Organelle, bead, and microtubule translocations promoted by soluble factors from the squid giant axon

A reconstituted system for examining directed organelle movements along purified microtubules has been developed. Axoplasm from the squid giant axon was separated into soluble supernatant and organelle-enriched fractions. Movement of axoplasmic organelles along MAP-free microtubules occurred consistently only after addition of axoplasmic supernatant and ATP. The velocity of such organelle movement (1.6 micron/sec) was the same as in dissociated axoplasm. The axoplasmic supernatant also supported movement of microtubules along a glass surface and movement of carboxylated latex beads along microtubules at 0.5 micron/sec. The direction of microtubule movement on glass was opposite to that of organelle and bead movement on microtubules. The factors supporting movements of microtubules, beads, and organelles were sensitive to heat, trypsin, AMP-PNP and 100 microM vanadate. All of these movements may be driven by a single, soluble ATPase that binds reversibly to organelles, beads, or glass and generates a translocating force on a microtubule.     

Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells

To understand the interactions between the microtubule-based motor protein kinesin and intracellular components, we have expressed the kinesin heavy chain and its different domains in CV-1 monkey kidney epithelial cells and examined their distributions by immunofluorescence microscopy. For this study, we cloned and sequenced cDNAs encoding a kinesin heavy chain from a human placental library. The human kinesin heavy chain exhibits a high level of sequence identity to the previously cloned invertebrate kinesin heavy chains; homologies between the COOH-terminal domain of human and invertebrate kinesins and the nonmotor domain of the Aspergillus kinesin-like protein bimC were also found. The gene encoding the human kinesin heavy chain also contains a small upstream open reading frame in a G-C rich 5' untranslated region, features that are associated with translational regulation in certain mRNAs. After transient expression in CV-1 cells, the kinesin heavy chain showed both a diffuse distribution and a filamentous staining pattern that coaligned with microtubules but not vimentin intermediate filaments. Altering the number and distribution of microtubules with taxol or nocodazole produced corresponding changes in the localization of the expressed kinesin heavy chain. The expressed NH2-terminal motor and the COOH-terminal tail domains, but not the alpha-helical coiled coil rod domain, also colocalized with microtubules. The finding that both the kinesin motor and tail domains can interact with cytoplasmic microtubules raises the possibility that kinesin could crossbridge and induce sliding between microtubules under certain circumstances.     

Activities of Ca2+ pump and low affinity Ca2+/H+ antiport in plasma membrane vesicles of corn roots

ATP-dependent Ca₂₊-uptake was investigated in sealed plasmamembrane vesicles isolated from corn roots (Zea mays L. cv.Hybrid-3352/Palma-Pioneer). In a chloride-containing medium,at high calcium concentrations, about 30% of the total Ca²⁺accumulation ({small tilde}4 nmol Ca²⁺ mg^–1 protein) wasshown to be protonophore-sensitive and corresponded to the fractionof Ca²⁺ not accumulated in a sulphate-containing medium. Furthermore,vesicles in the presence of nitrate, which stimulates H⁺ transport,or vesicles preloaded with H⁺, take up Ca²⁺ more rapidly, suggestingthat, at high calcium concentrations, there is a mechanism forCa²⁺ transport which depends on the magnitude of the protongradient across the membrane. The fraction of Ca²⁺ uptake shownto be sensitive to the protonophore CCCP increased by about150–200% as the Ca²⁺ concentration in the medium increasedfrom 50µM to 250µM. Under the same conditions, theCCCP-insensitive fraction of Ca²⁺ accumulated was reduced byabout 25–30% suggesting that different Ca²⁺ affinitiesexist in the two Ca²⁺ uptake processes. Although calmodulinstimulation was not observed, the sensitivity to Ca²⁺ and externalpH indicates that H⁺ gradient-independent Ca²⁺ accumulationreflects activity of the Ca²⁺–pump. These results indicatethat the plasma membrane of corn roots contain two distinctmechanisms of Ca²⁺ transport: a high Ca²⁺ affinity, proton gradient-independentCa²⁺ pump and a low Ca²⁺ affinity, proton gradient-dependentCa²⁺/H⁺ antiport, which have greatest activity at concentrationsof Ca²⁺ below and above 50+M, respectively. Key words: Ca²⁺/H+ antiport, Ca²⁺ pump, plasmalemma, roots, Zea mays L.     

The influence of plants on concentration and fractionation of Zn, Pb, and Cu in salt marsh sediments (Tagus Estuary, Portugal)

Sediment cores were collected from two sites of the Tagus estuary salt marshes which differed in degree of metal contamination. At each site, six 60-cm-long cores were taken, three from a non-vegetated intertidal zone, and one from each of areas colonized by salt marsh plants, Spartina maritima, Halimione portulacoides and Arthrocnemum fruticosum, respectively. Total concentrations and concentrations in sequential extractions of Zn, Pb, and Cu were determined in several sediment layers. Sediment slices containing most of the roots (5–15-cm depth) were enriched in metals in comparison with other depths in the core and with non-vegetated cores. Additionally, metals in sediment slices with roots were preferentially linked to the residual fraction. These results are evidence that aquatic plant roots can have a strong influence on metal concentration and speciation in sediments. Since metals become immobilized in vegetated sediments, the preservation of salt marshes or the creation of artificial wetlands could be considered as an efficient natural means for maintaining ecosystem health or restoring ecosystem quality.     

Soil charcoal as long‐term pyrogenic carbon storage in Amazonian seasonal forests

Forest fires (paleo + modern) have caused charcoal particles to accumulate in the soil vertical profile in Amazonia. This forest compartment is a long‐term carbon reservoir with an important role in global carbon balance. Estimates of stocks remain uncertain in forests that have not been altered by deforestation but that have been impacted by understory fires and selective logging. We estimated the stock of pyrogenic carbon derived from charcoal accumulated in the soil profile of seasonal forest fragments impacted by fire and selective logging in the northern portion of Brazilian Amazonia. Sixty‐nine soil cores to 1‐m depth were collected in 12 forest fragments of different sizes. Charcoal stocks averaged 3.45 ± 2.17 Mg ha^?1 (2.24 ± 1.41 Mg C ha^?1). Pyrogenic carbon was not directly related to the size of the forest fragments. This carbon is equivalent to 1.40% (0.25% to 4.04%) of the carbon stocked in aboveground live tree biomass in these fragments. The vertical distribution of pyrogenic carbon indicates an exponential model, where the 0–30 cm depth range has 60% of the total stored. The total area of Brazil's Amazonian seasonal forests and ecotones not altered by deforestation implies 65–286 Tg of pyrogenic carbon accumulated along the soil vertical profile. This is 1.2–2.3 times the total amount of residual pyrogenic carbon formed by biomass burning worldwide in 1 year. Our analysis suggests that the accumulated charcoal in the soil vertical profile in Amazonian forests is a substantial pyrogenic carbon pool that needs to be considered in global carbon models.     

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.     

Design of biologically active, conformationally constrained GnRH antagonists

The introduction of conformational constraints into a flexible peptide hormone can be exploited to develop models for the conformation required for receptor binding and activity. In this review, we illustrate this approach to analog design using our work on antagonists of gonadotropin-releasing hormone (GnRH). Design of a conformationally constrained, competitive antagonist of GnRH, cyclo[delta 3,4 Pro-D4ClPhe-DTrp-Ser-Tyr-DTrp-NMeLeu-Arg-Pro-bet a Ala] led to the prediction of its bioactive conformation. Template forcing experiments show that this conformation is accessible to other active GnRH analogs. Two-dimensional NMR studies verified the predicted conformation in solution. The predicted binding conformation has recently been used to design two new analogs incorporating side chain-side chain linkages suggested by the conformational model: Ac-delta 3,4Pro-D4FPhe-DTrp-Dap-Tyr-DTrp-Leu-Arg-Asp-Gly- NH2 and Ac-delta 3,4Pro-D4FPhe-DTrp-Dap-Tyr-D2Nal-Leu-Arg-Pro-Asp -NH2. These analogs were synthesized and the one predicted to be most similar to the parent conformation had equivalent potency while the second, designed to refine the conformational hypothesis, was found to exhibit enhanced potency, thus confirming the original binding conformation hypothesis. These compounds and their derivatives now provide a new class of GnRH antagonists possessing both high biological potency and limited conformational flexibility, thus making them ideal for both biophysical and structure-activity studies.