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.     

Factors affecting entomophilic activity of Neoaplectana feltiae in mushroom compost

The host-searching ability of Neoaplectana feltiae Filipjev (= S. bibionis Bovien) (Nematoda: Steinernematidae) in response to larvae of a mushroom fly, Lycoriella solani Winn. was examined in a mushroom substrate. Individuals of L. solani were less attractive for the parasite than larvae of Galleria mellonella L. The nematode juveniles penetrated a 22 cm layer of casing mixture within 2–4 days. In the casing alone nematode effectiveness was better than in mushroom compost or in compost and casing together. In the casing mixture parasite dosages of 20 and 100 juveniles per cm² led to 22% and 45% parasitization of L. solani respectively, while all G. mellonella larvae were parasitized at both dosages. The prevalence of nematode infection depended on the content of water in the mushroom substrate. The highest N. feltiae infectivity was observed, when the ratio of the dry casing weight to the weight of water content was 1: 2.5. The practical aspects of the observed phenomena, essential for the use of N. feltiae in the protection of commercial mushroom cultivation are discussed.     

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.     

Evolutionary dynamics of the kinetochore network in eukaryotes as revealed by comparative genomics

During eukaryotic cell division, the sister chromatids of duplicated chromosomes are pulled apart by microtubules, which connect via kinetochores. The kinetochore is a multiprotein structure that links centromeres to microtubules, and that emits molecular signals in order to safeguard the equal distribution of duplicated chromosomes over daughter cells. Although microtubule‐mediated chromosome segregation is evolutionary conserved, kinetochore compositions seem to have diverged. To systematically inventory kinetochore diversity and to reconstruct its evolution, we determined orthologs of 70 kinetochore proteins in 90 phylogenetically diverse eukaryotes. The resulting ortholog sets imply that the last eukaryotic common ancestor (LECA) possessed a complex kinetochore and highlight that current‐day kinetochores differ substantially. These kinetochores diverged through gene loss, duplication, and, less frequently, invention and displacement. Various kinetochore components co‐evolved with one another, albeit in different manners. These co‐evolutionary patterns improve our understanding of kinetochore function and evolution, which we illustrated with the RZZ complex, TRIP13, the MCC, and some nuclear pore proteins. The extensive diversity of kinetochore compositions in eukaryotes poses numerous questions regarding evolutionary flexibility of essential cellular functions.     

Simulation of human atherosclerotic femoral plaque tissue: the influence of plaque material model on numerical results

Background

Due to the limited number of experimental studies that mechanically characterise human atherosclerotic plaque tissue from the femoral arteries, a recent trend has emerged in current literature whereby one set of material data based on aortic plaque tissue is employed to numerically represent diseased femoral artery tissue. This study aims to generate novel vessel-appropriate material models for femoral plaque tissue and assess the influence of using material models based on experimental data generated from aortic plaque testing to represent diseased femoral arterial tissue.

Methods

Novel material models based on experimental data generated from testing of atherosclerotic femoral artery tissue are developed and a computational analysis of the revascularisation of a quarter model idealised diseased femoral artery from a 90% diameter stenosis to a 10% diameter stenosis is performed using these novel material models. The simulation is also performed using material models based on experimental data obtained from aortic plaque testing in order to examine the effect of employing vessel appropriate material models versus those currently employed in literature to represent femoral plaque tissue.

Results

Simulations that employ material models based on atherosclerotic aortic tissue exhibit much higher maximum principal stresses within the plaque than simulations that employ material models based on atherosclerotic femoral tissue. Specifically, employing a material model based on calcified aortic tissue, instead of one based on heavily calcified femoral tissue, to represent diseased femoral arterial vessels results in a 487 fold increase in maximum principal stress within the plaque at a depth of 0.8 mm from the lumen.

Conclusions

Large differences are induced on numerical results as a consequence of employing material models based on aortic plaque, in place of material models based on femoral plaque, to represent a diseased femoral vessel. Due to these large discrepancies, future studies should seek to employ vessel-appropriate material models to simulate the response of diseased femoral tissue in order to obtain the most accurate numerical results.

     

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.     

Improved Cultivation Systems for Isolation of the Colorado Potato Beetle Spiroplasma

In North America, the Colorado potato beetle, Leptinotarsa decemlineata, is often infected with the host-specific, gut-inhabiting Colorado potato beetle spiroplasma (CPBS). CPBS is apparently a commensal, but it may be useful in biocontrol if it can be transformed to express an insect-lethal gene. Difficulty in cultivating the organism, however, has hindered the development of a suitable transformation system. In this study, we eliminated the need for coculturing CPBS with insect cells. CPBS was reliably isolated with the BBL Anaerobic GasPak Jar system (low redox, enhanced CO(inf2)), which was easier to use and less expensive than insect cell coculture methods. A further advantage is a reduction in contaminating insect cell components. Use of anaerobiosis should facilitate early-passage screening of isolates for extrachromosomal elements, for use in gene vector constructs. The unique spiral (decreasing amplitude of coils) morphology of CPBS was preserved by anaerobiosis. The use of low-pH (6.0 to 6.5) media allowed aerobic adaptation of CPBS to M1D and SP-4 broth media. These formulations permitted the first cultivation of CPBS on solid media, an accomplishment that will simplify the selection of molecular transformants. Potato beetles collected at four sites in Poland yielded CPBS strains similar to those previously obtained from populations in North America.     

Postconditioning for salvage of ischemic skeletal muscle from reperfusion injury: efficacy and mechanism

We tested our hypothesis that postischemic conditioning (PostC) is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP). In bilateral 8x13 cm pig latissimus dorsi muscle flaps subjected to 4 h ischemia, muscle infarction increased from 22+/-4 to 41+/-1% between 2 and 24 h reperfusion and remained unchanged at 48 (38+/-6%) and 72 (40+/-1%) h reperfusion (P<0.05; n=4 pigs). PostC induced by four cycles of 30-s reperfusion/reocclusion at the onset of reperfusion after 4 h ischemia reduced muscle infarction from 44+/-2 to 22+/-2% at 48 h reperfusion. This infarct protective effect of PostC was mimicked by intravenous injection of the mPTP opening inhibitor cyclosporin A or NIM-811 (10 mg/kg) at 5 min before the end of 4 h ischemia and was abolished by intravenous injection of the mPTP opener atractyloside (10 mg/kg) at 5 min before PostC (P<0.05; n=4-5 pigs). PostC or intravenous cyclosporin A injection at 5 min before reperfusion caused a decrease in muscle myeloperoxidase activity and mitochondrial free Ca2+ concentration and an increase in muscle ATP content after 4 h ischemia and 2 h reperfusion compared with the time-matched controls. These effects of PostC were abolished by intravenous injection of atractyloside at 5 min before PostC (P<0.05; n=6 pigs). These observations support our hypothesis that PostC is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mPTP.     

Highly reactive cysteine residues are part of the substrate binding site of mammalian dipeptidyl peptidases III

Dipeptidyl peptidase III (DPP III) is a cytosolic zinc-exopeptidase involved in the intracellular protein catabolism of eukaryotes. Although inhibition by thiol reagents is a general feature of DPP III originating from various species, the function of activity important sulfhydryl groups is still inadequately understood. The present study of the reactivity of these groups was undertaken in order to clarify their biological significance.The inactivation kinetics of human and rat DPP III by sulfhydryl reagent p-hydroxy-mercuribenzoate (pHMB) was monitored by determination of the enzyme's residual activity with fluorimetric detection.Inactivation of this human enzyme exhibited pseudo-first-order kinetics, suggesting that all reactive SH-groups have equivalent reactivity, and the second-order rate constant was calculated to be 3523+/-567M(-1)min(-1). Rat DPP III was hyperreactive to pHMB and showed biphasic kinetics indicating two classes of reactive SH-groups. The second-order rate constants of 3540M(-1)s(-1) for slower reacting sulfhydryl, and 21,855M(-1)s(-1) for faster reacting sulfhydryl were obtained from slopes of linear plots of pseudo-first-order constants versus reagent concentration. Peptide substrates protected both mammalian DPPs III from inactivation by pHMB. Physiological concentrations of biological thiols and H(2)O(2) inactivated the rat DPP III. Human enzyme was resistant to H(2)O(2) attack and less affected by reduced glutathione (GSH) than the rat homologue. A significantly lower DPP III level, determined by activity measurement and Western blotting, was found in the cytosols of highly oxygenated rat tissues.These results provide kinetic evidence that cysteine residues are involved in substrate binding of mammalian DPPs III.