The influence of spatial scale on the genetic structure of a widespread tropical wetland tree, Pterocarpus officinalis (Fabaceae)

Identifying factors that cause genetic differentiation in plant populations and the spatial scale at which genetic structuring can be detected will help to understand plant population dynamics and identify conservation units. In this study, we determined the genetic structure and diversity of Pterocarpus officinalis, a widespread tropical wetland tree, at three spatial scales: (1) drainage basin “watershed” (<10 km), (2) within Puerto Rico (<100 km), and (3) Caribbean-wide (>1000 km) using AFLP. At all three spatial scales, most of the genetic variation occurred within populations, but as the spatial scale increased from the watershed to the Caribbean region, there was an increase in the among population variation (Φ_ST=0.19 to Φ_ST=0.53). At the watershed scale, there was no significant differentiation (P=0.77) among populations in the different watersheds, although there was some evidence that montane and coastal populations differed (P<0.01). At the island scale, there was significant differentiation (P<0.001) among four populations in Puerto Rico. At the regional scale (>1000 km), we found significant differentiation (P<0.001) between island and continental populations in the Caribbean region, which we attributed to factors associated with the colonization history of P. officinalis in the Neotropics. Given that genetic structure can occur from local to regional spatial scales, it is critical that conservation recommendations be based on genetic information collected at the appropriate spatial scale.     

ATP augments von Willebrand factor-dependent shear-induced platelet aggregation through Ca2+-calmodulin and myosin light chain kinase activation

Shear stress triggers von Willebrand factor (VWF) binding to platelet glycoprotein Ibalpha and subsequent integrin alpha(IIb)beta(3)-dependent platelet aggregation. Concomitantly, nucleotides are released from plateletdense granules, and ADP is known to contribute to shear-induced platelet aggregation (SIPA). We found that the impaired SIPA of platelets from a Hermansky-Pudlak patient lacking dense granules was restored by exogenous l-beta,gamma-methylene ATP, a stable P2X(1) agonist, as well as by ADP, confirming that in addition to ADP (via P2Y(1) and P2Y(12)), ATP (via P2X(1)) also contributes to SIPA. Likewise, SIPA of apyrase-treated platelets was restored upon P2X(1) activation with l-beta,gamma-methylene ATP, which promoted granule centralization within platelets and stimulated P-selectin expression, which is a marker of alpha-granule release. In addition, during SIPA, platelet degranulation required both extracellular Ca(2+) and VWF-glycoprotein Ibalpha interactions without involving alpha(IIb)beta(3). Neither platelet release nor SIPA was affected by protein kinase C inactivation, even though protein kinase C blockade inhibits platelet responses to collagen and thrombin in stirring conditions. In contrast, inhibiting myosin light chain (MLC) kinase with ML-7 reduced platelet release and SIPA by 30%. Accordingly, the potentiating effect of P2X(1) stimulation on the aggregation of apyrase-treated platelets coincided with intensified phosphorylation of MLC and was abrogated by ML-7. SIPA-induced MLC phosphorylation occurred exclusively through released nucleotides and selective antagonism of P2X(1) with MRS2159-reduced SIPA, ATP release, and potently inhibited MLC phosphorylation. We conclude that the P2X(1) ion channel induces MLC-mediated cytoskeletal rearrangements, thus contributing to SIPA and degranulation during VWF-triggered platelet activation.     

The first total synthesis of the marine fatty acid (+/-)-2-methoxy-13-methyltetradecanoic acid: a cytotoxic fatty acid to leukemia cells

The recently discovered marine fatty acid (+/-)-2-methoxy-13-methyltetradecanoic acid was synthesized for the first time in six steps (26% overall yield) starting from commercially available methyl 12-methyltridecanoate. The synthetic approach provided enough material to corroborate the structure of the acid, which was recently identified in the sponge Amphimedon complanata from Aguadilla, Puerto Rico, and to test its cytotoxicity to three leukemia cell lines. The key step in the synthesis was the addition of trimethylsilyl cyanide to 12-methyltridecanal under triethylamine catalysis. Nuclear magnetic resonance data are provided for the first time for this methoxylated fatty acid and the synthetic approach utilized is of general applicability since it can be used in the synthesis of other methyl-branched 2-methoxylated fatty acids. We also report that the acid (+/-)-2-methoxy-13-methyltetradecanoic acid is cytotoxic to human chronic myelogenous leukemia K-562 (EC50=238 microM), histiocytic lymphoma U-937 (EC50=250 microM), and promielocytic leukemia HL-60 (EC50=476 microM) in RPMI 1640 medium.     

Calcium amelioration of cadmium-induced cytotoxicity in cultured rat hepatocytes

Summary Parenchymal hepatocytes from neonatal rats were isolated, cultured about 24 h, exposed to cadmium with or without calcium, and processed for scanning electron microscopy. To assess the severity of cadmium-induced changes, exposed hepatocytes were categorized based upon the extent of morphological damage. Differences in surface blebbing, alterations in microvilli, variations in the degree of swelling, and changes in cell shape were used to categorize the severity of cell damage. A double-blind morphometric analysis (a geometricostatistical processing of two-dimensional data for the collection of three-dimensional information) of cellular changes was conducted for each exposure time and for each concentration of cadmium in the presence or absence of calcium. Significant decreases occurred in the percent relative volume of normal, flattened cells present in cultures exposed for 30 min to 50 or 100 μM cadmium in the absence of calcium. In contrast, the percent relative volume of severely damaged spherical cells was significantly increased after exposure to solutions containing 50 or 100 μM cadmium and lacking calcium. Percent relative volume of intermediate cells (which were slightly swollen and showed changes in microvillar number) was significantly increased following a 30 min exposure to all cadmium concentrations in the absence of calcium. The examination of hepatocytes exposed for 60 min showed that the degree of cadmium-induced cytotoxicity was more severe in the absence of calcium than was the case for the hepatocyte cultures exposed for 30 min: approximately 30% more spherical cells and 30% fewer flattened cells were present if cultures were exposed in the absence of calcium for 60 min compared to those exposed for 30 min. The degree of blebbing was significantly greater at all cadmium concentrations in the absence of calcium. The presence of calcium, therefore, reduced cadmium-induced cytotoxicity in primary cultures of rat hepatocytes subjected to morphometric analysis after scanning electron microscopy.     

Changes in Aldose Reductase After Crush Injury of Normal Rat Sciatic Nerve

The response of aldose reductase (AR) to crush injury was studied in normal rat sciatic nerve. Enzyme activity and immunoreactivity of AR were determined at intervals of 1, 5, 14, 28, and 35 days after crush and correlated with histologic and immunocytochemical observations. During nerve degeneration in the distal segments of crushed nerves, a significant reduction in AR activity was detected. At 5 and 14 days, coincident with Schwann cell proliferation, enzyme activity decreased by nearly two- and fourfold, respectively. Although activity of AR increased by 28 days during nerve regeneration, it was not restored to normal levels at 35 days. Similar reductions were observed with the immunoblotting of the enzyme. Quantitative analysis of immunogold labelling on electron micrographs confirmed that proliferating as well as remyelinating Schwann cells contained reduced gold particle density compared to Schwann cells of noncrushed myelinated fibers. Immunoblots of P0, a marker for the degree of Schwann cell differentiation or myelination, showed that the temporal sequence of changes in P0 paralleled that of AR. Thus expression of AR is a function of differentiated or mature Schwann cells. The putative volume regulatory role of AR in Schwann cells may become superfluous during Wallerian degeneration.     

Characterization of D-amino-acid-containing excitatory conotoxins and redefinition of the I-conotoxin superfamily

Post-translational isomerization of l-amino acids to d-amino acids is a subtle modification, not detectable by standard techniques such as Edman sequencing or MS. Accurate predictions require more sequences of modified polypeptides. A 46-amino-acid-long conotoxin, r11a, belonging to the I-superfamily was previously shown to have a d-Phe residue at position 44. In this report, we characterize two related peptides, r11b and r11c, with d-Phe and d-Leu, respectively, at the homologous position. Electrophysiological tests show that all three peptides induce repetitive activity in frog motor nerve, and epimerization of the single amino acid at the third position from the C-terminus attenuates the potency of r11a and r11b, but not that of r11c. Furthermore, r11c (but neither r11a nor r11b) also acts on skeletal muscle. We identified more cDNA clones encoding conopeptide precursors with Cys patterns similar to r11a/b/c. Although the predicted mature toxins have the same cysteine patterns, they belong to two different gene superfamilies. A potential correlation between the identity of the gene superfamily to which the I-conotoxin belongs and the presence or absence of a d-amino acid in the primary sequence is discussed. The great diversity of I-conopeptide sequences provides a rare opportunity for defining parameters that may be important for this most stealthy of all post-translational modifications. Our results indicate that neither the chemical nature of the side chain nor the precise vicinal sequence around the modified residue seem to be critical, but there may be favored loci for isomerization to a d-amino acid.     

Definition of the M-conotoxin superfamily: characterization of novel peptides from molluscivorous Conus venoms

Most of the >50,000 different pharmacologically active peptides in Conus venoms belong to a small number of gene superfamilies. In this work, the M-conotoxin superfamily is defined using both biochemical and molecular criteria. Novel excitatory peptides purified from the venoms of the molluscivorous species Conus textile and Conus marmoreus all have a characteristic pattern of Cys residues previously found in the mu-, kappaM-, and psi-conotoxins (CC-C-C-CC). The new peptides are smaller (12-19 amino acids) than the mu-, kappaM-, and psi-conotoxins (22-24 amino acids). One peptide, mr3a, was chemically synthesized in a biologically active form. Analysis of the disulfide bridges of a natural peptide tx3c from C. textile and synthetic peptide mr3a from C. marmoreus showed a novel pattern of disulfide connectivity, different from that previously established for the mu- and psi-conotoxins. Thus, these peptides belong to a new group of structurally and pharmacologically distinct conotoxins that are particularly prominent in the venoms of mollusc-hunting Conus species. Analysis of cDNA clones encoding the novel peptides as well as those encoding mu-, kappaM-, and psi-conotoxins revealed highly conserved amino acid residues in the precursor sequences; this conservation in both amino acid sequence and in the Cys pattern defines a gene superfamily, designated the M-conotoxin superfamily. The peptides characterized can be provisionally assigned to four distinct groups within the M-superfamily based on sequence similarity within and divergence between each group. A notable feature of the superfamily is that two distinct structural frameworks have been generated by changing the disulfide connectivity on an otherwise conserved Cys pattern.     

Post-translational amino acid isomerization: a functionally important D-amino acid in an excitatory peptide

The post-translational modification of an L- to a D-amino acid has been documented in relatively few gene products, mostly in small peptides under 10 amino acids in length. In this report, we demonstrate that a 46-amino acid polypeptide toxin has one D-phenylalanine at position 44, and that the epimerization from an L-Phe to a D-Phe has a dramatic effect on the excitatory effects of the peptide. In one electrophysiological assay carried out, the D-Phe-containing peptide was extremely potent, whereas the unmodified polypeptide had no biological activity, demonstrating that the chirality of the post-translationally modified amino acid is functionally significant. The peptide toxin analyzed, r11a, belongs to the I-gene superfamily of conotoxins that has four disulfide cross-links. The D-Phe in r11a is at the third amino acid from the C terminus, the same relative position from the C-terminal end as the d-amino acid in omega-agatoxin TK from a spider, an unrelated peptide. Thus, although post-translational amino acid isomerization appears to have no strong specificity for the chemical nature of the amino acid side chain, the few peptides where this modification has been established suggest that there may be favored positions near the N or C terminus that are preferential sites for isomerization to a D-amino acid.