Temporal characteristics of activation,deactivation, and restimulation of signal transduction following depolarization in the pheochromocytoma cell line PC12

This study focuses on the transient and dynamic activation of intracellular signal transduction following different protocols of depolarization. During chronic depolarization, phosphorylation of extracellular signal-regulated kinases (ERKs) was observed to peak and subsequently fall to low levels within 10 min of depolarization. Short periods of depolarization, from 1 to 5 min in duration, also led to phosphorylation of ERK, and the rate of ERK dephosphorylation was not affected by the duration of depolarization. Phosphorylation of the cyclic AMP response element binding protein (CREB) also peaked as a result of chronic depolarization but decreased to intermediate levels that were maintained for more than 1 h. Pulsatile depolarization was explored as a means to circumvent the deactivation of intracellular signaling activity during chronic depolarization. Both ERK and CREB were rephosphorylated by a second period of depolarization that followed a recovery period of 10 min or more. The effects of the durations of depolarization and interpulse recovery on reactivation of ERK and CREB were characterized. Measurements of free cytoplasmic Ca(2+) confirmed the transient rise in the intracellular calcium concentration ([Ca(2+)](i)) during chronic depolarization and the pulsatile increase in [Ca(2+)](i) that can be achieved with short periods of depolarization. This study characterizes the dynamic activities of signal transduction following depolarization. Electrical stimulation of neurons induces many cellular changes that unfold over time, and the influx of Ca(2+) ions that mediate these events is transient. This study suggests that pulsatile activity may be a means of maintaining signaling activity over long periods of time.     

Catabolism of an isolated, purified intermediate of prostaglandin biosynthesis by regions of the adult rat kidney.

1. A heat labile, cold-stable, stannous chloride-reducible intermediate of prostaglandin biosynthesis was formed in good yield (greater than 60%) from 3H-labeled arachidonic acid during brief incubations (30--90 s, 37 degrees C) with sheep seminal vesicle microsomes in the presence of p-hydroxymercuribenzoate (4 mM). This intermediate appears to have properties similar to one of the endoperoxides (15-hydroxyprostaglandin-9,11-endoperoxide) recently isolated by Hamberg and Samuelsson (Proc. Natl. Acad. Sci. U.S. (1973) 70, 889-903) AND Nugteren and Hazelhof (Biochem. Biophys. Acta. (1973) 326, 448-461). 2. Treatment of the purified intermediate with homogenates of rat kidney cortex, medulla and papilla resulted within 2 min (37 degrees C) in complete conversion into several compounds including prostaglandins E2 and F2alpha. The main product (40-50% yield formed by papilla homogenates was prostaglandin E2. The conversion into prostaglandin E2 was largely abolished by previous bo9ling of the homogenate whereas the conversion into prostaglandin F2alpha was not. The intermediate was stable in buffer for the same period of incubation. 3. The ratio of tritiated prostaglandins E2: F2alpha obtained were: papilla, 1.90; medulla, 0.76; cortex, 0.48. 4. These observations indicate that both types of prostaglandins can be formed by all three regions of the rat kidney and that regional differences exist in the proportion of E2 : F2alpha that is formed. Whereas prostaglandin E2 is mostly formed by an enzymatic process, prostaglandin F2alpha is not.     

The use of a GIS-based multi-criteria evaluation technique for the development of a zoning plan for a seasonally variable Ramsar wetland site in Syria: Sabkhat Al-Jabboul

Zoning is an important tool used in the management of protected areas and wetlands. It has been developed to apply conservation objectives at different levels of management where conflicting uses can be taking place. GIS is increasingly used to solve land allocation problem with techniques based on zoning, varying from simple overlays to complex multi-criteria evaluation techniques (MCE). This study presents a new approach to the development of zoning plans for Ramsar sites that exhibit major seasonal variations in conservation value and human use and threats. It employs a well-defined GIS-based MCE technique which involves both expert judgment and a set of criteria based on ecological and potential threats. A GIS model using the additive approach in MCE was developed to evaluate Sabkhat Al-Jabboul the only Ramsar site in Syria. The model was applied separately for the spring and winter to accommodate seasonal changes in the intensity and spatial distribution of conservation areas and threats across the site. Separate zoning plans were developed for each of the two main seasons. The zoning plan of the spring season, which is the birds breeding season, included three management zones; the special conservation zone, the conservation buffer zone, and the wise use zone. The winter zoning plan, where the bird feeding activity was the major activity includes two zones: a special conservation zone and a wise use zone. Thus, in cases like Sabkhat Al-Jabboul, where the wetlands are subject to significant seasonal variations, in their ecology and human utilization, it is recommended that flexible seasonal zoning plans are developed to assist in the organization of permitted activities, and to reduce the potential human impacts throughout the year.     

CATABOLISM OF PROSTAGLANDIN ENDOPEROXIDES INTO PROSTAGLANDIN E2 AND F2x BY THE RAT BRAIN

Abstract— Tritium labeled prostaglandin (PG) endoperoxides PGG₂ and PGH₂ were rapidly transformed (2 min, 37°C) in good yield (> 50%) by homogenates of whole rat brain into a mixture of products including prostaglandin E₂ and F_2x: under similar conditions (10min. 37°C) tritium labeled arachidonic acid remained essentially unoxidised. The ratio of PGE-like products: PGF_2x formed was approx 0.5 as determined by radio thin layer chromatography. This ratio changed to unity when homogenates of cerebral cortex or cerebral hemispheres were employed. On the other hand cerebellar homogenates formed PGF_2x in much greater amounts. The structures of the products were confirmed by mass spectrometry and were further supported by experiments using octadeuterio-endoperoxides. In the latter experiments the resulting PGE₂ and PGF_2x contained the expected seven and eight deuterium atoms respectively. Evidence for the formation of heptadeuterio PGD₂. heptadeuterio-6-keto-PGF₁, and hexadeuterio 12-hydroxyheptadecatrienoic acid was also obtained by mass spectrometry. These experiments demonstrate for the first time in brain tissue the biosynthesis of labeled prostaglandins from exogenous tritiated and deuterated precursors.     

Detection of functional groups and antibodies on microfabricated surfaces by confocal microscopy

Fluorescence confocal microscopy was used to characterize micron-sized microfabricated silicon particles and planar oxide surfaces after silanization and immobilization of IgG antibody. Surfaces treated with amino- and mercaptosilanes were tested for the presence of amine and sulfhydryl groups by labeling with specific fluorescein probes. In addition, human antibody (IgG) was immobilized to the thiol-coated microparticles using the heterobifunctional crosslinker succinimidyl 4-(N-maleimidolmethyl)-cyclohexane-1-carboxylate. Estimates of the surface density of IgG were consistent with 8.3% of a monolayer of covalently-bound antibody. Confocal images confirmed uniform layers of both silanes and antibodies on the microparticles. The sensitivity limit for the confocal measurements was determined to be as low as 1.5 x 10(-5) fluors per nm2.     

Vaccination of castration-resistant prostate cancer patients with TroVax (MVA–5T4) in combination with docetaxel: a randomized phase II trial

The attenuated vaccinia virus, modified vaccinia Ankara, has been engineered to deliver the tumor antigen 5T4 (TroVax^®). Here, we report results from a randomized open-label phase II trial in castration-resistant prostate cancer patients in which TroVax was administered in combination with docetaxel and compared against docetaxel alone. The aim was to recruit 80 patients (40 per arm), but the study was terminated early due to recruitment challenges. Therefore, this paper reports the comparative safety and immunological and clinical efficacy in 25 patients, 12 of whom were treated with TroVax plus docetaxel and 13 with docetaxel alone. 5T4-specific immune responses were monitored throughout the study. Clinical responses were assessed by measuring changes in tumor burden by CT and bone scan and by quantifying PSA concentrations. TroVax was well tolerated in all patients. Of 10 immunologically evaluable patients, 6 mounted 5T4-specific antibody responses. Patients treated with TroVax plus docetaxel showed a greater median progression-free survival of 9.67 months compared with 5.10 months for patients on the docetaxel alone arm (P = 0.097; HR = 0.31; 95 % CI 0.08–1.24). Importantly, a pre-treatment biomarker previously demonstrated to predict 5T4 immune response and treatment benefit showed a strong association with 5T4 antibody response and a statistically significant association with progression-free survival in patients treated with TroVax plus docetaxel, but not docetaxel alone.