The transformation of a normal cell into a cancer cell has been correlated with alterations in gene regulation and protein expression. To identify altered proteins and link them to the tumorigenesis of breast cancer, we have distinguished normal breast cells (MCF-10A) from noninvasive breast cancer cells (MCF-7) and invasive breast cancer cells (MB-MDA-231) to identify potential breast cancer markers in transformed breast cells. Using the 2D-DIGE and MALDI-TOF MS techniques, we quantified and identified differentially expressed extracellular secreted proteins and total cellular proteins across MCF-7, MB-MDA-231 and MCF-10A. The proteomic analysis of the secreted proteins identified 50 unique differentially expressed proteins from three different media. In addition, 133 unique differentially expressed proteins from total cellular proteins were also identified. Note that 14 of the secreted proteins and 51 of the total cellular proteins have not been previously reported in breast cancer research. Some of these unreported proteins have been examined in other breast cancer cell lines and have shown positive correlations with 2D-DIGE data. In summary, this study identifies numerous putative breast cancer markers from various stages of breast cancer. The results of this study may aid in developing proteins identified as useful diagnostic and therapeutic candidates in research on cancer and proteomics.

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The structure of a novel plant defensin isolated from the seeds of the mung bean, Vigna radiate, has been determined by (1)H nuclear magnetic resonance spectroscopy. The three-dimensional structure of VrD2, the V. radiate plant defensin 2 protein, comprises an alpha-helix and one triple-stranded anti-parallel beta-sheet stabilized by four disulfide bonds. This protein exhibits neither insecticidal activity nor alpha-amylase inhibitory activity in spite of showing a similar global fold to that of VrD1, an insecticidal plant defensin that has been suggested to function by inhibiting insect alpha-amylase. Our previous study proposed that loop L3 of plant defensins is important for this inhibition. Structural analyses and surface charge comparisons of VrD1 and VrD2 revealed that the charged residues of L3 correlate with the observed difference in inhibitory activities of these proteins. A VrD2 chimera that was produced by transferring the proposed functional loop of VrD1 onto the structurally equivalent loop of VrD2 supported this hypothesis. The VrD2 chimera, which differs by only five residues compared with VrD2, showed obvious activity against Tenebrio molitor alpha-amylase. These results clarify the mode of alpha-amylase inhibition of plant defensins and also represent a possible approach for engineering novel alpha-amylase inhibitors. Plant defensins are important constituents of the innate immune system of plants, and thus the application of protein engineering to this protein family may provide an efficient method for protecting against crop losses.

Bacillus megaterium can produce poly-␤-hydroxybutyrate (PHB) as carbon and energy storage materials. We now report that the phaQ gene, which is located upstream of the phasin-encoding phaP gene, codes for a new class of transcriptional regulator that negatively controls expression of both phaQ and phaP. A PhaQ binding site that plays a role in this control has been identified by gel mobility shift assays and DNase I footprinting analysis. We have also provided evidence that PhaQ could sense the presence of PHB in vivo and that artificial PHB granules could inhibit the formation of PhaQ-DNA complex in vitro by binding to PhaQ directly. These suggest that PhaQ is a PHB-responsive repressor.Poly-␤-hydroxybutyrate (PHB) and other polyhydroxyalkanoates (PHAs) are biodegradable polyesters that are produced by a wide variety of bacteria as intracellular carbon and energy storage materials (1, 13, 31). PHB synthases (PhaC) and phasins (PhaP) are proteins that play important roles in PHB production and granule formation. Phasin, an abundant granule-associated protein, forms a boundary layer on the PHB surface to sequester hydrophobic PHB from the cytoplasm. Thus, phasin can inhibit individual granules from coalescing and promotes PHB synthesis by regulating the ratio of surface area to volume of PHB granules (27)(28)(29). PhaP may also have a protective function to reduce the passive attachment of cytoplasmic proteins to PHB surface (16,27). It is generally thought that the synthesis of phasin proteins is highly regulated (8). In Ralstonia eutropha, expression of the phaP gene is negatively controlled by the autoregulated repressor PhaR (21,30). The accumulation of PhaP in the R. eutropha cells is strictly dependent on PHB production (28). After the onset of PHB biosynthesis, PhaR can sense the presence of PHB and bind to nascent PHB granules, leading to derepression of phaP. In Paracoccus denitrificans the phaR gene, which is located immediately downstream of phaP, encodes a repressor that regulates phaP expression. PhaR can sense the presence of PHA and interact with PHA granules (14,15).Although Bacillus megaterium is already known to be able to synthesize PHB and accumulate PHB granules, genes that are involved in PHB synthesis and encode PHB granule-associated proteins were not cloned until recently (17). Among a cluster of five pha genes of B. megaterium, phaP and phaQ are transcribed in one direction, whereas phaR, phaB, and phaC are divergently transcribed as a tricistronic operon. phaP codes for a phasin protein. phaB and phaC encode NADPH-dependent acetoacetyl coenzyme A reductase and a novel PHB synthase, respectively (17). This novel PHB synthase requires both PhaC and PhaR for activity (18). It should be noted that the designation of the B. megaterium phaR gene does not follow the conventional rule. Rather than encoding a transcriptional regulator like the PhaR protein of R. eutropha or P. denitrificans, the B. megaterium phaR gene probably encodes a subunit of a heterodimeric PHB synthase (18,25). Neverthel...