Metal-Induced Stabilization of Trypsin Modified with α-Oxoglutaric Acid†

Alpha-oxoglutaric acid was attached to trypsin via reductive alkylation with NaBH4 thereby introducing metal-chelating groups at the protein surface. The thermostability of the modified enzyme was increased by 6.5-13 degrees C and its resistance to autolytic degradation was improved 2- to 4-fold in 5 mM ZnCl2, MnCl2 or MgCl2.     

Calmodulin regulates intracellular trafficking of epidermal growth factor receptor and the MAPK signaling pathway

The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family involved in signal transduction and the regulation of cellular proliferation and differentiation. It is also a calmodulin-binding protein. To examine the role of calmodulin in the regulation of EGFR, the effect of calmodulin antagonist, W-13, on the intracellular trafficking of EGFR and the MAPK signaling pathway was analyzed. W-13 did not alter the internalization of EGFR but inhibited its recycling and degradation, thus causing the accumulation of EGF and EGFR in enlarged early endosomal structures. In addition, we demonstrated that W-13 stimulated the tyrosine phosphorylation of EGFR and consequent recruitment of Shc adaptor protein with EGFR, presumably through inhibition of the calmodulin-dependent protein kinase II (CaM kinase II). W-13-mediated EGFR phosphorylation was blocked by metalloprotease inhibitor, BB94, indicating a possible involvement of shedding in this process. However, MAPK activity was decreased by W-13; dissection of this signaling pathway showed that W-13 specifically interferes with Raf-1 activity. These data are consistent with the regulation of EGFR by calmodulin at several steps of the receptor signaling and trafficking pathways.     

Transgenic crops,biotechnology and ownership rights: what scientists need to know

Ownership of intellectual and tangible property (IP/TP) rights in agricultural biotechnology (ag-biotech) and transgenic plants has become critically important. For scientists in all institutions, whether industrialized or developing country, public or private sector, an understanding of IP/TP rights is fundamental in both research and development. Transgenic plants and ag-biotech products embody numerous components and processes, each of which may have IP/TP rights attached. To identify these rights, a transgenic plant or ag-biotech product must be dissected into its essential components and processes, with each 'piece' analysed under the IP/TP 'microscope'. This product deconstruction is an integral step in product clearance (PC) analysis leading to freedom to operate (FTO). To facilitate a PC analysis, the following points are important: (1) knowing what one has and where it's from, (2) organizing material transfer agreements and licences, (3) researching scientific and patent databases and relevant literature, (4) instituting a laboratory notebook policy, (5) keeping track of ownership of germplasm and plant genetic resources, and (6) promoting ongoing IP/TP management, awareness and training. However, a FTO opinion does not solve the IP/TP issues of releasing a transgenic plant or ag-biotech product; rather, it is a management tool for assessing the risks of litigation. When transferring transgenic plants or ag-biotech to developing nations, scientists from industrialized countries have the heightened responsibility of verifying that IP/TP issues are fully addressed and documented. Successful technology transfer goes beyond research, development and licensing; it is an holistic package leading to long-term partnerships in international development. Managing IP/TP requires capacity-building in scientists and technology transfer offices, in both industrialized and developing countries.     

Definition of appropriate temperature and storage conditions in the detection of Leishmania DNA with PCR in phlebotomine flies

For epidemiological studies and control programs of leishmaniasis, taxonomic identification of the etiologic agent of the disease in the insect vector is of critical importance. The implementation of molecular techniques such as the polymerase chain reaction (PCR) has permitted great advances in the efficacy and sensitivity of parasite identification. Previously, these investigations involved labor-intensive dissections and required expert personnel. The present work evaluates the effects of storage methods of phlebotomine samples in the optimization of PCR identification of Leishmania. Females of Lutzomyia longipalpis, from the colony of the Instituto Nacional de Salud, were experimentally infected with Leishmania chagasi (= L. infantum), from the upper Magdalena Valley (Quipile, Cundinamarca, Colombia). The infected insects were preserved in three solutions: 100% ethanol, 70% ethanol, and TE; subsamples of each class were stored at -80 degrees C, -20 degrees C and room temperature. To determine infection rates, samples were dissected and screened microscopically. Chelex 100 was used for extraction of total Leishmania DNA. For PCR amplification, the kinetoplastic minicircle DNA primers OL1 and OL2 of Leishmania were used, and the products were visualized by electrophoresis in 1% agarose gels. For each of the 3 storage conditions, amplifications were successful, producing a approximately 120 base pair product unique to Leishmania. The results demonstrated the advantage of PCR as a routine screening method for detecting infected flies in endemic foci of visceral leishmaniasis. Since storage method did not affect PCR amplification success, the most cost effective method -70% ethanol at room temperature--is the option recommended for storing entomological samples in vector incrimination studies.     

The kinetics of oligonucleotide replacements

The formation of a duplex between two nucleic acid strands is restricted if one of the strands forms an intra- or intermolecular secondary structure. The formation of the new duplex requires the dissociation and replacement of the initial structure. To understand the mechanism of this type of kinetics we studied the replacement of a labeled DNA oligonucleotide probe bound to a complementary DNA target with an unlabeled probe of the same sequence. The replacement kinetics were measured using a gel-shift assay for 12, 14 and 16-nucleotide probes as a function of temperature and concentration of the unlabeled probe. The results demonstrate that the overall replacement rate is a combination of two kinetic pathways: dissociative and sequential displacement. The dissociative pathway occurs by the spontaneous dissociation of the initial duplex followed by association of the target and unlabeled probe. The sequential displacement pathway requires only the partial melting of the initial duplex to allow for the formation of a branched nucleation complex with the unlabeled probe, followed by the complete displacement of the labeled probe by migration of the branch point. The contribution from the dissociative pathway is predominant at temperatures close to the melting point of the labeled probe, whereas the contribution from the displacement pathway prevails at lower temperatures and when the concentration of the replacing unlabeled probe is high. The results show that at physiological conditions, duplex formation between a single-stranded oligonucleotide probe and a structured region of a target molecule occurs mainly by the sequential-displacement mechanism.     

RhoE function is regulated by ROCK I-mediated phosphorylation

The Rho GTPase family member RhoE regulates actin filaments partly by binding to and inhibiting ROCK I, a serine/threonine kinase that induces actomyosin contractility. Here, we show that ROCK I can phosphorylate multiple residues on RhoE in vitro. In cells, ROCK I-phosphorylated RhoE localizes in the cytosol, whereas unphosphorylated RhoE is primarily associated with membranes. Phosphorylation has no effect on RhoE binding to ROCK I, but instead increases RhoE protein stability. Using phospho-specific antibodies, we show that ROCK phosphorylates endogenous RhoE at serine 11 upon cell stimulation with platelet-derived growth factor, and that this phosphorylation requires an active protein kinase C signalling pathway. In addition, we demonstrate that phosphorylation of RhoE correlates with its activity in inducing stress fibre disruption and inhibiting Ras-induced transformation. This is the first demonstration of an endogenous Rho family member being phosphorylated in vivo and indicates that phosphorylation is an important mechanism to control the stability and function of this GTPase-deficient Rho protein.     

Delay-Dependent Response in Weakly Electric Fish under Closed-Loop Pulse Stimulation

In this paper, we apply a real time activity-dependent protocol to study how freely swimming weakly electric fish produce and process the timing of their own electric signals. Specifically, we address this study in the elephant fish, Gnathonemus petersii, an animal that uses weak discharges to locate obstacles or food while navigating, as well as for electro-communication with conspecifics. To investigate how the inter pulse intervals vary in response to external stimuli, we compare the response to a simple closed-loop stimulation protocol and the signals generated without electrical stimulation. The activity-dependent stimulation protocol explores different stimulus delivery delays relative to the fish’s own electric discharges. We show that there is a critical time delay in this closed-loop interaction, as the largest changes in inter pulse intervals occur when the stimulation delay is below 100 ms. We also discuss the implications of these findings in the context of information processing in weakly electric fish.     

New Chinchillid Rodents (Hystricognathi: Caviomorpha) from Northern Chile and Bolivia Fill a 17-Million-Year Gap in the Pan-Chinchilline Fossil Record

Journal of Mammalian Evolution - The fossil record of chinchillid rodents (Hystricomorpha: Caviomorpha) begins in the early Miocene. However, nearly all remains have thus far been limited to the...     

Spatial and temporal variability of pCO2 and CO2 efflux in seven Amazonian Rivers

Current estimates of CO₂ outgassing from Amazonian rivers and streams have considerable uncertainty since they are based on limited-time surveys of pCO₂ measurements along the Amazon mainstem and mouths of major tributaries, using conservative estimates of gas exchange velocities. In order to refine basin-scale CO₂ efflux estimates from Amazonian rivers, we present a long time (5-year) dataset of direct measurements of CO₂ fluxes, gas transfer velocities and pCO₂ measurements in seven representative rivers of the lowland Amazon basin fluvial network, six non-tidal (Negro, Solimões, Teles Pires, Cristalino, Araguaia and Javaés) and one tidal river (Caxiuanã), with sizes ranging from 4th to 9th order. Surveys were conducted from January 2006 to December 2010, in a total of 389 campaigns covering all stages of their hydrographs. CO₂ fluxes and gas transfer velocities (k) were measured using floating chambers and pCO₂ was measured simultaneously by headspace extraction followed by gas chromatography analysis. Results show high CO₂ flux rate variability among rivers and hydrograph stages, ranging from ?0.8 to 15.3 μmol CO₂ m^?2 s^?1, with unexpected negative fluxes in clear-water rivers during low waters. Non-tidal rivers showed marked seasonal CO₂ flux patterns, with significantly higher exchange during high waters. Seasonality was modulated by pCO₂, which was positive and strongly correlated with discharge. In these rivers k was well correlated with wind speed, which allowed the use of wind data to model k. We estimate a release of 360 ± 60 Tg C year^?1 from Amazonian rivers and streams within a 1.47 million km² quadrant in the central lowland Amazon. Extrapolating these values to the basin upstream of Óbidos, results in an outgassing of 0.8 Pg C to the atmosphere each year. Our results are a step forward in achieving more accurate gas emission values for Amazonian rivers and their role in the annual carbon budget of the Amazon basin.     

Intra and interannual variability in the Madeira River water chemistry and sediment load

Concentrations of cations (Na⁺, Ca²⁺, Mg²⁺, K⁺, NH₄ ⁺), anions (HCO₃ ^?, Cl^?,?NO₃ ^?, SO₄ ^2?, PO₄ ^3?) and suspended sediments in the Madeira River water were?determined near the city of Porto Velho (RO), in order to assess variation in?water chemistry from 2004 to 2007. Calcium and bicarbonate were the dominant?cation and anion, respectively. Significant seasonal differences were found,?with highest concentrations occurring during the dry season, as expected from?the drainage of Andean carbonate-rich substratum. Interannual variations were?also observed, but became significant only when annual average discharge was?25% less than normal. Under this atypical discharge condition, bicarbonate was?replaced by sulfate, and higher suspended sediment concentrations and loads?were also observed. Compared to previously published studies, it appears that?no significant changes in water chemistry have occurred during the last 20?C30?years, although differences in approaches and sampling designs among this and previous studies may not allow detection of modest changes.?The calculated suspended sediment load reported here is close to the values presented elsewhere, reinforcing the relative importance of this river as a?sediment supplier for the Amazon Basin. Seasonality has a significant control?on the chemistry of Madeira River waters, and severe decrease in discharge due?to anthropogenic changes, such as construction of reservoirs or the occurrence?of drier years??a plausible consequence of global climate change??may lead?to modification in the chemical composition as well in the sediment deliver to?the Amazon River.