Polymerization of Horseradish Peroxidase by a Laccase‐Catalyzed Tyrosine Coupling Reaction

The polymerization of proteins can create newly active and large bio‐macromolecular assemblies that exhibit unique functionalities depending on the properties of the building block proteins and the protein units in polymers. Herein, the first enzymatic polymerization of horseradish peroxidase (HRP) is reported. Recombinant HRPs fused with a tyrosine‐tag (Y‐tag) through a flexible linker at the N‐ and/or C‐termini are expressed in silkworm, Bombyx mori. Trametes sp. laccase (TL) is used to activate the tyrosine of Y‐tagged HRPs with molecular O₂ to form a tyrosyl‐free radical, which initiates the tyrosine coupling reaction between the HRP units. A covalent dityrosine linkage is also formed through a HRP‐catalyzed self‐crosslinking reaction in the presence of H₂O₂. The addition of H₂O₂ in the self‐polymerization of Y‐tagged HRPs results in lower activity of the HRP polymers, whereas TL provides site‐selectivity, mild reaction conditions and maintains the activity of the polymeric products. The cocrosslinking of Y‐tagged HRPs and HRP‐protein G (Y‐HRP‐pG) units catalyzed by TL shows a higher signal in enzyme‐linked immunosorbent assay (ELISA) than the genetically pG‐fused HRP, Y‐HRP‐pG, and its polymers. This new enzymatic polymerization of HRP promises to provide highly active and functionalized polymers for biomedical applications and diagnostics probes.     

Responses of Adult Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) to Light and Combinations of Attractants and Light

Responses of adult Indianmeal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) to light alone and to a combination of attractant(s) and green/UV lights were studied. When P. interpunctella adults were given a choice between dark areas and areas illuminated with UV, green, or white light, they rested preferentially on surfaces in the illuminated areas. UV light elicited the strongest of the positive phototactic responses. Light traps were not as effective as traps baited with pheromones or food lures in capturing adult moths, and combining green or UV light with these attractants did not significantly increase the trap catches. Gravid females required a period of darkness to realize maximum oviposition, and illumination above 8 lx during the scotophase of a 24-hr light–dark cycle inhibited oviposition.     

Deriving how far structural information is transmitted through parallel homodimeric coiled‐coils: A correlation analysis of helical staggers

How local conformation is affected by local sequence is fairly well understood for alpha‐helical coiled‐coils, but less is known about how local conformation is influenced by distant features. Here, I describe an approach to detect such an effect, based on computing correlation coefficients of local out‐of‐register alignments, or so‐called “staggers” between the helices, as a function of the axial distance between the staggers. This approach requires parallel homodimers, in which each stagger can occur with two “signs,” where either one helix or the other is shifted towards the N terminus. The signs of such staggers separated by up to 12 residues are strongly correlated, indicating that the conformations of the ends of coiled‐coils are commonly influenced by attached structures. Thus, the structures of coiled‐coil residues aberrantly attached to alternative proteins, such as those resulting from leukemogenic chromosomal rearrangements, may be distinguishable from those in normal tissues, and in turn serve as targets of selective drug design. The signs of helical staggers separated by between 13 and 30 residues are moderately yet significantly correlated, indicating that some of the coiled‐coils transmit this conformational feature axially for at least 45 Å. A positive, albeit noisy, correlation also exists among tropomyosin coiled‐coils for signed staggers separated by the 40‐residue actin repeat distance, consistent with the semi‐flexible tropomyosin filament binding F‐actin and regulating skeletal muscle contraction in a partially cooperative manner. Communication of the signs of axial staggers is explained in part by minimization of main‐chain hydrogen bond deformations. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Galactomannanases Man2 and Man5 from Thermotoga species: growth physiology on galactomannans, gene sequence analysis, and biochemical properties of recombinant enzymes

The enzymatic hydrolysis of mannan-based hemicelluloses is technologically important for applications ranging from pulp and paper processing to food processing to gas and oil well stimulation. In many cases, thermostability and activity at elevated temperatures can be advantageous. To this end, the genes encoding beta-mannosidase (man2) and beta-mannanase (man5) from the hyperthermophilic bacteria Thermotoga neapolitana 5068 and Thermotoga maritima were isolated, cloned, and expressed in Escherichia coli. The amino acid sequences for the mannosidases from these organisms were 77% identical and corresponded to proteins with an M(r) of approximately 92 kDa. The translated nucleotide sequences for the beta-mannanase genes (man5) encoded polypeptides with an M(r) of 76 kDa that exhibited 84% amino acid sequence identity. The recombinant versions of Man2 and Man5 had similar respective biochemical and biophysical properties, which were also comparable to those determined for the native versions of these enzymes in T. neapolitana. The optimal temperature and pH for the recombinant Man2 and Man5 from both organisms were approximately 90 degrees C and 7.0, respectively. The presence of Man2 and Man5 in these two Thermotoga species indicates that galactomannan is a potential growth substrate. This was supported by the fact that beta-mannanase and beta-mannosidase activities were significantly stimulated when T. neapolitana was grown on guar or carob galactomannan. Maximum cell densities increased by at least tenfold when either guar or carob galactomannan was added to the growth medium. For T. neapolitana grown on guar at 83 degrees C, Man5 was secreted into the culture media, whereas Man2 was intracellular. These localizations were consistent with the presence and lack of signal peptides for Man5 and Man2, respectively. The identification of the galactomannan-degrading enzymes in these Thermotoga species adds to the list of biotechnologically important hemicellulases produced by members of this hyperthermophilic genera.     

Multivariate multiple test procedures based on nonparametric copula estimation

Multivariate multiple test procedures have received growing attention recently. This is due to the fact that data generated by modern applications typically are high‐dimensional, but possess pronounced dependencies due to the technical mechanisms involved in the experiments. Hence, it is possible and often necessary to exploit these dependencies in order to achieve reasonable power. In the present paper, we express dependency structures in the most general manner, namely, by means of copula functions. One class of nonparametric copula estimators is constituted by Bernstein copulae. We extend previous statistical results regarding bivariate Bernstein copulae to the multivariate case and study their impact on multiple tests. In particular, we utilize them to derive asymptotic confidence regions for the family‐wise error rate (FWER) of multiple test procedures that are empirically calibrated by making use of Bernstein copulae approximations of the dependency structure among the test statistics. This extends a similar approach by Stange et al. (2015) in the parametric case. A simulation study quantifies the gain in FWER level exhaustion and, consequently, power that can be achieved by exploiting the dependencies, in comparison with common threshold calibrations like the Bonferroni or ?idák corrections. Finally, we demonstrate an application of the proposed methodology to real‐life data from insurance.     

Variation in the life history pattern of Tetranychus urticae (Acari: Tetranychidae) after selection for dispersal

The spider mite Tetranychus urticae shows variation in its dispersal capacity (i.e., the leaf quality at which a female decides to disperse). We were able to artificially select mites that had either a high or a low dispersal capacity, indicating that this trait was genetically controlled. We then compared correlated responses to this selection. Mites with a genetically high dispersal capacity (‘HD’ strains) had a higher diapause incidence and a lower performance compared to mites with a low dispersal capacity (‘LD’ strains). A possible effect of random genetic drift during the selection was negligible. Our results suggest that differential dispersal capacity is associated with contrasting life history patterns as a result of natural selection. This revised version was published online in July 2006 with corrections to the Cover Date.     

The evolution of sex determination systems in dipteran insects other than Drosophila

The multitude of sex determination mechanisms displayed in dipteran insects has usually been described in terms of variations on a single principle in which the primary signal of the primitive pathway consists of a single allelic difference at one locus. Evolution of sex determination mechanisms is thought to have occurred by the addition of genes below the top gene of the pathway. The elucidation of the complex sex determination pathway of Drosophila melanogaster, as well as recent evidence that the basal genes of the pathway seem to be conserved across metazoan genera both in structure and, to a lesser degree, in function, points towards the possibility that sex determination pathways may have evolved from the bottom-up. Further to this is the question of whether the dominant male-determining factor, M, which is found in a number of insect species, represents part of the ancient sex determination pathway or is a later addition to the pathway. This, together with the possibility that the Mfactors found in numerous dipteran insect species may have a common origin, is discussed. The similarities of the sex determination pathways under the control of Mand the implications in relation to the construction of genetic sexing strains for biological control are also discussed.     

Automated in situ measurement of cell-specific antibody secretion and laser-mediated purification for rapid cloning of highly-secreting producers

Cloning of highly-secreting recombinant cells is critical for biopharmaceutical manufacturing, but faces numerous challenges including the fact that secreted protein does not remain associated with the producing cell. A fundamentally new approach was developed combining in situ capture and measurement of individual cell protein secretion followed by laser-mediated elimination of all non- and poorly-secreting cells, leaving only the highest-secreting cell in a well. Recombinant cells producing humanized antibody were cultured serum-free on a capture matrix, followed by staining with fluorescently-labeled anti-human antibody fragment. A novel, automated, high-throughput instrument (called LEAP) was used to image and locate every cell, quantify the cell-associated and secreted antibody (surrounding each cell), eliminate all undesired cells from a well via targeted laser irradiation, and then track clone outgrowth and stability. Temporarily sparing an island of helper cells around the clone of interest improved cloning efficiency (particularly when using serum-free medium), and helper cells were easily eliminated with the laser after several days. The in situ nature of this process allowed several serial sub-cloning steps to be performed within days of one another, resulting in rapid generation of clonal populations with significantly increased and more stable, homogeneous antibody secretion. Cell lines with specific antibody secretion rates of > 50 pg/cell per day (in static batch culture) were routinely obtained as a result of this cloning approach, often times representing up to 20% of the clones screened.     

Relationship of stigma behaviors and breeding system in three Mazus (Phrymaceae) species with bilobed stigma

Sensitive stigma has been recognized to facilitate outcrossing. We hypothesized that species with different levels of sensitivity might have corresponding differences in components of their breeding system. In this study, three Mazus species with bilobed stigmas were used to test the hypothesis. We explored stigma behaviors of the species in reaction time, recovery time, permanent closing time, and the minimum pollen load causing permanent closure. We investigated floral traits, pollinator type and behavior, pollination intensity, and natural schedule of pollen deposition on stigma. Moreover, we evaluated the mating system of the species by checking seed set after controlled pollination treatments, namely, natural flowers with open pollination, enclosed flowers without pollination, and enclosed flowers with self and outcross hand pollination. Results indicated that stigma of M. pumilus (N. L. Burman) Steenis was not sensitive, whereas stigmas of M. miquelii Makino and M. stachydifolius (Turcz.) Maxim. closed and reopened quickly in response to pollination. Accordingly, hand pollination treatments revealed that seed set of self-spontaneous pollination in M. pumilus was similar to the other treatments. For M. miquelii, outcross pollen resulted in significantly higher seed set than self-pollen.Mazus stachydifolius was self-incompatible. Additionally, the corresponding characteristics in other components of the breeding system for each species were found. Our study indicated that the sensitivity of bilobed stigma might be linked with floral traits and the mating system in a given species. Sensitive stigma should be regarded as an evolutionary mechanism for enhancement of outcrossing.