Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Homogeneity of the 16S rDNA sequence among geographically disparate isolates of Taylorella equigenitalis

Background

At present, six accessible sequences of 16S rDNA from Taylorella equigenitalis (T. equigenitalis) are available, whose sequence differences occur at a few nucleotide positions. Thus it is important to determine these sequences from additional strains in other countries, if possible, in order to clarify any anomalies regarding 16S rDNA sequence heterogeneity. Here, we clone and sequence the approximate full-length 16S rDNA from additional strains of T. equigenitalis isolated in Japan, Australia and France and compare these sequences to the existing published sequences.

Results

Clarification of any anomalies regarding 16S rDNA sequence heterogeneity of T. equigenitalis was carried out. When cloning, sequencing and comparison of the approximate full-length 16S rDNA from 17 strains of T. equigenitalis isolated in Japan, Australia and France, nucleotide sequence differences were demonstrated at the six loci in the 1,469 nucleotide sequence. Moreover, 12 polymorphic sites occurred among 23 sequences of the 16S rDNA, including the six reference sequences.

Conclusion

High sequence similarity (99.5% or more) was observed throughout, except from nucleotide positions 138 to 501 where substitutions and deletions were noted.     

Phylogenomic species delimitation and host‐symbiont coevolution in the fungus‐farming ant genus Sericomyrmex Mayr (Hymenoptera: Formicidae): ultraconserved elements (UCEs) resolve a recent radiation

Ants in the Neotropical genus Sericomyrmex Mayr cultivate fungi for food. Both ants and fungi are obligate, coevolved symbionts. The taxonomy of Sericomyrmex is problematic because the morphology of the worker caste is generally homogeneous across all of the species within the genus, species limits are vague, and the relationships between them are unknown. We used ultraconserved elements (UCEs) as genome‐scale markers to reconstruct evolutionary history and to infer species boundaries in Sericomyrmex. We recovered an average of ～990 UCE loci for 88 Sericomyrmex samples from across the geographical range of the genus as well as for five outgroup taxa. Using maximum likelihood and species‐tree approaches, we recovered nearly identical topologies across datasets with 50–95% matrix completeness. We identify nine species‐level lineages in Sericomyrmex, including two new species. This is less than the previously described 19 species, even accounting for two species for which we had no UCE samples, which brings the total number of Sericomyrmex species to 11. Divergence‐dating analyses recovered 4.3 Ma as the crown‐group age estimates for Sericomyrmex, indicating a recent, rapid radiation. We also sequenced mitochondrial cytochrome oxidase subunit I (COI) for 125 specimens. Resolution and support for clades in our COI phylogeny are weak, indicating that COI is not an appropriate species‐delimitation tool. However, taxa within species consistently cluster together, suggesting that COI is useful as a species identification (‘DNA barcoding’) tool. We also sequenced internal transcribed spacer (ITS) and large subunit (LSU) for 32 Sericomyrmex fungal cultivars. The fungal phylogeny confirms that Sericomyrmex fungi are generalized higher‐attine cultivars, interspersed with Trachymyrmex‐associated fungal species, indicating cultivar sharing and horizontal transfer between these two genera. Our results indicate that UCEs offer immense potential for delimiting and resolving relationships of problematic, recently diverged species.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Modeling <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> metabolism: from genome to metabolic fluxes

Background  

Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. In general, most of the recent work on N. meningitidis focuses on potential antigens and their functions, immunogenicity, and pathogenicity mechanisms. Very little work has been carried out on Neisseria primary metabolism over the past 25 years.     

The cold‐water climate shield: delineating refugia for preserving salmonid fishes through the 21st century

The distribution and future fate of ectothermic organisms in a warming world will be dictated by thermalscapes across landscapes. That is particularly true for stream fishes and cold‐water species like trout, salmon, and char that are already constrained to high elevations and latitudes. The extreme climates in those environments also preclude invasions by most non‐native species, so identifying especially cold habitats capable of absorbing future climate change while still supporting native populations would highlight important refugia. By coupling crowd‐sourced biological datasets with high‐resolution stream temperature scenarios, we delineate network refugia across >250 000 stream km in the Northern Rocky Mountains for two native salmonids—bull trout (BT) and cutthroat trout (CT). Under both moderate and extreme climate change scenarios, refugia with high probabilities of trout population occupancy (>0.9) were predicted to exist (33–68 BT refugia; 917–1425 CT refugia). Most refugia are on public lands (>90%) where few currently have protected status in National Parks or Wilderness Areas (<15%). Forecasts of refuge locations could enable protection of key watersheds and provide a foundation for climate smart planning of conservation networks. Using cold water as a ‘climate shield’ is generalizable to other species and geographic areas because it has a strong physiological basis, relies on nationally available geospatial data, and mines existing biological datasets. Importantly, the approach creates a framework to integrate data contributed by many individuals and resource agencies, and a process that strengthens the collaborative and social networks needed to preserve many cold‐water fish populations through the 21st century.     

Review and classification of variability analysis techniques with clinical applications

Background

Representation of independent biophysical sources using Fourier analysis can be inefficient because the basis is sinusoidal and general. When complex fractionated atrial electrograms (CFAE) are acquired during atrial fibrillation (AF), the electrogram morphology depends on the mix of distinct nonsinusoidal generators. Identification of these generators using efficient methods of representation and comparison would be useful for targeting catheter ablation sites to prevent arrhythmia reinduction.

Method

A data-driven basis and transform is described which utilizes the ensemble average of signal segments to identify and distinguish CFAE morphologic components and frequencies. Calculation of the dominant frequency (DF) of actual CFAE, and identification of simulated independent generator frequencies and morphologies embedded in CFAE, is done using a total of 216 recordings from 10 paroxysmal and 10 persistent AF patients. The transform is tested versus Fourier analysis to detect spectral components in the presence of phase noise and interference. Correspondence is shown between ensemble basis vectors of highest power and corresponding synthetic drivers embedded in CFAE.

Results

The ensemble basis is orthogonal, and efficient for representation of CFAE components as compared with Fourier analysis (p ≤ 0.002). When three synthetic drivers with additive phase noise and interference were decomposed, the top three peaks in the ensemble power spectrum corresponded to the driver frequencies more closely as compared with top Fourier power spectrum peaks (p ≤ 0.005). The synthesized drivers with phase noise and interference were extractable from their corresponding ensemble basis with a mean error of less than 10%.

Conclusions

The new transform is able to efficiently identify CFAE features using DF calculation and by discerning morphologic differences. Unlike the Fourier transform method, it does not distort CFAE signals prior to analysis, and is relatively robust to jitter in periodic events. Thus the ensemble method can provide a useful alternative for quantitative characterization of CFAE during clinical study.     

Aromatic ring cleavage by homoprotocatechuate 2,3-dioxygenase: role of His200 in the kinetics of interconversion of reaction cycle intermediates

Homoprotocatechuate 2,3-dioxygenase (WT 2,3-HPCD) isolated from Brevibacterium fuscum utilizes an active site Fe(II) and O(2) to catalyze proximal extradiol cleavage of the aromatic ring of the substrate (HPCA). Here, the conserved active site residue His200 is changed to Gln, Glu, Ala, Asn, and Phe, and the reactions of the mutant enzymes are probed using steady-state and transient kinetic techniques. Each mutant catalyzes ring cleavage of HPCA to yield the normal product. H200Q and H200N retain 30-40% of the WT 2,3-HPCD activity at 24 degrees C, but the other mutants reduce the k(cat) to less than 9% of normal. The origin of the reduced activity is unlikely to be the substrate binding phase of the catalytic cycle, because the multistep anaerobic binding reaction of the chromophoric substrate 4-nitrocatechol (4NC) is shown to proceed with rate constants similar to those observed for WT 2,3-HPCD. In contrast, the rate constants of several steps in the multistep O(2) binding/insertion and product release half of the reaction cycle are substantially slowed, in particular the steps in which activated oxygen attacks the organic substrate and in which product is released. In the case of the H200N mutant, the product of 4NC oxidation is not the usual ring cleavage product, but rather the 4NC quinone. These results suggest that the main role of His200 is in facilitating the steps in the second half of the reaction cycle. The decreased rate constants for the O(2) insertion steps in the catalytic cycles of the mutant enzymes allow the oxygen adduct of an extradiol dioxygenase to be detected for the first time.     

Morphological Changes in the Midgut of Aedes aegypti L. (Diptera: Culicidae) Larvae Following Exposure to an Annona coriacea (Magnoliales: Annonaceae) Extract

Bioinsecticides are important in the control of disease vectors, but data regarding their physiological effects on target insects are incomplete. This study describes morphological changes that occur in the midgut of third instar Aedes aegypti L. (Diptera: Culicidae) following treatment with a methanolic extract of Annona coriacea (Magnoliales: Annonaceae). Dissected midguts were subdivided into anterior and posterior regions and analyzed by light and scanning electron microscopy. Insects exposed to the extract displayed intense, destructive cytoplasmic vacuolization in columnar and regenerative midgut cells. The apical surfaces of columnar cells exhibited cytoplasmic protrusions oriented toward the lumen, suggesting that these cells could be involved in apocrine secretory processes and/or apoptosis. We report that A. coriacea extracts induced morphological alterations in the midgut of A. aegypti midgut larvae, supporting the use of plant extracts for control of the dengue vector.