Phenoloxidase Activity of Helix aspersa Maxima (Garden Snail, Gastropod) Hemocyanin

The oxygen-transporting protein, hemocyanin (Hc), of the garden snail Helix aspersa maxima (HaH) was isolated and kinetically characterized. Kinetic parameters of the reaction of catalytic oxidation of catechol to quinone, catalyzed by native HaH were determined: the V _max value amounted to 22 nmol min^?1 mg^?1, k _cat to 1.1 min^?1. Data were compared to those reported for other molluscan Hcs and phenoloxidases (POs). The o-diphenoloxidase activity of the native HaH is about five times higher than the activity determined for the Hcs of the terrestrial snail Helix pomatia and of the marine snail Rapana thomasiana (k _cat values of 0.22 and 0.25 min^?1, respectively). The K _m values obtained for molluscan Hcs from different species are comparable to those for true POs, but the low catalytic efficiency of Hcs is probably related to inaccessibility of the active sites to potential substrates. Upon treatment of HaH with subtilisin DY, the enzyme activity against substrate catechol was considerably increased. The relatively high proteolytically induced o-diPO activity of HaH allowed using it for preparation of a biosensor for detection of catechol.     

Optimized assay and storage conditions for enzyme activity profiling of ectomycorrhizae

The aim of a joint effort by different research teams was to provide an improved procedure for enzyme activity profiling of field-sampled ectomycorrhizae, including recommendations on the best conditions and maximum duration for storage of ectomycorrhizal samples. A more simplified and efficient protocol compared to formerly published procedures was achieved by using manufactured 96-filter plates in combination with a vacuum manifold and by optimizing incubation times. Major improvements were achieved by performing the series of eight enzyme assays with a single series of root samples instead of two series, reducing the time needed for sample preparation, minimizing error-prone steps such as pipetting and morphotyping, and facilitating subsequent DNA analyses due to the reduced sequencing effort. The best preservation of samples proved to be storage in soil at 4?C6°C in the form of undisturbed soil cores containing roots. Enzyme activities were maintained for up to 4?weeks under these conditions. Short-term storage of washed roots and ectomycorrhizal tips overnight in water did not cause substantial changes in enzyme activity profiles. No optimal means for longer-term storage by freezing at ?20°C or storage in 100% ethanol were recommended.     

Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics

In response to alphabeta1 integrin signaling, transducers such as focal adhesion kinase (FAK) become activated, relaying to specific machineries and triggering distinct cellular responses. By conditionally ablating Fak in skin epidermis and culturing Fak-null keratinocytes, we show that FAK is dispensable for epidermal adhesion and basement membrane assembly, both of which require alphabeta1 integrins. FAK is also dispensible for proliferation/survival in enriched medium. In contrast, FAK functions downstream of alphabeta1 integrin in regulating cytoskeletal dynamics and orchestrating polarized keratinocyte migration out of epidermal explants. Fak-null keratinocytes display an aberrant actin cytoskeleton, which is tightly associated with robust, peripheral focal adhesions and microtubules. We find that without FAK, Src, p190RhoGAP, and PKL-PIX-PAK, localization and/or activation at focal adhesions are impaired, leading to elevated Rho activity, phosphorylation of myosin light chain kinase, and enhanced tensile stress fibers. We show that, together, these FAK-dependent activities are critical to control the turnover of focal adhesions, which is perturbed in the absence of FAK.     

Involving undergraduates in the annotation and analysis of global gene expression studies: creation of a maize shoot apical meristem expression database

Through a multi-university and interdisciplinary project we have involved undergraduate biology and computer science research students in the functional annotation of maize genes and the analysis of their microarray expression patterns. We have created a database to house the results of our functional annotation of >4400 genes identified as being differentially regulated in the maize shoot apical meristem (SAM). This database is located at http://sam.truman.edu and is now available for public use. The undergraduate students involved in constructing this unique SAM database received hands-on training in an intellectually challenging environment, which has prepared them for graduate and professional careers in biological sciences. We describe our experiences with this project as a model for effective research-based teaching of undergraduate biology and computer science students, as well as for a rich professional development experience for faculty at predominantly undergraduate institutions.     

The vascular niche and its basement membrane

Over the past few years, scientists have realized that many cellular and developmental processes, including pancreatic beta-cell growth and differentiation, stem cell and progenitor cell proliferation and cancer cell metastasis, occur in what are known as 'vascular niches'. Despite increasing numbers of reports on these niches, few common mechanisms have been identified to explain their various effects. Here, we define the term 'vascular niche' and suggest that a common and conserved feature of this niche is to provide a basement membrane to cells that are unable to form their own. We further propose that these cells require a vascular niche when they retain a high degree of plasticity.     

The chromosome 11 region from strain 129 provides protection from sex reversal in XYPOS mice

C57BL/6J (B6) mice containing the Mus domesticus poschiavinus Y chromosome, YPOS, develop ovarian tissue, whereas testicular tissue develops in DBA/2J or 129S1/SvImJ (129) mice containing the YPOS chromosome. To identify genes involved in sex determination, we used a congenic strain approach to determine which chromosomal regions from 129Sl/SvImJ provide protection against sex reversal in XYPOS mice of the C57BL/6J.129-YPOS strain. Genome scans using microsatellite and SNP markers identified a chromosome 11 region of 129 origin in C57BL/6J.129-YPOS mice. To determine if this region influenced testis development in XYPOS mice, two strains of C57BL/6J-YPOS mice were produced and used in genetic experiments. XYPOS adults homozygous for the 129 region had a lower incidence of sex reversal than XYPOS adults homozygous for the B6 region. In addition, many homozygous 129 XYPOS fetuses developed normal-appearing testes, an occurrence never observed in XYPOS mice of the C57BL/6J-YPOS strain. Finally, the amount of testicular tissue observed in ovotestes of heterozygous 129/B6 XYPOS fetuses was greater than the amount observed in ovotestes of homozygous B6 XYPOS fetuses. We conclude that a chromosome 11 locus derived from 129Sl/SvImJ essentially protects against sex reversal in XYPOS mice. A number of genes located in this chromosome 11 region are discussed as potential candidates.     

Whole cell biocatalysis in nonconventional media

Summary In this paper biocatalytic reactions carried out by whole cells in nonconventional media are reviewed. Similar relationships are observed between solvent hydrophobicity and catalytic activity in reactions carried out by isolated enzymes and whole cells. In addition to the effect of organic solvent on biocatalyst stability, microbial cells are susceptible to damaging effects caused by the organic phase. In general, more hydrophobic solvents manifest lower toxicity towards the cells. Whole cell biocatalysts require more water than isolated enzymes and two-phase systems have been most widely used to study whole cell biocatalysis. Immobilization makes cell biocatalysts more resistant to organic solvents and helps achieve homogeneous biocatalyst dispersion. Cell entrapment methods have been widely used with organic solvent systems and mixtures of natural and/or synthetic polymers allow adjustment of the hydrophobicity-hydrophilicity balance of the support matrix. Some examples of stereoselective catalysis using microbial cells in organic solvent media are presented.     

Calcium-mediated DNA adsorption to yeast cells and kinetics of cell transformation by electroporation.

The interfacial elastic packing interactions of different galactosylceramides (GalCers), sphingomyelins (SMs), and phosphatidylcholines (PC) were compared by determining their elastic area compressibility moduli (Cs-1) as a function of lateral packing pressure (pi) in a Langmuir-type film balance. To assess the relative contributions of the lipid headgroups as well as those of the ceramide and diacylglycerol hydrocarbon regions, we synthesized various GalCer and SM species with identical, homogeneous acyl residues and compared their behavior to that of PCs possessing similar hydrocarbon structures. For PCs, this meant that the sn-1 acyl chain was long and saturated (e.g., palmitate) and the sn-2 chain composition was varied to match that of GalCer or SM. When at equivalent pi and in either the chain-disordered (liquid-expanded) or chain-ordered (liquid-condensed) state, GalCer films were less elastic than either SM or PC films. When lipid headgroups were identical (SM and PC), Cs-1 values (at equivalent pi) for chain-disordered SMs, but not chain-ordered SMs, were 25-30% higher than those of PCs. Typical values for fluid phase (liquid-expanded) GalCer at 30 mN/m and 24 degrees C were 158 (+/- 7) mN/m, whereas those of SM were 135 (+/- 7) mN/m and those of PC were 123 (+/- 2) mN/m. Pressure-induced transitions to chain-ordered states (liquid-condensed) resulted in significant increases (two- to fourfold) in the "in-plane" compressibility for all three lipid types. Typical Cs-1 values for chain-ordered GalCers at 30 mN/m and 24 degrees C were between 610 and 650 mN/m, whereas those of SM and of PC were very similar and were between 265 and 300 mN/m. Under fluid phase conditions, the pi-Cs-1 behavior for each lipid type was insensitive to whether the acyl chain was saturated or monounsaturated. Measurement of the Cs-1 values also provided an effective way to evaluate the two-dimensional phase transition region of SMs, GalCers, and PCs. Modest heterogeneity in the acyl composition led to transitional broadening. Our findings provide useful information regarding the in-plane elasticity of lipids that are difficult to investigate by alternative methods, i.e., micropipette aspiration technique. The results also provide insight into the stability of sphingolipid-enriched, membrane microdomains that are thought to play a role in the sorting and trafficking of proteins containing glycosylphosphatidylinositol anchors with cells.     

Reductive biotransformation by wild type and mutant strains of Saccharomyces cerevisiae in aqueous-organic solvent biphasic systems

Whole cells of Saccharomyces cerevisiae analyzed the conversion of benzaldehyde to benzyl alcohol in aqueous-organic biphasic media. Reaction rate increased dramatically as moisture content of the solvent was increased in the range 0% to 2%. The highest biotransformation rates were observed when hexane was used as organic solvent. Benzaldehyde was also converted to benzyl alcohol by a cell-free crude extract in biphasic systems containing hexane, although the rate of product formation was much lower. Mutant strains of S. cerevisiae lacking some or all of the ADH isoenzymes, ADH I, II, and III, manifested similar rates for bioconversion of benzaldehyde to benzyl alcohol in both aqueous and two-phase systems. In general, conversion rates observed in aqueous media were 2 to 3 times higher than those observed in hexane containing 2% moisture.