Estimating the Location and Time Course of Synaptic Input from Multi-Site Potential Recordings

A method is introduced that permits accurate and robust extraction of the location and time course of synaptic conductance from potentials recorded on either side of, and perhaps at some distance from, the synapse in question. It is shown that such data permits one to fully overcome the problems typically associated with lack of spaceclamp. The method does not presume anything about the nature of the time course and yet is applicable to branched, active cells receiving simultaneous input from a number of synapses.     

Bacterial Diversity in Mine Tailings Compared by Cultivation and Cultivation-independent Methods and their Resistance to Lead and Cadmium

To examine bacterial community composition in rhizosphere of plants colonizing on mine tailings and phylogenetic differences between subcommunities resistant to different metals, we constructed four clone libraries of 16S rDNA sequences. One was amplified directly from tailing microbial DNA (named as Ci library) and three from cultures on the plates containing of 0.5 mM CdCl(2) (Cd library), 2 mM Pb (NO(3))(2) (Pb library), and without any metals (Cw library). In total, nine bacterial divisions and two unclassified groups were identified from 352 clones of these libraries. Ci clones covered eight divisions, whereas all cultivable clones only covered four divisions. Thus, Ci library provided more phylogenetic diversity than cultivable libraries. However, the microbes represented by the cultivable clones were more similar to previously described bacteria than those represented by Ci clones. All Ci clones were not found in three cultivable libraries. Cd library were exclusively Gram-negative bacteria of Acinetobacter, Ralstonia, Comamonas, and Chryseobacterium. Meanwhile, dominant Gram-positive bacteria in Pb library, Paenibacillus and Bacillus, were also not found in Cd library. Our data indicate that phylogenetic structure was very different from those in acid mine drainage. Meanwhile, tailings harbored phylogenetically distinct subcommunities resistant to Pb and Cd.     

A comparative study of endophytic and epiphytic fungal association with leaf of <Emphasis Type="Italic">Eucalyptus citriodora</Emphasis> Hook., and their antimicrobial activity

Eucalyptus citriodora Hook, is frequently cultivated tree in India for its wood and medicinal usages. The endophytic and epiphytic fungi were estimated from healthy leaves of E. citriodora growing in the premise of Banaras Hindu University, Varanasi, India. A total of 33 fungal species were isolated from leaf segments. Of 33 taxa, 20 were recorded as endophytes, while 22 as epiphytes. Nine, out of 33 species were found to be common in leaf tissues and surfaces (Alternaria alternata, Aspergillus fumigatus, A. terreus, Cladosporium cladosporioides, Drechslera rostrata, Humicola grisea, Nigrospora oryzae, Penicillium cristata, and Pestalotia sp.). Out of 478 fungal isolates, 279 were epiphytic while only 199 were endophytic. Most isolates were anamorphic filamentous fungi which often don’t produce sexual spores. The Sorensen’s index of similarity between endophytes and epiphytes (leaf surface colonizers) was found to be at 0.300. Diversity index of fungal species was higher in endophytes than epiphytes. The frequency of colonization differs greatly in both myco-populations. Cladosporium cladosporioides (26%) was dominant species on leaf surfaces while Botrytis cinerea (10.5%) was dominant in leaf tissues. Out of 16 endophytic isolates evaluated for antagonistic test, 8 (50%) gave the antagonistic activity against variety of fungi representing pathogens to both humans and plants.     

Investigation of fullerenol-induced changes in poroelasticity of human hepatocellular carcinoma by AFM-based creep tests

In this study, atomic force microscopy (AFM) is used to investigate the alterations of the poroelastic properties of hepatocellular carcinoma (SMMC-7721) cells treated with fullerenol. The SMMC-7721 cells were subject to AFM-based creep tests, and a corresponding poroelastic indentation model was used to determine the poroelastic parameters by curve fitting. Comparative analyses indicated that the both permeability and diffusion of fullerenol-treated cells increased significantly while their elastic modulus decreased by a small amount. From the change in the trend of the determined parameter, we verified the corresponding alternations of cytoskeleton (mainly filaments actin), which was reported by the previous study using confocal imaging method. Our investigation on SMMC-7721 cell reveals that the poroelastic properties could provide a better understanding how the cancer cells are affected by fullerenol or potentially other drugs which could find possible applications in drug efficacy test, cancer diagnosis and secure therapies.     

Differences in the chitinolytic activity of mammalian chitinases on soluble and insoluble substrates

Chitin is an abundant polysaccharide used by many organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Acidic mammalian chitinase, a processive glycosyl hydrolase, is the primary enzyme involved in the degradation of environmental chitin in mammalian lungs. Mutations to acidic mammalian chitinase have been associated with asthma, and genetic deletion in mice increases morbidity and mortality with age. We initially set out to reverse this phenotype by engineering hyperactive acidic mammalian chitinase variants. Using a screening approach with commercial fluorogenic substrates, we identified mutations with consistent increases in activity. To determine whether the activity increases observed were consistent with more biologically relevant chitin substrates, we developed new assays to quantify chitinase activity with insoluble chitin, and identified a one‐pot fluorogenic assay that is sufficiently sensitive to quantify changes to activity due to the addition or removal of a carbohydrate‐binding domain. We show that the activity increases from our directed evolution screen were lost when insoluble substrates were used. In contrast, naturally occurring gain‐of‐function mutations gave similar results with oligomeric and insoluble substrates. We also show that activity differences between acidic mammalian chitinase and chitotriosidase are reduced with insoluble substrate, suggesting that previously reported activity differences with oligomeric substrates may have been driven by differential substrate specificity. These results highlight the need for assays against physiological substrates when engineering metabolic enzymes, and provide a new one‐pot assay that may prove to be broadly applicable to engineering glycosyl hydrolases.     

Nested Canalyzing Depth and Network Stability

We introduce the nested canalyzing depth of a function, which measures the extent to which it retains a nested canalyzing structure. We characterize the structure of functions with a given depth and compute the expected activities and sensitivities of the variables. This analysis quantifies how canalyzation leads to higher stability in Boolean networks. It generalizes the notion of nested canalyzing functions (NCFs), which are precisely the functions with maximum depth. NCFs have been proposed as gene regulatory network models, but their structure is frequently too restrictive and they are extremely sparse. We find that functions become decreasingly sensitive to input perturbations as the canalyzing depth increases, but exhibit rapidly diminishing returns in stability. Additionally, we show that as depth increases, the dynamics of networks using these functions quickly approach the critical regime, suggesting that real networks exhibit some degree of canalyzing depth, and that NCFs are not significantly better than functions of sufficient depth for many applications of the modeling and reverse engineering of biological networks.     

γ<Subscript>1</Subscript>-Dependent Down-regulation of Recombinant Voltage-gated Ca<Superscript>2+</Superscript> Channels

(1) Voltage-gated Ca²⁺ (Ca_V) channels are multi-subunit membrane complexes that allow depolarization-induced Ca²⁺ influx into cells. The skeletal muscle L-type Ca_V channels consist of an ion-conducting Ca_V1.1 subunit and auxiliary α₂δ−1, β₁ and γ₁ subunits. This complex serves both as a Ca_V channel and as a voltage sensor for excitation–contraction coupling. (2) Though much is known about the mechanisms by which the α₂δ−1 and β₁ subunits regulate Ca_V channel function, there is far less information on the γ₁ subunit. Previously, we characterized the interaction of γ₁ with the other components of the skeletal Ca_V channel complex, and showed that heterologous expression of this auxiliary subunit decreases Ca²⁺ current density in myotubes from γ₁ null mice. (3) In the current report, using Western blotting we show that the expression of the Ca_V1.1 protein is significantly lower when it is heterologously co-expressed with γ₁. Consistent with this, patch-clamp recordings showed that transient transfection of γ₁ drastically inhibited macroscopic currents through recombinant N-type (Ca_V2.2/α₂δ−1/β₃) channels expressed in HEK-293 cells. (4) These findings provide evidence that co-expression of the auxiliary γ₁ subunit results in a decreased expression of the ion-conducting subunit, which may help to explain the reduction in Ca²⁺ current density following γ₁ transfection.     

Anciently duplicated <Emphasis Type="Italic">Broad Complex</Emphasis> exons have distinct temporal functions during tissue morphogenesis

Broad Complex (BRC) is an essential ecdysone-pathway gene required for entry into and progression through metamorphosis in Drosophila melanogaster. Mutations of three BRC complementation groups cause numerous phenotypes, including a common suite of morphogenesis defects involving central nervous system (CNS), adult salivary glands (aSG), and male genitalia. These defects are phenocopied by the juvenile hormone mimic methoprene. Four BRC isoforms are produced by alternative splicing of a protein-binding BTB/POZ-encoding exon (BTB _BRC ) to one of four tandemly duplicated, DNA-binding zinc-finger-encoding exons (Z1 _BRC , Z2 _BRC , Z3 _BRC , Z4 _BRC ). Highly conserved orthologs of BTB _BRC and all four Z _BRC were found among published cDNA sequences or genome databases from Diptera, Lepidoptera, Hymenoptera, and Coleoptera, indicating that BRC arose and underwent internal exon duplication before the split of holometabolous orders. Tramtrack subfamily members, abrupt, tramtrack, fruitless, longitudinals lacking (lola), and CG31666 were characterized throughout Holometabola and used to root phylogenetic analyses of Z _BRC exons, which revealed that the Z _BRC clade includes Z _abrupt . All four Z _BRC domains, including Z4 _BRC , which has no known essential function, are evolving in a manner consistent with selective constraint. We used transgenic rescue to explore how different BRC isoforms contribute to shared tissue-morphogenesis functions. As predicted from earlier studies, the common CNS and aSG phenotypes were rescued by BRC-Z1 in rbp mutants, BRC-Z2 in br mutants, and BRC-Z3 in 2Bc mutants. However, the isoforms are required at two different developmental stages, with BRC-Z2 and -Z3 required earlier than BRC-Z1. The sequential action of BRC isoforms indicates subfunctionalization of duplicated Z _BRC exons even when they contribute to common developmental processes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

What is known about phytohormones in halophytes? A review

Phytohormones participate in many aspects of the plant life cycle, including responses to biotic and abiotic stresses. They play a key role in plant responses to the environment with direct bearing on a plant’s fitness for adaptation and reproduction. In recent years, there have been major advances in our understanding of the role of phytohormones in halophytic plants. The variability in maximal salinity level that halophytes can tolerate makes it difficult to characterize the specific traits responsible for salt tolerance. However, the most evident effect of salinity is growth disturbance, and growth is directly governed by phytohormones. Phytohormones such as abscisic acid, salicylic acid ethylene and jasmonates are traditionally related to stress responses, while the involvement of cytokinins, gibberellins and auxins has started to be analyzed. Polyamines, although they can’t be considered phytohormones because of the high concentrations required for cell responses, have been proposed as a new category of plant growth regulators involved in several plant processes and stress responses. This review integrates the advances in the knowledge about phytohormones in halophytes and their participation in salt tolerance.