Ethnobotanical ground‐truthing: indigenous knowledge,floristic inventories and satellite imagery in the upper Rio Negro,Brazil

Aim To assess the utility of indigenous habitat knowledge in studies of habitat diversity in Amazonia. Location Baniwa indigenous communities in Rio Içana, upper Rio Negro, Brazil. Methods Six campinarana vegetation types, recognized and named by a consensus of Baniwa indigenous informants according to salient indicator species, were studied in 15 widely distributed plots. Floristic composition (using Baniwa plant nomenclature only, after frustrated attempts to obtain botanical collection permits), quantitative measures of forest structure and GPS waypoints of the 4‐ha composite plot contours were registered, permitting their location on Landsat satellite images. Non‐metric multidimensional scaling (NMDS) ordination was carried out using pc‐ord software. Results The NMDS ordinations of the plot data revealed a clear gradient of floristic composition that was highly correlated with three quantitative measures of forest structure: basal area, canopy height and satellite reflectance. Main conclusions Baniwa‐defined forest types are excellent predictors of habitat diversity along the structural gradient comprising distinctive white‐sand campinarana vegetation types. Indigenous ecological knowledge, as revealed by satellite imagery and floristic analyses, proves to be a powerful and efficient shortcut to assessing habitat diversity, promoting dialogue between scientific and indigenous worldviews, and promoting joint study and conservation of biodiversity.     

Evidence for polynuclear iron(III) clusters in the root nodule bacterium, Rhizobium leguminosarum bv. viciae WSM710

Cells of the root nodule bacterium Rhizobium leguminosarum bv. viciae WSM710 were cultured in a medium containing 20 M ⁵⁷Fe. Mössbauer spectra of the cells at 5.5 and 3.7 K indicated that the major form of iron present in the cells was in the form of polynuclear iron(III) clusters. At 5.5 K the spectral component associated with these clusters was in the form of a superposition of a broad feature (large magnetic hyperfine field distribution) and a doublet. On lowering the temperature of the cells to 3.7 K, the spectral component was transformed into resolved magnetic hyperfine field splitting which yielded a magnetic hyperfine field of 42.4 T when fitted with broad Lorentzian peaks. These spectral characteristics are typical of the hydrated iron(III) phosphate cores of several bacterioferritins. A small fraction (11%) of the Mössbauer spectral area of the cells was in the form of a doublet which yielded parameters ( = 1.35 mm/s; E_Q = 3.15 mm/s) indicative of iron(II). The parameters are very similar to those of a spectral component previously observed in several other microbes (R. Böhnke and B.F. Matzanke (1995) BioMetals 8, 223-230) and which has been associated with a 2.2 kDa oligomeric iron(II) carbohydrate phosphate     

Differential hydrolysis of erythrocyte and mitochondrial membrane phospholipids by two phospholipase A2 isoenzymes (NK-PLA2-I and NK-PLA2-II) from the venom of the Indian monocled cobra Naja kaouthia

We previously demonstrated that venom from the Indian monocled cobra Naja kaouthia is a rich source of phospholipase A2 enzymes, and we purified and characterized a major PLA2 isoenzyme (NK-PLA2-I) from N. kaouthia venom. In the present study, we report the purification and biochemical characterization of a second PLA2 isoenzyme (NK-PLA2-II) from the same venom. A comparison of the membrane phospholipid hydrolysis patterns by these two PLA2s has revealed that they cause significantly more damage to mitochondrial membranes (NK-PLA2-I > NK-PLA2-II) as compared to erythrocyte membranes due to more efficient binding of the enzymes to mitochondrial membranes. Fatty acid release patterns by these PLA2s from the membrane phospholipid PC-pools indicate that NK-PLA2-I does not discriminate between saturated and unsaturated fatty acids whereas NK-PLA2-II shows a preference for unsaturated fatty acids during the initial phase of attack. The current investigation provides new insight into the molecular arrangement of NK-PLA2-sensitive domains in erythrocyte and mitochondrial membranes and highlights the contribution of polar, but uncharged, amino acids such as serine and cysteine in NK-PLA2 induced membrane damage.     

Extracellular proteolytic activity plays a central role in swarming motility in Bacillus subtilis

Natural isolates of Bacillus subtilis exhibit a robust multicellular behavior known as swarming. A form of motility, swarming is characterized by a rapid, coordinated progression of a bacterial population across a surface. As a collective bacterial process, swarming is often associated with biofilm formation and has been linked to virulence factor expression in pathogenic bacteria. While the swarming phenotype has been well documented for Bacillus species, an understanding of the molecular mechanisms responsible remains largely isolated to gram-negative bacteria. To better understand how swarming is controlled in members of the genus Bacillus, we investigated the effect of a series of gene deletions on swarm motility. Our analysis revealed that a strain deficient for the production of surfactin and extracellular proteolytic activity did not swarm or form biofilm. While it is known that surfactin, a lipoprotein surfactant, functions in swarming motility by reducing surface tension, this is the first report demonstrating that general extracellular protease activity also has an important function. These results not only help to define the factors involved in eliciting swarm migration but support the idea that swarming and biofilm formation may have overlapping control mechanisms.     

Lipopolysaccharide 3-deoxy-D-manno-octulosonic acid (Kdo) core determines bacterial association of secreted toxins

In contrast to cholera toxin (CT), which is secreted solubly by Vibrio cholerae across the outer membrane, heat-labile enterotoxin (LT) is retained on the surface of enterotoxigenic Escherichia coli (ETEC) via an interaction with lipopolysaccharide (LPS). We examined the nature of the association between LT and LPS. Soluble LT binds to the surface of LPS deep-rough biosynthesis mutants but not to lipid A, indicating that only the Kdo (3-deoxy-d-manno-octulosonic acid) core is required for binding. Although capable of binding truncated LPS and Kdo, LT has a higher affinity for longer, more complete LPS species. A putative LPS binding pocket is proposed based on the crystal structure of the toxin. The ability to bind LPS and remain associated with the bacterial surface is not unique to LT, as CT also binds to E. coli LPS. However, neither LT nor CT is capable of binding to the surface of Vibrio. The core structures of Vibrio and E. coli LPS differ in that Vibrio contains a phosphorylated single Kdo-lipid A, and E. coli LPS contains unphosphorylated Kdo2-lipid A. We determined that the phosphate group on the Kdo core of Vibrio LPS prevents CT from binding, resulting in the secretion of soluble toxin. Because LT binds E. coli LPS, it remains associated with the extracellular bacterial surface and is released in association with outer membrane vesicles. We propose that difference in the extracellular fates of LT and CT contribute to the differences in disease caused by ETEC and Vibrio cholerae.     

Selective production of rat mutant selenoprotein W with and without bound glutathione

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and electrospray ionization mass spectrometry (ESI MS) analysis of a 6x His-tagged recombinant form of rat mutant selenoprotein W (RMSW) reveals that aerobic growth conditions primarily produce a form of RMSW without bound glutathione (10,305 Da) whereas anaerobic conditions produce a glutathione-bound (305 Da) form (10,610 Da). Purification of RMSW was achieved with a procedure employing acetone precipitation and DEAE-cellulose chromatography, in addition to Ni-NTA agarose chromatography. Additional steps, including polyvalent metal ion binding (PMIB) resin chromatography and CM-cellulose chromatography, were necessary after elution from the Ni-NTA agarose column, in order to maintain solubility of the purified protein.     

Spacrcan binding to hyaluronan and other glycosaminoglycans. Molecular and biochemical studies

Photoreceptors project from the outer retinal surface into a specialized glycocalyx, the interphotoreceptor matrix (IPM), which contains hyaluronan (HA) and two novel proteoglycans, Spacr and Spacrcan. This matrix must be stable enough to function in the attachment of the retina to the outer eye wall yet porous enough to allow movement of metabolites between these tissues. How this matrix is organized is not known. HA is a potential candidate in IPM organization since biochemical studies show that these proteoglycans bind HA. RHAMM (receptor for HA-mediated motility)-type HA binding motifs (HABMs) are present in their deduced amino acid sequence and may be the sites of this HA interaction. To test this hypothesis, we subcloned three fragments of mouse Spacrcan that contain the putative HABMs. We found that each recombinant fragment binds HA. Binding decreased when residues in the HABMs were mutated. This provides direct evidence that the RHAMM-type HABMs in Spacrcan are involved in hyaluronan binding. Since chondroitin sulfate and heparan sulfate proteoglycans are important for retinal development and function, we also evaluated the binding of these recombinant proteins to heparin and chondroitin sulfates, the glycosaminoglycan side chain of these proteoglycans. We found that each recombinant protein bound to both heparin and chondroitin sulfates. Binding to chondroitin sulfates involved these HABMs, because mutagenesis reduced binding. Binding to heparin was probably not mediated through these HABMs since heparin binding persisted following their mutagenesis. These studies provide the first evidence defining the sites of protein-carbohydrate interaction of molecules present in the IPM.     

Subdiaphragmatic murine electrophysiological studies: sequential determination of ventricular refractoriness and arrhythmia induction

Programmed electrical stimulation (PES) is a crucial aspect of the evaluation of the risk of arrhythmias in cardiac patients and provides a powerful tool for understanding the mechanisms of arrhythmia in experimental models. Whereas PES in the mouse is well characterized, the procedures allowing for follow-up studies in the same animal have not been developed. In this report, we describe a novel subdiaphragmatic approach that allows for repeat electrophysiological studies in the mouse. Under inhaled anesthesia, PES was performed in 36 wild-type mice via a stimulating electrode introduced through an epigastric incision and placed directly into the diaphragmatic surface of the heart. The procedure was repeated 7 days later. Ventricular effective refractory periods (VERP) did not change significantly between the initial and follow-up trials. Chronic treatment with amiodarone, however, was associated with a 70% prolongation in VERP from initial to follow-up studies (P < or = 0.001). In addition, PES of a genetically modified strain with sudden cardiac death, the connexin43 conditional knockout mouse consistently induced lethal polymorphic ventricular tachycardia. Thus sequential PES in mice is feasible with the use of a subdiaphragmatic approach, yields reproducible VERP values, and can be used to follow pharmacologically induced changes in VERP and identify mice at risk of lethal ventricular arrhythmias.     

The gene-orientation structure of eukaryotes

It is shown that the sequence of gene orientations of four eukaryotes-for those that are presently known-are well modeled by a two-state, two-parameter (first order) Markov chain. These include the six chromosomes of nematodes (C. elegans), the 16 chromosomes of yeast (S. cerevisiae), the five chromosomes of Arabidopsis (A. thaliana), and the 19 scaffolds of fruit flies (D. melanogaster). Moreover, they are reasonably well modeled, more simply, by a one-parameter symmetric version of the Markov chain. Further, compelling statistical evidence is presented which suggests that the parameters particularizing the Markov chain are organism dependent rather than merely chromosome dependent. This surprising observation begs an appropriate biological explanation. Does there exist some kind of mechanism of "communication" among a eukaryote's chromosomes that serves to maintain common values for all of the chromosomal parameters? Or are the common parameter values merely a consequence of a common environment of origin for all of the chromosomes of an organism? (If so, why so?) A third possible explanation is ruled out: while there exists within the class of Markov chain models under consideration, a case that could be described as equivalent to "flipping a fair coin," a thorough-random-shuffling-of-the-genes explanation, via mutations, does not hold up to statistical scrutiny.