Effect of germination temperature on characteristics of phytase production from barley

The effects of germination temperature on the growth of barley seedlings for phytase production were studied at 15, 20 and 25 degrees C for 6-10 days. The growth rate of the barley seedlings was increased as the germination temperature was increased. The initial rate of total protein production was closely coupled to that of the barley growth, and the rate of total protein production tended to increase as the germination temperature was increased. SDS-PAGE analysis of total protein from the barley seedlings showed time-dependent appearance and disappearance of protein bands. Although no significant phytase activity was detected at zero time of germination, a significant increase in phytase activity up to 7.9-fold occurred during the first several days of germination then decreased. Phosphate production (viz. phytate degradation) in the barley seedlings occurred rapidly at the beginning of germination. However, the rate of production continued to decrease with further germination. A time lag of about 1-2 days between the rate of total protein production and that of phytase production was observed. Unlike the extent of total protein production, that of phytase production was similar irrespective of germination temperature. Partial purification of a crude enzyme extract by hydrophobic interaction chromatography resulted in two phytase fractions (PI and PII). Zymogram analysis demonstrated that PI had two bands with molecular masses of about 66 and 123 kDa while PII had one band corresponding to a molecular mass of about 96 kDa. The optimal temperature for PI was found to be 55 degrees C, while it was 50 degrees C for PII. The enzyme fraction PI had a pH optimum at 6.0, whereas the optimum pH for PII was found to be 5.0. Addition of 0.1% (v/v) Tween 80 was found to increase enzyme activity significantly (i.e., 167% for PI and 137% for PII). Phytate in cereals including barley, rice, corn and soybean degraded effectively by the treatment of the barley phytases.     

Characterization of heparan sulfate from the unossified antler of Cervus elaphus

The antler is the most rapidly growing tissue in the animal kingdom. According to previous reports, antler glycosaminoglycans (GAGs) consist of all kinds GAGs except for heparan sulfate (HS). Chondroitin sulfate is the major antler GAG component comprising 88% of the total uronic acid content. In the current study, we have isolated HS from antler for the first time and characterized it based on both NMR spectroscopy and disaccharide composition analysis. Antler GAGs were isolated by protease treatment and followed by cetylpyridinium chloride precipitation. The sensitivity of antler GAGs to heparin lyase III showed that this sample contained heparan sulfate. After incubation of antler GAGs with chondroitin lyase ABC, the HS-containing fraction was recovered by ethanol precipitation. The composition of HS disaccharides in this fraction was determined by its complete depolymerization with a mixture of heparin lyase I, II, and III and analysis of the resulting disaccharides by the reversed-phase (RP) ion pairing-HPLC, monitored by the fluorescence detection using 2-cyanoacetamide as a post-column labeling reagent. Eight unsaturated disaccharides (DeltaUA-GlcNAc, DeltaUA-GlcNS, DeltaUA-GlcNAc6S, DeltaUA2S-GlcNAc, DeltaUA-GlcNS6S, DeltaUA2S-GlcNS, DeltaUA2S-GlcNAc6S, DeltaUA2S-GlcNS6S) were produced from antler HS by digestion with the mixture of heparin lyases. The total content of 2-O-sulfo disaccharide units in antler HS was higher than that of heparan sulfate from most other animal sources.     

Fabrication of degradable carboxymethyl cellulose (CMC) microneedle with laser writing and replica molding process for enhancement of transdermal drug delivery

Transdermal drug delivery system (TDDS) may provide a more reliable method of drug delivery than oral delivery by avoiding gut absorption and first-pass metabolism, but needs a method for efficiently crossing the epidermal barrier. To enhance the delivery through the skin, we have developed a biocompatible, dissolvable microneedle array made from carboxymethyl cellulose (CMC). Using laser ablation for creating the mold greatly improved the efficiency and reduced the cost of microneedle fabrication. Mixing CMC with amylopectin (AP) enhanced the mechanical and tunable dissolution properties of the microneedle for controlled release of model compounds. Using the CMC microneedle array, we observed significant enhancement in the skin permeability of a fluorescent model compound, and also increase in the anti-oxidant activity of ascorbic acid after crossing the skin. Our dissolvable microneedle array provides a new and biocompatible method for delivery of drugs and cosmetic compounds through the skin.     

Activated PAK4 regulates cell adhesion and anchorage-independent growth

The serine/threonine kinase PAK4 is an effector molecule for the Rho GTPase Cdc42. PAK4 differs from other members of the PAK family in both sequence and function. Previously we have shown that an important function of this kinase is to mediate the induction of filopodia in response to activated Cdc42. Since previous characterization of PAK4 was carried out only with the wild-type kinase, we have generated a constitutively active mutant of the kinase to determine whether it has other functions. Expression of activated PAK4 in fibroblasts led to a transient induction of filopodia, which is consistent with its role as an effector for Cdc42. In addition, use of the activated mutant revealed a number of other important functions of this kinase that were not revealed by studying the wild-type kinase. For example, activated PAK4 led to the dissolution of stress fibers and loss of focal adhesions. Consequently, cells expressing activated PAK4 had a defect in cell spreading onto fibronectin-coated surfaces. Most importantly, fibroblasts expressing activated PAK4 had a morphology that was characteristic of oncogenic transformation. These cells were anchorage independent and formed colonies in soft agar, similar to what has been observed previously in cells expressing activated Cdc42. Consistent with this, dominant-negative PAK4 mutants inhibited focus formation by oncogenic Dbl, an exchange factor for Rho family GTPases. These results provide the first demonstration that a PAK family member can transform cells and indicate that PAK4 may play an essential role in oncogenic transformation by the GTPases. We propose that the morphological changes and changes in cell adhesion induced by PAK4 may play a direct role in oncogenic transformation by Rho family GTPases and their exchange factors.     

Design and application of highly responsive fluorescence resonance energy transfer biosensors for detection of sugar in living Saccharomyces cerevisiae cells

A protein sensor with a highly responsive fluorescence resonance energy transfer (FRET) signal for sensing sugars in living Saccharomyces cerevisiae cells was developed by combinatorial engineering of the domain linker and the binding protein moiety. Although FRET sensors based on microbial binding proteins have previously been created for visualizing various sugars in vivo, such sensors are limited due to a weak signal intensity and a narrow dynamic range. In the present study, the length and composition of the linker moiety of a FRET-based sensor consisting of CFP-linker(1)-maltose-binding protein-linker(2)-YFP were redesigned, which resulted in a 10-fold-higher signal intensity. Molecular modeling of the composite linker moieties, including the connecting peptide and terminal regions of the flanking proteins, suggested that an ordered helical structure was preferable for tighter coupling of the conformational change of the binding proteins to the FRET response. When the binding site residue Trp62 of the maltose-binding protein was diversified by saturation mutagenesis, the Leu mutant exhibited an increased binding constant (82 microM) accompanied by further improvement in the signal intensity. Finally, the maltose sensor with optimized linkers was redesigned to create a sugar sensor with a new specificity and a wide dynamic range. When the optimized maltose sensors were employed as in vivo sensors, highly responsive FRET images were generated from real-time analysis of maltose uptake of Saccharomyces cerevisiae (baker's yeast).     

Tetraspanin CD151 Stimulates Adhesion-dependent Activation of Ras, Rac, and Cdc42 by Facilitating Molecular Association between β1 Integrins and Small GTPases

Tetraspanin CD151 associates with laminin-binding α(3)β(1)/α(6)β(1) integrins in epithelial cells and regulates adhesion-dependent signaling events. We found here that CD151 plays a role in recruiting Ras, Rac1, and Cdc42, but not Rho, to the cell membrane region, leading to the formation of α(3)β(1)/α(6)β(1) integrin-CD151-GTPases complexes. Furthermore, cell adhesion to laminin enhanced CD151 association with β(1) integrin and, thereby, increased complex formation between the β(1) family of integrins and small GTPases, Ras, Rac1, and Cdc42. Adhesion receptor complex-associated small GTPases were activated by CD151-β(1) integrin complex-stimulating adhesion events, such as α(3)β(1)/α(6)β(1) integrin-activating cell-to-laminin adhesion and homophilic CD151 interaction-generating cell-to-cell adhesion. Additionally, FAK and Src appeared to participate in this adhesion-dependent activation of small GTPases. However, engagement of laminin-binding integrins in CD151-deficient cells or CD151-specific siRNA-transfected cells did not activate these GTPases to the level of cells expressing CD151. Small GTPases activated by engagement of CD151-β(1) integrin complexes contributed to CD151-induced cell motility and MMP-9 expression in human melanoma cells. Importantly, among the four tetraspanin proteins that associate with β(1) integrin, only CD151 exhibited the ability to facilitate complex formation between the β(1) family of integrins and small GTPases and stimulate β(1) integrin-dependent activation of small GTPases. These results suggest that CD151 links α(3)β(1)/α(6)β(1) integrins to Ras, Rac1, and Cdc42 by promoting the formation of multimolecular complexes in the membrane, which leads to the up-regulation of adhesion-dependent small GTPase activation.     

Identification and properties of type I-signal peptidases of Bacillus amyloliquefaciens.

The use of Bacillus amyloliquefaciens for enzyme production and its exceptional high protein export capacity initiated this study where the presence and function of multiple type I signal peptidase isoforms was investigated. In addition to type I signal peptidases SipS(ba) [Meijer, W.J.J., de Jong, A., Bea, G., Wisman, A., Tjalsma, H., Venema, G., Bron, S. & van Dijl, J.M. (1995) Mol. Microbiol. 17, 621-631] and SipT(ba) [Hoang, V. & Hofemeister, J. (1995) Biochim. Biophys. Acta 1269, 64-68] which were previously identified, here we present evidence for two other Sip-like genes in B. amyloliquefaciens. Same map positions as well as sequence motifs verified that these genes encode homologues of Bacillus subtilis SipV and SipW. SipU-encoding DNA was not found in B. amyloliquefaciens. SipW-encoding DNA was also found for other Bacillus strains representing different phylogenetic groups, but not for Bacillus stearothermophilus and Thermoactinomyces vulgaris. The absence of these genes, however, could have been overlooked due to sequence diversity. Sequence alignments of 23 known Sip-like proteins from Bacillus origin indicated further branching of the P-group signal peptidases into clusters represented by B. subtilis SipV, SipS-SipT-SipU and B. anthracis Sip3-Sip5 proteins, respectively. Each B. amyloliquefaciens sip(ba) gene was expressed in an Escherichia coli LepBts mutant and tested for genetic complementation of the temperature sensitive (TS) phenotype as well as pre-OmpA processing. Although SipS(ba) as well as SipT(ba) efficiently restored processing of pre-OmpA in E. coli, only SipS(ba) supported growth at TS conditions, indicating functional diversity. Changed properties of the sip(ba) gene disruption mutants, including cell autolysis, motility, sporulation, and nuclease activities, seemed to correlate with specificities and/or localization of B. amyloliquefaciens SipS, SipT and SipV isoforms.     

Molecular weight standards for calibration of gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis: ferritin and apoferritin

Ferritin and apoferritin are widely used for the calibration of gel filtration columns and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and are commercially offered for these purposes as part of molecular weight calibration kits. Many of the reported applications are severely in error as presented in leading references and application manuals. The manufacturers have based their recommendations on incorrect physicochemical parameters in the literature and incorrect or inadmissible assumptions about the protein subunit composition and architecture and have not taken into account the unusual resistance of these proteins to denaturation in SDS. Here the relevant physicochemical parameters of horse spleen apoferritin as reported in the literature are critically reevaluated and the best current estimates are identified as the following: weight average molecular weight of apoferritin, Mw = 481,200; molecular weight of subunits, major subunit, ML = 19,889; minor subunit, MH = 22,200; apparent specific volumes in 0.02 M acetate buffer, pH 5.5, and 0.1 M NaCl, phi = 0.721 ml g-1 and phi' = 0.743 ml g-1; partial specific volume at 20 degrees C, v = 0.738 ml g-1; viscosimetric molar volume, M[n] = 1.78 X 10(6) ml mol-1; Stokes radius, RSt = 67.1 A; viscosimetric radius, Rvis = 65.6 A; sedimentation coefficient S degrees 20, w = 16.6 S; translational diffusion coefficient, D20, w = 3.24 X 10(-7) cm2 s-1. Recommendations are provided for proper application of ferritin and apoferritin for calibration purposes in gel filtration and SDS-polyacrylamide gel electrophoresis.     

Development of a novel spatiotemporal depletion system for cellular cholesterol

Cholesterol is an essential component of mammalian cell membranes whose subcellular concentration and function are tightly regulated by de novo biosynthesis, transport, and storage. Although recent reports have suggested diverse functions of cellular cholesterol in different subcellular membranes, systematic investigation of its site-specific roles has been hampered by the lack of a methodology for spatiotemporal manipulation of cellular cholesterol levels. Here, we report the development of a new cholesterol depletion system that allows for spatiotemporal manipulation of intracellular cholesterol levels. This system utilizes a genetically encoded cholesterol oxidase whose intrinsic membrane binding activity is engineered in such a way that its membrane targeting can be controlled in a spatiotemporally specific manner via chemically induced dimerization. In combination with in situ quantitative imaging of cholesterol and signaling activity measurements, this system allows for unambiguous determination of site-specific functions of cholesterol in different membranes, including the plasma membrane and the lysosomal membrane.