Modification of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells by N-bromoacetamide

Summary Ca²⁺-activated K⁺ channels were studied in cultured medullary thick ascending limb (MTAL) cells using the patch-clamp technique in the inside-out configuration. The Ca²⁺ activation site was modified using N-bromoacetamide (NBA). 1mm NBA in the bath solution, at 2.5 m Ca²⁺ reduces the open probability,P _o , of the channel to <0.01, without an effect on single-channel conductance. NBA-modified channels are still Ca²⁺-sensitive, requiring 25mm Ca²⁺ to raiseP _o to 0.2. Both before and after NBA modification channel openings display at least two distributions, indicative of more than one open state. High Ca²⁺ (1mm) protects the channels from modification. Also presented is a second class of Ca²⁺-activated K⁺ channels which are normally present in MTAL cells which open infrequently at 10 m Ca²⁺ (P _o =0.01) but have aP _o of 0.08 at 1mm Ca²⁺. We can conclude (i) that NBA modifies the channel by shifting Ca²⁺-sensitivity to very high Ca²⁺, (ii) that NBA acts on a site involved in Ca²⁺ gating, and (iii) that a low affinity channel is present in the apical cell membrane with characteristics similar to those of normal channels modified with NBA.     

Effect of distance,aggregation, and habitat on levels of seed predation for two mammal — dispersed neotropical rain forest tree species

The effect of seed aggregation and distance from conspecific trees on seed predation was experimentally examined for two neotropical tree species, Macoubea guianensis (Apocynaceae) and Pouteria sp. (Sapotaceae) in a lowland tropical rain forest in northeastern Peru. Results of these experiments are discussed in the context of the Janzen-Connell model (Janzen 1970; Connell 1971), which predicts decreased seed survival near parent trees due to either density-or distance-responsive mortality, and Howe's model (Howe 1989) which predicts that trees with seeds dispersed in clumps (aggregated) will not suffer density-dependent predation, and will have higher survival of seeds near the parent tree than other trees. We also examined whether predation on seeds of these species was affected by seed placement in or near 30-m-wide strips regenerating after clear-cutting. Both species appeared to be mammal-dispersed but differed in how frugivores handled seeds, seed size, overall fruit crop size, and gemination time. Neither of the two species studied appeared to suffer seed predation in a manner predicted by the Janzen-Connell model, and patterns of seed predation for only one of the species was similar to predictions of Howe's model. For neither species did seed predation along the edge of, or in the center of, regenerating clear cuts differ from predation 15 m into the primary forest. For Pouteria, seed predation in and near regnerating strips was significantly greater than around forest trees, but the opposite pattern held for Macoubea. Overall, seed predation was much greater on Macoubea. The difference in seed predation for these two species was most likely a result of differences in the types of seed predators that attacked these two species.     

The action of luteinizing hormone on the testis

Luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) receptors are coupled to intracellular effector systems, most notably adenylate cyclase, through guanyl nucleotide-binding proteins or G-proteins. The molecular mechanism involved in the dynamic coupling of the LH/hCG receptor however, are not known. It has been postulated that receptor aggregation at the molecular level plays a critical role in this process. There have been attempts to understand the receptor association and dissociation phenomena at the molecular level. One of them involves the participation of the major histocompatibility complex (MHC) class I antigen in the mechanism of receptor activation and/or expression. One molecular basis for these mechanisms consists of a physical interaction between MHC proteins and receptors to form "compound receptors" able to transfer a hormonal signal to the cell. Using a photo-reactive probe we demonstrated that the LH/hCG receptors and the class I antigens are closely associated in the membrane. Thus, it is possible to form covalent complexes of hCG and class I antigens through the binding of the hormone to specific receptors. These findings imply that LH/hCG receptors and the MHC class I antigens may interact at the level of the plasma membrane in the mechanism of LH action. We also performed experiments using a single cell and limiting stimulation to a patch of membrane. The results stimulating the cell in a localized area suggested that even if all components are entirely free to float there is a constraint in the localization of the receptor, G-protein, and/or the effector, supporting the constraint dissociation model. Within a limited area subunits could dissociate, but they would not be free to diffuse throughout the membrane. Moreover the concept of compartmentalization that has been utilized to explain some inconsistencies in second-messenger action now can be proved by experimental design.     

Phytochrome assembly. The structure and biological activity of 2(R),3(E)-phytochromobilin derived from phycobiliproteins.

The unicellular rhodophyte, Porphyridium cruentum, and the filamentous cyanobacterium, Calothrix sp. PCC 7601, contain phycobiliproteins that have covalently bound phycobilin chromophores. Overnight incubation of solvent-extracted cells at 40 degrees C with methanol liberates free phycobilins that are derived from the protein-bound bilins by methanolytic cleavage of the thioether linkages between bilin and apoprotein. Two of the free bilins were identified as 3(E)-phycocyanobilin and 3(E)-phycoerythrombilin by comparative spectrophotometry and high pressure liquid chromatography. Methanolysis also yields a third bilin free acid whose absorption and 1H NMR spectra support the assignment of the 3(E)-phytochromobilin structure. This novel bilin is the major pigment isolated from cells that are pre-extracted with acetone-containing solvents. Since phytochrome- or phytochromobilin-containing proteins are not present in either organism, the 3(E)-phytochromobilin must arise by oxidation of phycobilin chromophores. This pigment is not obtained by similar treatment of a cyanobacterium and a rhodophyte that lack phycoerythrin. Therefore, 3(E)-phytochromobilin appears to be derived from phycoerythrobilin-containing proteins. Comparative CD spectroscopy of 3(E)-phytochrombilin and 3(E)-phycocyanobilin suggests that the two bilins share the R stereochemistry at the 2-position in the reduced pyrrole ring. Incubation of 2(R),3(E)-phytochromobilin with recombinant oat apophytochrome yields a covalent bilin adduct that is photoactive and spectrally indistinguishable from native oat phytochrome isolated from etiolated seedlings. These results establish that the phycobiliprotein-derived 2(R),3(E)-phytochromobilin is a biologically active phytochrome chromophore precursor.     

Early evolution of the megadiverse subtribe Philonthina (Staphylinidae: Staphylininae: Staphylinini) and its Neotropical lineage

Philonthina (2864 species in 74 genera) represents almost one-half of the diversity of Staphylinini and is the largest of its subtribes. Most Philonthina species are found in tropical areas, but the origin of this diversity is still not well understood, mainly because their systematics belongs to a past era of taxonomy. Such is the case of a group of genera, most of them endemic to the Neotropical region (NT), whose monophyly has been repeatedly confirmed and which constitutes the so-called Neotropical lineage (NL). However, basal relationships have not been clarified, neither for Philonthina nor its NL. The NL includes ∼300 species and 26 genera, but two of them (Belonuchus Nordmann and Paederomimus Sharp) account for two-thirds of its species. Here, using the largest molecular-based phylogeny of Philonthina and its NL to date, a time-calibrated phylogeny, and ancestral range reconstructions for the NL, we explore the evolutionary history of Philonthina with a focus on its NL to reveal their early evolution and diversification in the NT. We show that Philonthina originated during the Late Cretaceous ∼ 67.6 Ma and diversified into five main lineages mostly during the Eocene. The NL originated in northwestern South America (SA) and the Andes not earlier than 64.2 Ma from a Laurasian lineage present in SA ∼49.1–69.9 Ma. Shortly afterward, that is, 39.9–56.9 Ma, the NL diversified into the Andean clade and the most species-rich Belonuchus-Paederomimus group. Our analyses recover northwestern SA and the Andes as the primary centers of diversification. Dispersal events to the northern landmasses took place at least three times during the Miocene in the early evolution of the NL.     

Dammarane triterpenes targeting α-synuclein: biological activity and evaluation of binding sites by molecular docking

Parkinson''s disease (PD) is a neurodegenerative disorder that affects adult people whose treatment is palliative. Thus, we decided to test three dammarane triterpenes 1, 1a, 1b, and we determined that 1 and 1a inhibit β-aggregation through thioflavine T rather than 1b. Since compound 1 was most active, we determined the interaction between α-synuclein and 1 at 50 µM (Kd) through microscale thermophoresis. Also, we observed differences in height and diameter of aggregates, and α-synuclein remains unfolded in the presence of 1. Also, aggregates treated with 1 do not provoke neurites'' retraction in N2a cells previously induced by retinoic acid. Finally, we studied the potential sites of interaction between 1 with α-synuclein fibrils using molecular modelling. Docking experiments suggest that 1 preferably interact with the site 2 of α-synuclein through hydrogen bonds with residues Y39 and T44.     

Two new species (Gustavia johnclarkii and G. hubbardiorum) and other contributions to the systematics of Gustavia (Lecythidaceae)

Gustavia johnclarkii and G. hubbardiorum, two new species from the wet forests of northwestern and Amazonian Ecuador, are described and illustrated. In addition, the two subspecies of both G. speciosa and G. macarenensis are elevated to species, and the new combination Gustavia paucisperma is provided. A justification for these changes is presented.     

Proliferation of Murine Midbrain Neural Stem Cells Depends upon an Endogenous Sonic Hedgehog (Shh) Source

The Sonic Hedgehog (Shh) pathway is responsible for critical patterning events early in development and for regulating the delicate balance between proliferation and differentiation in the developing and adult vertebrate brain. Currently, our knowledge of the potential role of Shh in regulating neural stem cells (NSC) is largely derived from analyses of the mammalian forebrain, but for dorsal midbrain development it is mostly unknown. For a detailed understanding of the role of Shh pathway for midbrain development in vivo, we took advantage of mouse embryos with cell autonomously activated Hedgehog (Hh) signaling in a conditional Patched 1 (Ptc1) mutant mouse model. This animal model shows an extensive embryonic tectal hypertrophy as a result of Hh pathway activation. In order to reveal the cellular and molecular origin of this in vivo phenotype, we established a novel culture system to evaluate neurospheres (nsps) viability, proliferation and differentiation. By recreating the three-dimensional (3-D) microenvironment we highlight the pivotal role of endogenous Shh in maintaining the stem cell potential of tectal radial glial cells (RGC) and progenitors by modulating their Ptc1 expression. We demonstrate that during late embryogenesis Shh enhances proliferation of NSC, whereas blockage of endogenous Shh signaling using cyclopamine, a potent Hh pathway inhibitor, produces the opposite effect. We propose that canonical Shh signaling plays a central role in the control of NSC behavior in the developing dorsal midbrain by acting as a niche factor by partially mediating the response of NSC to epidermal growth factor (EGF) and fibroblast growth factor (FGF) signaling. We conclude that endogenous Shh signaling is a critical mechanism regulating the proliferation of stem cell lineages in the embryonic dorsal tissue.     

Diversity and trophic structure of insects associated with grains of three maize landraces in San Agustín Loxicha,Oaxaca, Mexico

Diversity and trophic structure of grain insect communities were examined in Olotillo, Nal‐Tel and Comiteco maize landraces cultivated within a milpa agroecosystem by Zapotec ethnic groups in Mexico. Higher insect diversity was expected in Olotillo, whose cultivation comprises a wide variety of agroecosystems, and low insect abundance in Nal‐Tel with small grains and thick testa. Forty Olotillo cobs were collected at low, medium and high elevations, and 40 each of Nal‐Tel at low elevation and Comiteco at high elevation. Cobs were monitored for 30 days under controlled laboratory conditions until all insects emerged. Thickness of testa of 400 grains from each landrace was measured. Community composition and trophic structure were described and standard diversity indices were estimated. A total of 9,708 insects, corresponding to five orders, 24 families and 36 species, were recorded, with six species not previously reported in this region. Insect guilds were composed of 70% phytophages, 22% parasitoids and 8% predators. Species richness was S = 27, 16 and 8 in Olotillo, Comiteco and Nal‐Tel, respectively. Nal‐Tel and Olotillo had the highest diversity index values (H′ = 1.32 and 1.2, respectively) and no significant differences; Comiteco had the lowest value (H′ = 0.65) and differed significantly from the other landraces. Comiteco and Olotillo, which have large grains and thin testa, showed higher insect abundance than Nal‐Tel, which has small grains and thick testa and showed lower abundance. Results support our hypotheses and highlight the role of traditional crop management in insect agrobiodiversity maintenance and conservation.