PKC-dependent stimulation of EAAT3 glutamate transporter does not require the integrity of actin cytoskeleton

The activity and the membrane expression of EAAT3 glutamate transporter are stimulated upon PKC activation by phorbol esters in C6 rat glioma cells. To investigate the role of cytoskeleton in these effects, we have employed actin-perturbing toxins and found that the perturbation of actin cytoskeleton inhibits basal but not phorbol-stimulated EAAT3 activity and membrane trafficking. In the absence of phorbols, latrunculin A, a toxin that disassembles actin cytoskeleton, produced a rapid inhibition of EAAT3 activity, due to a decrease in transport V(max). The inhibitory effect was fully reversible and was not detected for other sodium dependent transport systems for amino acids. However, latrunculin did not prevent the increase in transport caused by phorbol esters and, moreover, cells pre-treated with phorbols were resistant to the inhibitory effect of the toxin on EAAT3 activity. Biotinylation experiments indicated that the inhibitory effect of latrunculin was attributable to a decreased expression of the carrier on the membrane, while the toxin did not suppress the PKC-dependent increase in EAAT3 membrane abundance. Latrunculin A effects on EAAT3 were shared by cytochalasin D, a toxin that disorganizes actin filaments with a distinct mechanism of action. On the contrary, a small, but significant, increase of EAAT3 activity was observed upon incubation with jasplakinolide, a drug that stabilizes actin microfilaments. Also jasplakinolide, however, did not hinder phorbol-dependent stimulation of aspartate transport. Colchicine, a toxin that disrupts microtubules, also lowered EAAT3 activity without preventing transport stimulation by phorbols, while microtubule stabilization by paclitaxel led to an increase in aspartate transport. It is concluded that, in C6 cells, the PKC-mediated stimulatory effects on EAAT3 are cytoskeleton-independent, while in the absence of phorbols, the transporter is partially inhibited by the disorganization of either actin microfilaments or microtubules. These results suggest that EAAT3 trafficking in C6 cells involves different pools of transporters.     

Identification of the sex pheromone of Copitarsia decolora (Lepidoptera: Noctuidae)

Chemical and electrophysiological analyses and field trials were used to identify the female sex pheromone of Copitarsia decolora (Guenée) (Lepidoptera: Noctuidae). Coupled gas chromatographic-electroantennographic detection (GC-EAD) analysis of the female gland extract showed the presence of two EAD-active peaks, which were identified by GC-mass spectrometric (MS) analysis as (Z)-9-tetradecenyl acetate (Z9-14:Ac) and (Z)-9-tetradecenol (Z9-14:OH). The field evaluation of the EAD-active compounds indicated that traps baited with either Z9-14:Ac or Z9-14:OH caught few males. In contrast, traps baited with the binary blend of both components caught significantly more males than traps baited with the single compounds. Captures in traps baited with a mixture of Z9-14:Ac and Z9 -14:OH at 4:1, 10:1, and 100:1 ratios were not significantly different from the catches in traps baited with virgin females. Few males were captured in traps baited with a blend of Z9-14:Ac and Z9-14:OH at 1:4, 1:10, and 1:100 ratios.     

The characterization of the soybean polygalacturonase-inhibiting proteins (Pgip) gene family reveals that a single member is responsible for the activity detected in soybean tissues

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins that inhibit fungal endopolygalacturonases (PGs). They are encoded by multigene families whose members show functional redundancy and subfunctionalization for recognition of fungal PGs. In order to expand the information on the structure and functional features of legume PGIP, we have isolated and characterized four members of the soybean Pgip gene family and determined the properties of the encoded protein products. Sequence analysis showed that these genes form two clusters: one cluster of about 5 kbp containing Gmpgip1 and Gmpgip2, and the other containing Gmpgip3 and Gmpgip4 within a 60 kb fragment of a separate BAC clone. Sequence diversification of the four members resides mainly in the xxLxLxx region that includes residues forming the β-sheet B1. When compared with other legume Pgip genes, Gmpgip3 groups with the bean genes Pvpgip1 and Pvpgip2, suggesting that these genes are closer to the ancestral gene. At the protein level, only GmPGIP3 shows the capability to inhibit fungal PGs. The spectrum of inhibition of GmPGIP3 against eight different fungal PGs mirrors that of the PGIP purified from soybean tissues and is similar to that of the bean PvPGIP2, one of the most efficient inhibitors so far characterized. We also report that the four Gmpgip genes are differentially regulated after wounding or during infection with the fungal pathogen Sclerotinia sclerotiorum. Following fungal infection Gmpgip3 is up regulated promptly, while Gmpgip2 is delayed.     

Extracranial stereotactic body radiotherapy. Review of main SBRT features and indications in primary tumors

Aim

Review of main SBRT features and indications in primary tumors.

Background

Stereotactic body radiotherapy has been developed in the last few years. SBRT allows the hypofractionated treatment of extra cranial tumors, using either a single or limited number of dose fractions, and resulting in the delivery of a high biological effective dose with low toxicity.

Material and methods

SBRT requires a high level of accuracy for all phases of the treatment process: effective patient immobilization, precise target localization, highly conformed dosimetry and image guided systems for treatment verification. The implementation of SBRT in routine requires a careful considering of organ motion. Gating and tracking are effective ways to do so, and less invasive technologies “fiducials free” have been developed. Due to the hypofractionated scheme, the physician must pay attention to new dosimetric constraints in organ at risk and new radiobiological models are needed to assess the optimal fractionation and dose schemes.

Results

Currently, SBRT is safe and effective to treat primary tumors, which are otherwise untreatable with conventional radiotherapy or surgery. SBRT has quickly developed because of its excellent results in terms of tolerance and its high locoregional control rates. SBRT indications in primary tumors, such as lung primary tumors, have become a standard of care for inoperable patients. SBRT seems to be effective in many others indications in curative or palliative intent such as liver primary tumors, and novel indications and strategies are currently emerging in prostate cancer, head and neck tumor recurrences or pelvis reirradiations.

Conclusion

Currently, SBRT is mainly used when there is no other therapeutic alternative for the patient. This is due to the lack of randomized trials in these settings. However, the results shown in retrospective studies let us hope to impose SBRT as a new standard of care for many patients in the next few years.     

A bit of quiet between the migrations: the resting life of the European eel during their freshwater growth phase in a small stream

The movements and habitat use of resident yellow eels were studied in a stream stretch having both natural and minimum flow zones. N = 12 individuals (total length 505–802 mm) were surgically tagged with radio transmitters and released at their capture sites. They were located using manual radio receivers during the daytime from 2 to 5 days/week over periods ranging from 200 to 329 days, for a total of 1,098 positions. Eels showed home ranges ranging from 33 to 341 m (median value, 62 m), displayed strong fidelity to sites and demonstrated a great degree of plasticity in habitat use. Eels were slightly mobile throughout the year, but their movements were season and temperature dependent, with a maximum during the spring (mean water temperature, 12 °C) and a minimum in winter (3 °C). Stones and roots (utilization rate greater than 50 % of eels for more than 30 % of location days) were significantly the most frequently used habitats. Between the two flow zones, the natural flow was the most occupied, with a significantly higher proportion of resident eels (66.7 % of radio-tagged yellow eels) and longer occupation (81 % of location days) than the minimum flow zone with less suitable habitats.     

Constitutive expression of the embryonic stem cell marker OCT4 in bovine somatic donor cells influences blastocysts rate and quality after nucleus transfer

Nuclear transfer (NT) is associated with epigenetic reprogramming of donor cells. Expression of certain genes in these cells might facilitate their expression in the NT embryo. This research was aimed to investigate the effect of constitutive expression of OCT4 in bovine somatic cells used for NT on the developmental potential of derived cloned embryos as well as in the expression of pluripotency markers in the Day-7 resulting embryos. Cloned blastocysts were generated from five cell lines that expressed OCT4. Pools of blastocysts were screened to detect OCT4, SOX2, and NANOG by qPCR. In vitro-fertilized time-matched blastocysts were used as controls. The development potential was assessed on the basis of blastocysts rate; grading and total cell counts at Day 7. OCT4 expression in the cell lines positively correlates with blastocysts rate (r?=?0.92; p?=?0.02), number of grade I blastocysts (r?=?0.96; p?=?0.01), and total cell number (r?=?0.98; p?=?0.002). The high expression of OCT4 in the cell line did not improve the final outcome of cloning. Somatic expression of OCT4 lead to increased expression of OCT4 and SOX2 in cloned grade I blastocysts; however, there was a bigger variability in OCT4 and SOX2 (p?=?0.03; p?=?0.02) expression in the embryos generated from cells expressing highest levels of OCT4. Probably the higher variability in OCT4 expression in cloned embryos is due to incorrect reprogramming and incapability of the oocyte to correct for higher OCT4 levels. For that reason, we concluded that OCT4 expression in somatic cells is not a good prognosis marker for selecting cell lines.