Synthesis and Enzymatic Deprotection of Fully Protected 2′‐5′ Oligoadenylates (2‐5A): Towards a Prodrug Strategy for Short 2‐5A

Fully protected pA2′p5′A2′p5′A trimers 1a and 1b have been prepared as prodrug candidates for a short 2′‐5′ oligoadenylate, 2‐5A, and its 3′‐O‐Me analog, respectively. The kinetics of hog liver carboxyesterase (HLE)‐triggered deprotection in HEPES buffer (pH 7.5) at 37° has been studied. The deprotection of 1a turned out to be very slow, and 2‐5A never appeared in a fully deprotected form. By contrast, a considerable proportion of 1b was converted to the desired 2‐5A trimer, although partial removal of the 3′‐O‐[(acetyloxy)methyl] group prior to exposure of the adjacent phosphodiester linkage resulted in 2′,5′→3′,5′ phosphate migration and release of adenosine as side reactions.     

Reversible pegylation prolongs the hypotensive effect of atrial natriuretic peptide

Natriuretic peptides (NP), including atrial natriuretic peptide (ANP), induce potent natriuresis and vasodilation and thereby generate hypotension in vivo. Despite intensive efforts, clinical application of NP as an antihypertensive agent is limited because of their short biological half-life and poor bioavailability. Recently, we have developed a strategy that facilitates slow release of peptides from PEG-peptide inactive conjugates, based on reversible pegylation. Peptides prepared by this approach undergo slow, spontaneous chemical hydrolysis at physiological conditions, releasing the native active peptide/protein drug from the inactive conjugates over prolonged periods. A PEG chain of 30 kDa was linked covalently to the alpha-amino side chain of the hormone via a MAL-Fmoc-NHS spacer, yielding PEG 30-Fmoc-ANP, a prodrug that releases the native hormone upon incubation at physiological conditions. Bolus administration of native ANP to Wistar rats receiving adrenaline yields a short, transitory effect in lowering blood pressure (BP), reaching a maximum at 2 min, and then returning to control values after 12 to 25 min. In contrast, administration of PEG 30-Fmoc-ANP lowered BP following a lag period of 50 min, and maintained low BP for a period exceeding 60 min. Saline or PEG 30-Fmoc-Alanine were not effective in lowering BP in Wistar rats. These results show that the novel compound, PEG 30-Fmoc-ANP, is a reversible pegylated prodrug derivative that facilitates a prolonged BP lowering effect in rats and may be considered as a candidate for development into an antihypertensive drug.     

A p38 MAPK-CREB pathway functions to pattern mesoderm in Xenopus

Dorsal-ventral patterning is specified by signaling centers secreting antagonizing morphogens that form a signaling gradient. Yet, how morphogen gradient is translated intracellularly into fate decisions remains largely unknown. Here, we report that p38 MAPK and CREB function along the dorsal-ventral axis in mesoderm patterning. We find that the phosphorylated form of CREB (S133) is distributed in a gradient along the dorsal-ventral mesoderm axis and that the p38 MAPK pathway mediates the phosphorylation of CREB. Knockdown of CREB prevents chordin expression and mesoderm dorsalization by the Spemann organizer, whereas ectopic expression of activated CREB-VP16 chimera induces chordin expression and dorsalizes mesoderm. Expression of high levels of p38 activator, MKK6E or CREB-VP16 in embryos converts ventral mesoderm into a dorsal organizing center. p38 MAPK and CREB function downstream of maternal Wnt/β-catenin and the organizer-specific genes siamois and goosecoid. At low expression levels, MKK6E induces expression of lateral genes without inducing the expression of dorsal genes. Loss of CREB or p38 MAPK activity enables the expansion of the ventral homeobox gene vent1 into the dorsal marginal region, preventing the lateral expression of Xmyf5. Overall, these data indicate that dorsal-ventral mesoderm patterning is regulated by differential p38/CREB activities along the axis.     

Differential sorting and Golgi export requirements for raft-associated and raft-independent apical proteins along the biosynthetic pathway

Sorting signals for apically destined proteins are highly diverse and can be present within the luminal, membrane-associated, and cytoplasmic domains of these proteins. A subset of apical proteins partition into detergent-resistant membranes, and the association of these proteins with glycolipid-enriched microdomains or lipid rafts may be important for their proper targeting. Recently, we observed that raft-associated and raft-independent apical proteins take different routes to the apical surface of polarized Madin-Darby canine kidney cells (Cresawn, K. O., Potter, B. A., Oztan, A., Guerriero, C. J., Ihrke, G., Goldenring, J. R., Apodaca, G., and Weisz, O. A. (2007) EMBO J. 26, 3737-3748). Here we reconstituted in vitro the export of raft-associated and raft-independent markers staged intracellularly at 19 degrees C. Surprisingly, whereas release of the raft-associated protein influenza hemagglutinin was dependent on the addition of an ATP-regenerating system and cytosol, release of a yellow fluorescent protein (YFP)-tagged raft-independent protein (the 75-kDa neurotrophin receptor; YFP-p75) was efficient even in the absence of these constituents. Subsequent studies suggested that YFP-p75 is released from the trans-Golgi network in fragile tubules that do not withstand isolation procedures. Moreover, immunofluorescence analysis revealed that hemagglutinin and YFP-p75 segregate into distinct subdomains of the Golgi complex at 19 degrees C. Our data suggest that raft-associated and raft-independent proteins accumulate at distinct intracellular sites upon low temperature staging, and that upon warming, they exit these compartments in transport carriers that have very different membrane characteristics and morphologies.     

不同时辰电针对大鼠杏仁核一氧化氮合酶表达的影响

目的观察不同时辰电针对大鼠杏仁核一氧化氮合酶(NOS)表达的影响.方法采用还原型尼克酰胺腺嘌呤二核苷酸脱氢酶(NADPH-d)法,观察不同时辰电针大鼠一侧"足三里"穴对杏仁核NOS表达的影响.结果电针对大鼠杏仁皮质内侧核群、基底外侧核群NOS的表达有上调作用,并存在时辰差异(P<0.05);电针对大鼠杏仁中央核NOS表达无明显作用(P>0.05).结论电针对大鼠杏仁核NOS表达的影响有时辰差异.     

Transactivation of ErbB2 and ErbB3 by tumor necrosis factor-alpha and anisomycin leads to impaired insulin signaling through serine/threonine phosphorylation of IRS proteins.

The cellular pathways involved in the impairment of insulin signaling by cellular stress, triggered by the inflammatory cytokine tumor necrosis factor-alpha (TNF) or by translational inhibitors like cycloheximide and anisomycin were studied. Similar to TNF, cycloheximide and anisomycin stimulated serine phosphorylation of IRS-1 and IRS-2, reduced their ability to interact with the insulin receptor, inhibited the insulin-induced tyrosine phosphorylation of IRS proteins, and diminished their association with phosphatidylinositol 3-kinase (PI3K). These defects were partially reversed by wortmannin and LY294002, indicating that a PI3K-regulated step is critical for the impairment of insulin signaling by cellular stress. Induction of cellular stress resulted in complex formation between PI3K and ErbB2/ErbB3 and enhanced PI3K activity, implicating ErbB proteins as downstream effectors of stress-induced insulin resistance. Indeed, stimulation of ErbB2/ErbB3 by NDFbeta1, the ErbB3 ligand, inhibited IRS protein tyrosine phosphorylation and recruitment of downstream effectors. Specific inhibitors of the ErbB2 tyrosine kinase abrogated the activation of ErbB2/ErbB3 and in parallel prevented the reduction in IRS protein functions. Taken together, our results suggest a novel mechanism by which cellular stress induces cross-talk between two different signaling pathways. Stress-dependent transactivation of ErbB2/ErbB3 receptors triggers a PI3K cascade that induces serine phosphorylation of IRS proteins culminating in insulin resistance.     

A Synechococcus PglnA::luxAB Fusion for Estimation of Nitrogen Bioavailability to Freshwater Cyanobacteria

In contrast to extensive studies of phosphorus, widely considered the main nutrient limiting phytoplankton biomass in freshwater ecosystems, there have been few studies on the role of nitrogen in controlling phytoplankton populations. This situation may be due partly to the complexity in estimating its utilization and bioavailability. In an attempt to provide a novel tool for this purpose, we fused the promoter of the glutamine synthetase-encoding gene, P glnA, from Synechococcus sp. strain PCC7942 to the luxAB luciferase-encoding genes of the bioluminescent bacterium Vibrio harveyi. The resulting construct was introduced into a neutral site on the Synechococcus chromosome to yield the reporter strain GSL. Light emission by this strain was dependent upon ambient nitrogen concentrations. The linear response range of the emitted luminescence was 1 mM to 1 μM for the inorganic nitrogen species tested (ammonium, nitrate, and nitrite) and 10- to 50-fold lower for glutamine and urea. When water samples collected from along a depth profile in Lake Kinneret (Israel) were exposed to the reporter strain, the bioluminescence of the reporter strain mirrored the total dissolved nitrogen concentrations determined for the same samples and was shown to be a sensitive indicator of the concentration of bioavailable nitrogen.     

Biodegradation of BTEX by bacteria on powdered activated carbon

Aerobic degradation of a mixture of benzene, toluene, ethylbenzene and the mixed xylenes (BTEX) by a mixed bacterial population was studied in a continuously fed, completely mixed bioreactor in the presence of powdered activated carbon (PAC). Adsorption was characterized in the presence and in the absence of bacteria on PAC, and the affinity of virgin PAC to individual BTEX components was shown to be inversely correlated to their solubility in water. Bacteria colonizing the PAC particles are essential for simultaneous adsorption–biodegradation processes. In order to restrict biofilm formation and thereby mass transfer resistance of pollutants from the bulk to the PAC, the slurry was recirculated in the reactor system using a high shear pump. The bacteria on the PAC surface were constantly in a flux between the adsorbed and free phase, a phenomenon that prevented the formation of a biofilm on the PAC surface and thereby extended the life of the PAC.     

Demographic population structure of black howler monkeys in fragmented and continuous forest in Chiapas,Mexico: Implications for conservation

For wild primates, demography studies are increasingly recognized as necessary for assessing the viability of vulnerable populations experiencing rapid environmental change. In particular, anthropogenic changes such as habitat loss and fragmentation can cause ecological and behavioral changes in small, isolated populations, which may, over time, alter population density and demographic structure (age/sex classes and group composition) in fragment populations relative to continuous forest populations. We compared our study population of Endangered black howler monkeys (Alouatta pigra) in 34 forest fragments around Palenque National Park (PNP), Mexico (62 groups, 407 individuals), to the adjacent population in PNP, protected primary forest (21 groups, 134 individuals), and to previous research on black howlers in fragments in our study area (18 groups, 115 individuals). We used χ² and Mann–Whitney U tests to address the questions: (a) what is the current black howler demographic population structure in unprotected forest fragments around PNP? (b) How does it compare to PNP's stable, continuous population? (c) How has it changed over time? Compared to the PNP population, the fragment populations showed higher density, a significantly lower proportion of multimale groups, and significantly fewer adult males per group. The population's age/sex structure in the fragmented landscape has been stable over the last 17 years, but differed in a higher proportion of multifemale groups, higher density, and higher patch occupancy in the present. In the context of conservation, some of our results may be positive as they indicate possible population growth over time. However, long-term scarcity of adult males in fragments and associated effects on population demographic structure might be cause for concern, in that it may affect gene flow and genetic diversity. The scarcity of adult males might stem from males experiencing increased mortality while dispersing in the fragmented landscape, whereas females might be becoming more philopatric in fragments.     

LIMK2 Mediates Resistance to Chemotherapeutic Drugs in Neuroblastoma Cells through Regulation of Drug-Induced Cell Cycle Arrest

Drug resistance is a major obstacle for the successful treatment of many malignancies, including neuroblastoma, the most common extracranial solid tumor in childhood. Therefore, current attempts to improve the survival of neuroblastoma patients, as well as those with other cancers, largely depend on strategies to counter cancer cell drug resistance; hence, it is critical to understand the molecular mechanisms that mediate resistance to chemotherapeutics. The levels of LIM-kinase 2 (LIMK2) are increased in neuroblastoma cells selected for their resistance to microtubule-targeted drugs, suggesting that LIMK2 might be a possible target to overcome drug resistance. Here, we report that depletion of LIMK2 sensitizes SHEP neuroblastoma cells to several microtubule-targeted drugs, and that this increased sensitivity correlates with enhanced cell cycle arrest and apoptosis. Furthermore, we show that LIMK2 modulates microtubule acetylation and the levels of tubulin Polymerization Promoting Protein 1 (TPPP1), suggesting that LIMK2 may participate in the mitotic block induced by microtubule-targeted drugs through regulation of the microtubule network. Moreover, LIMK2-depleted cells also show an increased sensitivity to certain DNA-damage agents, suggesting that LIMK2 might act as a general pro-survival factor. Our results highlight the exciting possibility of combining specific LIMK2 inhibitors with anticancer drugs in the treatment of multi-drug resistant cancers.