following stimulation through classic NK cell pathways. Independent of HCV infection, the liver was enriched for the immunoregulatory CD56^bright NK cell population, which produced less IFNγ and CD107a but comparable levels of MIP1β, and was immunophenotypically distinct from their blood counterparts. This profile was mostly unaltered in chronic HCV infection, though different expression levels of NKp46 and NKG2D were associated with different grades of fibrosis. In contrast to the liver, chronic HCV infection associated with an enrichment of CD161^lowperforin^high NK cells in the blood correlated with increased AST and 2B4 expression. However, the association of relatively discrete changes in the NK cell phenotype in the liver with the fibrosis stage nevertheless suggests an important role for the NK response. Overall these data suggest that tissue localization has a more pervasive effect on NK cells than the presence of chronic viral infection, during which these cells might be mostly attuned to limiting immunopathology. It will be important to characterize NK cells during early HCV infection, when they should have a critical role in limiting infection.     

Mechanism of blue-light-induced plasma-membrane depolarization in etiolated cucumber hypocotyls

A large, transient depolarization of the plasma membrane precedes the rapid blue-light (BL)-induced growth suppression in etiolated seedlings of Cucumis sativus L. The mechanism of this voltage transient was investigated by applying inhibitors of ion channels and the plasma-membrane H⁺-ATPase, by manipulating extracellular ion concentrations, and by measuring cell input resistance and ATP levels. The depolarizing phase was not affected by Ca²⁺-channel blockers (verapamil, La³⁺) or by reducing extracellular free Ca²⁺ by treatment with ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA). However, these treatments did reduce the rate of repolarization, indicating an inward movement of Ca²⁺ is involved. No effects of the K⁺-channel blocker tetraethylammonium (TEA⁺) were detected. Vanadate and KCN, used to inhibit the H⁺-ATPase, reduced or completely inhibited the BL-induced depolarization. Levels of ATP increased by 11–26% after 1–2 min of BL. Input resistance of trichome cells, measured with double-barreled microelectrodes, remained constant during the onset of the depolarization but decreased as the membrane voltage became more positive than -90 mV. The results indicate that the depolarization mechanism initially involves inactivation of the H⁺-ATPase with subsequent transient activation of one or more types of ion channels.Abbreviations and Symbols BL blue light - CI current injection - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - TEA⁺ tetraethylammonium - V_m membrane voltage We wish to thank Drs. Adam Bertl and Clifford L. Slayman, Yale School of Medicine, New Haven, Conn., USA, for helpful discussions. This work was supported by a Natural Sciences and Engineering Research Council of Canada Scholarship (E.P.S.) and National Science Foundation Grant DMB-8351030 (D.J.C.).