Pulsed EPR studies of the type 2 copper binding site in the mercury derivative of laccase.

The nuclear modulation effect in pulsed EPR spectroscopy was used to study the type 2 copper binding site in the mercury derivative of laccase (MDL) in which the type 1 copper is substituted by Hg(II). By comparing the three-pulse electron spin-echo modulations and Fourier transform spectra of MDL and several model compounds, we conclude that the imidazole groups of two histidyl amino acid residues are equatorially coordinated to Cu(II) in the type 2 site. Computer simulations of these data suggest that the remote nonbonding nitrogens of the two imidazoles possess nuclear quadrupole parameters e2qQ = 1.47 MHz and eta = 0.83. A(iso) values of these two nitrogens are not identical, being 1.5 and 2.0 MHz. We have also used samples of the enzyme exchanged with D2O to examine the coordination of the water to the type 2 copper site. The deuterium modulation that is resolved by taking the ratio of the time domain ESEEM data from native and D2O-exchanged enzyme indicates that there is an equatorial water ligand, and further data show that this water is displaced by azide.     

Image analysis applied to proliferating cells in malignant lymphoma

This study describes a method for obtaining the distribution of a given cell type in histological sections, as revealed by a specific immunological marker. Two hundred fifty-six contiguous microscopic fields were analysed, and a cellular map was automatically constructed. The resulting images were stored on magnetic media for subsequent quantification and statistical tests. The method was applied to the analysis of 20 small cleaved-cell lymphomas in which the monoclonal antibody Ki-67 was used to mark proliferating cells. Clusters of proliferative cells were observed in both diffuse and nodular lymphomas. Cellularity and the percentage of marked cells were not significantly different in the two types of tumors.     

Stem cells in adult tissues

In recent years the concept of a stem cell has evolved to encompass the hypotheses that stem cells exist within many adult tissues, and that a common 'interchangeable' progenitor cell may exist within the bone marrow capable of regenerating and repairing tissues throughout the body. As more knowledge is gained about stem cells, their potential roles in disease processes, including the development and progression of cancer, have moved to the forefront. The underlying hypothesis of this review is that cell fate is determined by a combination of intrinsic and extrinsic factors; growth and differentiation are regulated through intracellular integration of a multitude of signals initiated by internal and external stimuli. The development of successful stem cell based therapies may depend on experimental approaches that consider both the intrinsic and extrinsic factors that control cell fate.     

Rapid shoot regeneration in industrial ‘high starch’ sweetpotato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> L.) genotypes

Sweetpotato (Ipomoea batatas L.) is an important crop in North Carolina with annual production of 0.33 million tons, accounting for 37% of total US supply (USDA, Louisiana Farm Reporter 8(12), August 2008). To target industrial use, novel high-starch industrial-type varieties that contain more than 30% dry matter were developed by conventional breeding methods. In vitro cultures from selected genotypes were established using meristem culture. To establish regeneration procedures that could be coupled with transformation experiments, conditions for the induction of rapid shoot-organogenesis in leaf explants were compared using varying concentrations of the auxins ‘NAA’, ‘IAA’, ‘2,4-D’, and ‘4-FA’ either alone or in combination with zeatin riboside. Regeneration efficiencies, defined as the number of explants developing shoots out of the total number tested, were as high as 57% for the best genotypes, with a significant genotype-dependent response observed in all the hormone regimes evaluated. In all treatments, shoot regeneration was observed within 2 months. Our results led to the establishment of optimized in vitro regeneration procedures for the novel high-starch sweetpotato (SP) genotypes ‘DM01-158’, ‘FTA94’, ‘FT489’, and ‘PDM P4’ that are rapid and reliable.     

Genome wide gene-expression analysis of facultative reproductive diapause in the two-spotted spider mite Tetranychus urticae

Background

Diapause or developmental arrest, is one of the major adaptations that allows mites and insects to survive unfavorable conditions. Diapause evokes a number of physiological, morphological and molecular modifications. In general, diapause is characterized by a suppression of the metabolism, change in behavior, increased stress tolerance and often by the synthesis of cryoprotectants. At the molecular level, diapause is less studied but characterized by a complex and regulated change in gene-expression. The spider mite Tetranychus urticae is a serious polyphagous pest that exhibits a reproductive facultative diapause, which allows it to survive winter conditions. Diapausing mites turn deeply orange in color, stop feeding and do not lay eggs.

Results

We investigated essential physiological processes in diapausing mites by studying genome-wide expression changes, using a custom built microarray. Analysis of this dataset showed that a remarkable number, 11% of the total number of predicted T. urticae genes, were differentially expressed. Gene Ontology analysis revealed that many metabolic pathways were affected in diapausing females. Genes related to digestion and detoxification, cryoprotection, carotenoid synthesis and the organization of the cytoskeleton were profoundly influenced by the state of diapause. Furthermore, we identified and analyzed an unique class of putative antifreeze proteins that were highly upregulated in diapausing females. We also further confirmed the involvement of horizontally transferred carotenoid synthesis genes in diapause and different color morphs of T. urticae.

Conclusions

This study offers the first in-depth analysis of genome-wide gene-expression patterns related to diapause in a member of the Chelicerata, and further adds to our understanding of the overall strategies of diapause in arthropods.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-815) contains supplementary material, which is available to authorized users.     

Arabidopsis thaliana Chromosome 4 Replicates in Two Phases That Correlate with Chromatin State

DNA replication programs have been studied extensively in yeast and animal systems, where they have been shown to correlate with gene expression and certain epigenetic modifications. Despite the conservation of core DNA replication proteins, little is known about replication programs in plants. We used flow cytometry and tiling microarrays to profile DNA replication of Arabidopsis thaliana chromosome 4 (chr4) during early, mid, and late S phase. Replication profiles for early and mid S phase were similar and encompassed the majority of the euchromatin. Late S phase exhibited a distinctly different profile that includes the remaining euchromatin and essentially all of the heterochromatin. Termination zones were consistent between experiments, allowing us to define 163 putative replicons on chr4 that clustered into larger domains of predominately early or late replication. Early-replicating sequences, especially the initiation zones of early replicons, displayed a pattern of epigenetic modifications specifying an open chromatin conformation. Late replicons, and the termination zones of early replicons, showed an opposite pattern. Histone H3 acetylated on lysine 56 (H3K56ac) was enriched in early replicons, as well as the initiation zones of both early and late replicons. H3K56ac was also associated with expressed genes, but this effect was local whereas replication time correlated with H3K56ac over broad regions. The similarity of the replication profiles for early and mid S phase cells indicates that replication origin activation in euchromatin is stochastic. Replicon organization in Arabidopsis is strongly influenced by epigenetic modifications to histones and DNA. The domain organization of Arabidopsis is more similar to that in Drosophila than that in mammals, which may reflect genome size and complexity. The distinct patterns of association of H3K56ac with gene expression and early replication provide evidence that H3K56ac may be associated with initiation zones and replication origins.     

Plant cell calcium‐rich environment enhances thermostability of recombinantly produced α‐amylase from the hyperthermophilic bacterium Thermotoga maritime

In the industrial processing of starch for sugar syrup and ethanol production, a liquefaction step is involved where starch is initially solubilized at high temperature and partially hydrolyzed with a thermostable and thermoactive α‐amylase. Most amylases require calcium as a cofactor for their activity and stability, therefore calcium, along with the thermostable enzyme, are typically added to the starch mixture during enzymatic liquefaction, thereby increasing process costs. An attractive alternative would be to produce the enzyme directly in the tissue to be treated. In a proof of concept study, tobacco cell cultures were used as model system to test in planta production of a hyperthermophilic α‐amylase from Thermotoga maritima. While comparable biochemical properties to recombinant production in Escherichia coli were observed, thermostability of the plant‐produced α‐amylase benefited significantly from high intrinsic calcium levels in the tobacco cells. The plant‐made enzyme retained 85% of its initial activity after 3 h incubation at 100°C, whereas the E. coli‐produced enzyme was completely inactivated after 30 min under the same conditions. The addition of Ca²⁺ or plant cell extracts from tobacco and sweetpotato to the E. coli‐produced enzyme resulted in a similar stabilization, demonstrating the importance of a calcium‐rich environment for thermostability, as well as the advantage of producing this enzyme directly in plant cells where calcium is readily available. Biotechnol. Bioeng. 2009; 104: 947–956. © 2009 Wiley Periodicals, Inc.     

Hemolytic porcine intestinal Escherichia coli without virulence-associated genes typical of intestinal pathogenic E. coli

Testing 1,666 fecal or intestinal samples from healthy and diarrheic pigs, we obtained hemolytic Escherichia coli isolates from 593 samples. Focusing on hemolytic E. coli isolates without virulence-associated genes (VAGs) typical for enteropathogens, we found that such isolates carried a broad variety of VAGs typical for extraintestinal pathogenic E. coli.