Unnecessary Explantation of IDEAL IMPLANT Structured Saline Breast Implants Prompted byRadiologic Misdiagnosis of Rupture.

ABSTRACT: The unique dual-lumen and baffle design of the IDEAL IMPLANT Structured Saline breast implant gives it specific advantages over both silicone gel-filled and the original saline-filled implants. This internal baffle structure also gives it an appearance on various radiologic imaging studies that may be misinterpreted as a rupture because of similarities to the well-known radiologic appearance of a ruptured silicone gel implant. Patients may present with various misinterpreted imaging studies, highlighting the need for plastic surgeons and radiologists to be familiar with the normal appearance of the intact IDEAL IMPLANT and be able to distinguish it from a ruptured IDEAL IMPLANT. The radiology findings must be correlated with the clinical findings, or an intact IDEAL IMPLANT misdiagnosed as ruptured, may cause unnecessary patient worry, and may prompt unnecessary surgery for removal or replacement.

Tissue-specific gene expression in maize seeds during colonization by Aspergillus flavus and Fusarium verticillioides.

Aspergillus flavus and Fusarium verticillioides are fungal pathogens that colonize maize kernels and produce the harmful mycotoxins aflatoxin and fumonisin, respectively. Management practice based on potential host resistance to reduce contamination by these mycotoxins has proven difficult, resulting in the need for a better understanding of the infection process by these fungi and the response of maize seeds to infection. In this study, we followed the colonization of seeds by histological methods and the transcriptional changes of two maize defence-related genes in specific seed tissues by RNA in situ hybridization. Maize kernels were inoculated with either A. flavus or F. verticillioides 21-22 days after pollination, and harvested at 4, 12, 24, 48, 72, 96 and 120 h post-inoculation. The fungi colonized all tissues of maize seed, but differed in their interactions with aleurone and germ tissues. RNA in situ hybridization showed the induction of the maize pathogenesis-related protein, maize seed (PRms) gene in the aleurone and scutellum on infection by either fungus. Transcripts of the maize sucrose synthase-encoding gene, shrunken-1 (Sh1), were observed in the embryo of non-infected kernels, but were induced on infection by each fungus in the aleurone and scutellum. By comparing histological and RNA in situ hybridization results from adjacent serial sections, we found that the transcripts of these two genes accumulated in tissue prior to the arrival of the advancing pathogens in the seeds. A knowledge of the patterns of colonization and tissue-specific gene expression in response to these fungi will be helpful in the development of resistance.

Aspergillus flavus infection induces transcriptional and physical changes in developing maize kernels.

Maize kernels are susceptible to infection by the opportunistic pathogen Aspergillus flavus. Infection results in reduction of grain quality and contamination of kernels with the highly carcinogenic mycotoxin, aflatoxin. To understanding host response to infection by the fungus, transcription of approximately 9000 maize genes were monitored during the host-pathogen interaction with a custom designed Affymetrix GeneChip® DNA array. More than 4000 maize genes were found differentially expressed at a FDR of 0.05. This included the up regulation of defense related genes and signaling pathways. Transcriptional changes also were observed in primary metabolism genes. Starch biosynthetic genes were down regulated during infection, while genes encoding maize hydrolytic enzymes, presumably involved in the degradation of host reserves, were up regulated. These data indicate that infection of the maize kernel by A. flavus induced metabolic changes in the kernel, including the production of a defense response, as well as a disruption in kernel development.

Localization, morphology and transcriptional profile of Aspergillus flavus during seed colonization.

Aspergillus flavus is an opportunistic fungal pathogen that infects maize kernels pre-harvest, creating major human health concerns and causing substantial agricultural losses. Improved control strategies are needed, yet progress is hampered by the limited understanding of the mechanisms of infection. A series of studies were designed to investigate the localization, morphology and transcriptional profile of A. flavus during internal seed colonization. Results from these studies indicate that A. flavus is capable of infecting all tissues of the immature kernel by 96 h after infection. Mycelia were observed in and around the point of inoculation in the endosperm and were found growing down to the germ. At the endosperm-germ interface, hyphae appeared to differentiate and form a biofilm-like structure that surrounded the germ. The exact nature of this structure remains unclear, but is discussed. A custom-designed A. flavus Affymetrix GeneChip® microarray was used to monitor genome-wide transcription during pathogenicity. A total of 5061 genes were designated as being differentially expressed. Genes encoding secreted enzymes, transcription factors and secondary metabolite gene clusters were up-regulated and considered to be potential effector molecules responsible for disease in the kernel. Information gained from this study will aid in the development of strategies aimed at preventing or slowing down A. flavus colonization of the maize kernel.

X-ray structure of ILL2, an auxin-conjugate amidohydrolase from Arabidopsis thaliana.

The plant hormone indole-3-acetic acid (IAA) is the most abundant natural auxin involved in many aspects of plant development and growth. The IAA levels in plants are modulated by a specific group of amidohydrolases from the peptidase M20D family that release the active hormone from its conjugated storage forms. Here, we describe the X-ray crystal structure of IAA-amino acid hydrolase IAA-leucine resistantlike gene 2 (ILL2) from Arabidopsis thaliana at 2.0 A resolution. ILL2 preferentially hydrolyses the auxin-amino acid conjugate N-(indol-3-acetyl)-alanine. The overall structure of ILL2 is reminiscent of dinuclear metallopeptidases from the M20 peptidase family. The structure consists of two domains, a larger catalytic domain with three-layer alpha beta alpha sandwich architecture and aminopeptidase topology and a smaller satellite domain with two-layer alphabeta-sandwich architecture and alpha-beta-plaits topology. The metal-coordinating residues in the active site of ILL2 include a conserved cysteine that clearly distinguishes this protein from previously structurally characterized members of the M20 peptidase family. Modeling of N-(indol-3-acetyl)-alanine into the active site of ILL2 suggests that Leu175 serves as a key determinant for the amino acid side-chain specificity of this enzyme. Furthermore, a hydrophobic pocket nearby the catalytic dimetal center likely recognizes the indolyl moiety of the substrate. Finally, the active site of ILL2 harbors an absolutely conserved glutamate (Glu172), which is well positioned to act as a general acid-base residue. Overall, the structure of ILL2 suggests that this enzyme likely uses a catalytic mechanism that follows the paradigm established for the other enzymes of the M20 peptidase family.

Endoplasmic reticulum quality control and the unfolded protein response: insights from plants.

Protein quality control (QC) within the endoplasmic reticulum and the related unfolded protein response (UPR) pathway of signal transduction are major regulators of the secretory pathway, which is involved in virtually any aspect of development and reproduction. The study of plant-specific processes such as pathogen response, seed development and the synthesis of seed storage proteins and of particular toxins is providing novel insights, with potential implications for the general recognition events and mechanisms of action of QC and UPR.

A new branch of endoplasmic reticulum stress signaling and the osmotic signal converge on plant-specific asparagine-rich proteins to promote cell death.

NRPs (N-rich proteins) were identified as targets of a novel adaptive pathway that integrates endoplasmic reticulum (ER) and osmotic stress signals based on coordinate regulation and synergistic up-regulation by tunicamycin and polyethylene glycol treatments. This integrated pathway diverges from the molecular chaperone-inducing branch of the unfolded protein response (UPR) in several ways. While UPR-specific targets were inversely regulated by ER and osmotic stresses, NRPs required both signals for full activation. Furthermore, BiP (binding protein) overexpression in soybean prevented activation of the UPR by ER stress inducers, but did not affect activation of NRPs. We also found that this integrated pathway transduces a PCD signal generated by ER and osmotic stresses that result in the appearance of markers associated with leaf senescence. Overexpression of NRPs in soybean protoplasts induced caspase-3-like activity and promoted extensive DNA fragmentation. Furthermore, transient expression of NRPs in planta caused leaf yellowing, chlorophyll loss, malondialdehyde production, ethylene evolution, and induction of the senescence marker gene CP1. This phenotype was alleviated by the cytokinin zeatin, a potent senescence inhibitor. Collectively, these results indicate that ER stress induces leaf senescence through activation of plant-specific NRPs via a novel branch of the ER stress response.

Diverse inhibitors of aflatoxin biosynthesis.

Pre-harvest and post-harvest contamination of maize, peanuts, cotton, and tree nuts by members of the genus Aspergillus and subsequent contamination with the mycotoxin aflatoxin pose a widespread food safety problem for which effective and inexpensive control strategies are lacking. Since the discovery of aflatoxin as a potently carcinogenic food contaminant, extensive research has been focused on identifying compounds that inhibit its biosynthesis. Numerous diverse compounds and extracts containing activity inhibitory to aflatoxin biosynthesis have been reported. Only recently, however, have tools been available to investigate the molecular mechanisms by which these inhibitors affect aflatoxin biosynthesis. Many inhibitors are plant-derived and a few may be amenable to pathway engineering for tissue-specific expression in susceptible host plants as a defense against aflatoxin contamination. Other compounds show promise as protectants during crop storage. Finally, inhibitors with different modes of action could be used in comparative transcriptional and metabolomic profiling experiments to identify regulatory networks controlling aflatoxin biosynthesis.

Expression profiling on soybean leaves reveals integration of ER- and osmotic-stress pathways.

BACKGROUND: Despite the potential of the endoplasmic reticulum (ER) stress response to accommodate adaptive pathways, its integration with other environmental-induced responses is poorly understood in plants. We have previously demonstrated that the ER-stress sensor binding protein (BiP) from soybean exhibits an unusual response to drought. The members of the soybean BiP gene family are differentially regulated by osmotic stress and soybean BiP confers tolerance to drought. While these results may reflect crosstalk between the osmotic and ER-stress signaling pathways, the lack of mutants, transcriptional response profiles to stresses and genome sequence information of this relevant crop has limited our attempts to identify integrated networks between osmotic and ER stress-induced adaptive responses. As a fundamental step towards this goal, we performed global expression profiling on soybean leaves exposed to polyethylene glycol treatment (osmotic stress) or to ER stress inducers. RESULTS: The up-regulated stress-specific changes unmasked the major branches of the ER-stress response, which include enhancing protein folding and degradation in the ER, as well as specific osmotically regulated changes linked to cellular responses induced by dehydration. However, a small proportion (5.5%) of total up-regulated genes represented a shared response that seemed to integrate the two signaling pathways. These co-regulated genes were considered downstream targets based on similar induction kinetics and a synergistic response to the combination of osmotic- and ER-stress-inducing treatments. Genes in this integrated pathway with the strongest synergistic induction encoded proteins with diverse roles, such as plant-specific development and cell death (DCD) domain-containing proteins, an ubiquitin-associated (UBA) protein homolog and NAC domain-containing proteins. This integrated pathway diverged further from characterized specific branches of ER-stress as downstream targets were inversely regulated by osmotic stress. CONCLUSION: The present ER-stress- and osmotic-stress-induced transcriptional studies demonstrate a clear predominance of stimulus-specific positive changes over shared responses on soybean leaves. This scenario indicates that polyethylene glycol (PEG)-induced cellular dehydration and ER stress elicited very different up-regulated responses within a 10-h stress treatment regime. In addition to identifying ER-stress and osmotic-stress-specific responses in soybean (Glycine max), our global expression-profiling analyses provided a list of candidate regulatory components, which may integrate the osmotic-stress and ER-stress signaling pathways in plants.

The maize Mucronate mutation is a deletion in the 16-kDa gamma-zein gene that induces the unfolded protein response.

Mucronate (Mc) was identified as a dominant maize (Zea mays L.) opaque kernel mutation that alters zein storage protein synthesis. Zein protein bodies in Mc endosperm are misshapen and are associated with increased levels of ER Lumenal Binding Protein (BiP). Using GeneCalling to profile endosperm RNA transcripts, we identified an aberrant RNA in Mc that encodes the 16-kDa gamma-zein protein. The transcript contains a 38-bp deletion (nucleotides 406-444 after the initiation codon) that creates a frame-shift mutation and an abnormal sequence for the last 63 amino acids. Genetic mapping revealed the Mc mutation is linked with the locus encoding the 16-kDa gamma-zein, and two-dimensional gel electrophoresis confirmed the 16-kDa gamma-zein protein is altered in Mc. The mutant protein exhibited changes in solubility properties and co-immunoprecipitated with the molecular chaperone, BiP. Transgenic maize plants expressing the Mc 16-kDa gamma-zein manifested an opaque kernel phenotype with enhanced levels of BiP in the endosperm, similar to the Mc mutant. Unlike the wild-type protein, the Mc 16-kDa gamma-zein interacted only weakly with the 22-kDa alpha-zein when expressed in the yeast two-hybrid system. These results indicate that the Mc phenotype results from a frame-shift mutation in the gene encoding the 16-kDa gamma-zein protein, leading to the unfolded protein response in developing endosperm.

Relative activity of a tobacco hybrid expressing high levels of a tobacco anionic peroxidase and maize ribosome-inactivating protein against Helicoverpa zea and Lasioderma serricorne.

Tobacco (Nicotiana tabacum) plants grown from seed obtained by crossing a tobacco line that expressed an activated maize ribosome-inactivating protein (RIP) with a line that overexpressed tobacco anionic peroxidase were tested for their effects on corn earworm Helicoverpa zea and cigarette beetle Lasioderma serricorne larvae as compared to the wild-type plant cross. Significant feeding reductions were noted for transgenic plants expressing both resistance proteins as compared to wild-type plants for both H. zea and L. serricorne. Significant increases in mortality were also noted for those insects fed on the transgenic cross as compared to wild-type plants in some cases. Levels of both peroxidase and maize RIP were significantly higher in transgenic as compared to wild-type plants (which did not produce maize RIP). The degree of feeding was significantly negatively correlated with the level of RIP or peroxidase individually.

Identification and characterization of endoplasmic reticulum-associated degradation proteins differentially affected by endoplasmic reticulum stress.

The disposal of misfolded proteins from the lumen of the endoplasmic reticulum (ER) is one of the quality control mechanisms present in the protein secretory pathway. Through ER-associated degradation, misfolded substrates are targeted to the cytosol where they are degraded by the proteasome. We have identified four maize (Zea mays) Der1-like genes (Zm Derlins) that encode homologs of Der1p, a yeast (Saccharomyces cerevisiae) protein implicated in ER-associated degradation. Zm Derlins are capable of functionally complementing a yeast Der1 deletion mutant. Such complementation indicates that the Der1p function is conserved among species. Zm Derlin genes are expressed at low levels throughout the plant, but appear prevalent in tissues with high activity of secretory protein accumulation, including developing endosperm cells. Expression of three of the four Zm Derlin genes increases during ER stress, with Zm Derlin1-1 showing the strongest induction. Subcellular fractionation experiments localized Zm Derlin proteins to the membrane fraction of microsomes. In maize endosperm, Zm Derlin proteins were found primarily associated with ER-derived protein bodies regardless of the presence of an ER stress response.

Phylogenetic analyses identify 10 classes of the protein disulfide isomerase family in plants, including single-domain protein disulfide isomerase-related proteins.

Protein disulfide isomerases (PDIs) are molecular chaperones that contain thioredoxin (TRX) domains and aid in the formation of proper disulfide bonds during protein folding. To identify plant PDI-like (PDIL) proteins, a genome-wide search of Arabidopsis (Arabidopsis thaliana) was carried out to produce a comprehensive list of 104 genes encoding proteins with TRX domains. Phylogenetic analysis was conducted for these sequences using Bayesian and maximum-likelihood methods. The resulting phylogenetic tree showed that evolutionary relationships of TRX domains alone were correlated with conserved enzymatic activities. From this tree, we identified a set of 22 PDIL proteins that constitute a well-supported clade containing orthologs of known PDIs. Using the Arabidopsis PDIL sequences in iterative BLAST searches of public and proprietary sequence databases, we further identified orthologous sets of 19 PDIL sequences in rice (Oryza sativa) and 22 PDIL sequences in maize (Zea mays), and resolved the PDIL phylogeny into 10 groups. Five groups (I-V) had two TRX domains and showed structural similarities to the PDIL proteins in other higher eukaryotes. The remaining five groups had a single TRX domain. Two of these (quiescin-sulfhydryl oxidase-like and adenosine 5'-phosphosulfate reductase-like) had putative nonisomerase enzymatic activities encoded by an additional domain. Two others (VI and VIII) resembled small single-domain PDIs from Giardia lamblia, a basal eukaryote, and from yeast. Mining of maize expressed sequence tag and RNA-profiling databases indicated that members of all of the single-domain PDIL groups were expressed throughout the plant. The group VI maize PDIL ZmPDIL5-1 accumulated during endoplasmic reticulum stress but was not found within the intracellular membrane fractions and may represent a new member of the molecular chaperone complement in the cell.

Maize ribosome-inactivating proteins (RIPs) with distinct expression patterns have similar requirements for proenzyme activation.

Ribosome-inactivating proteins (RIPs, EC 3.2.2.22) are potent naturally occurring toxins found in numerous and diverse plant species. The maize RIP is unusual among the plant RIPs because it is synthesized as an inactive precursor (also known as maize proRIP1 or b-32). The proenzyme undergoes proteolytic activation that results in the removal of the NH(2)-terminal, the COOH-terminal, and internal sequences to form a two-chain holoenzyme capable of irreversibly modifying the large rRNA. The characterization of a second maize RIP (RIP2), encoded by the gene designated Rip3:2 is described here. Low levels of Rip3:2 RNA were detected in roots, shoots, tassels, silks, and leaves, but the Rip3:2 gene, unlike the Rip3:1 gene, is not under the control of the transcriptional activator Opaque-2. Instead, its expression was up-regulated by drought. Rip3:2 encodes a 31.1 kDa polypeptide that is very similar to proRIP1 in regions corresponding to those found in the active protein and the NH(2)-terminal extension. A 19-amino-acid internal portion of proRIP2 has little similarity to the proRIP1 sequence except that both are very rich in acidic residues. RIP activity assays revealed that Rip3:2 encodes a polypeptide that acquires RNA-specific N-glycosidase activity after proteolytic cleavage. Accumulation as inactive proenzymes may therefore be a general feature of maize RIPs. Differential regulation of the two RIP genes suggests that the corresponding proteins may be involved in defence-related functions with one being regulated developmentally and the other being responsive to an environmental stimulus.

A defective signal peptide in a 19-kD alpha-zein protein causes the unfolded protein response and an opaque endosperm phenotype in the maize De*-B30 mutant.

Defective endosperm* (De*)-B30 is a dominant maize (Zea mays) mutation that depresses zein synthesis in the developing endosperm. The mutant kernels have an opaque, starchy phenotype, malformed zein protein bodies, and highly increased levels of binding protein and other chaperone proteins in the endosperm. Immunoblotting revealed a novel alpha-zein protein in De*-B30 that migrates between the 22- and 19-kD alpha-zein bands. Because the De*-B30 mutation maps in a cluster of 19-kD alpha-zein genes, we characterized cDNA clones encoding these proteins from a developing endosperm library. This led to the identification of a 19-kD alpha-zein cDNA in which proline replaces serine at the 15th position of the signal peptide. Although the corresponding gene does not appear to be highly expressed in De*-B30, it was found to be tightly linked with the mutant phenotype in a segregating F2 population. Furthermore, when the protein was synthesized in yeast cells, the signal peptide appeared to be less efficiently processed than when serine replaced proline. To test whether this gene is responsible for the De*-B30 mutation, transgenic maize plants expressing this sequence were created. T1 seeds originating from the transformants manifested an opaque kernel phenotype with enhanced levels of binding protein in the endosperm, similar to De*-B30. These results are consistent with the hypothesis that the De*-B30 mutation causes a defective signal peptide in a 19-kD alpha-zein protein.

A Chitinase from Tex6 Maize Kernels Inhibits Growth of Aspergillus flavus.

ABSTRACT The maize inbred Tex6 has resistance to colonization and aflatoxin accumulation by Aspergillus flavus. A protein inhibitory to growth of A. flavus has been identified from aqueous extracts of mature Tex6 seeds. This study reports the purification of a chitinase associated with this inhibitory activity to electrophoretic homogeneity and the further characterization of its properties. The inhibitory protein, which has an M(r) of 29,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is an endochitinase that is also capable of exochitinase activity. The enzyme has an optimal pH of 5.5 and a temperature optimum of 45 degrees C. Chitinase activity in maize kernels peaked approximately 36 days after pollination. The Tex6 chitinase purified in this study is capable of inhibiting the growth of A. flavus by 50% at a concentration of 20 mug/ml. Our data indicate that chitinase activity in Tex6 kernels makes a major contribution to the antifungal activity in this maize genotype. Partial peptide sequence of the chitinase showed it to differ from previously reported chitinases.

Co-expression of a modified maize ribosome-inactivating protein and a rice basic chitinase gene in transgenic rice plants confers enhanced resistance to sheath blight.

Chitinases, beta-1,3-glucanases, and ribosome-inactivating proteins are reported to have antifungal activity in plants. With the aim of producing fungus-resistant transgenic plants, we co-expressed a modified maize ribosome-inactivating protein gene, MOD1, and a rice basic chitinase gene, RCH10, in transgenic rice plants. A construct containing MOD1 and RCH10 under the control of the rice rbcS and Act1 promoters, respectively, was co-transformed with a plasmid containing the herbicide-resistance gene bar as a selection marker into rice by particle bombardment. Several transformants analyzed by genomic Southern-blot hybridization demonstrated integration of multiple copies of the foreign gene into rice chromosomes. Immunoblot experiments showed that MOD1 formed approximately 0.5% of the total soluble protein in transgenic leaves. RCH10 expression was examined using the native polyacrylamide-overlay gel method, and high RCH10 activity was observed in leaf tissues where endogenous RCH10 is not expressed. R1 plants were analyzed in a similar way, and the Southern-blot patterns and levels of transgene expression remained the same as in the parental line. Analysis of the response of R2 plants to three fungal pathogens of rice, Rhizoctonia solani, Bipolaris oryzae, and Magnaporthe grisea, indicated statistically significant symptom reduction only in the case of R. solani (sheath blight). The increased resistance co-segregated with herbicide tolerance, reflecting a correlation between the resistance phenotype and transgene expression.

Enhanced resistance to Helicoverpa zea in tobacco expressing an activated form of maize ribosome- inactivating protein.

Progeny of two transgenic tobacco (Nicotiana tabacum L.) lines that expressed an activated form of maize (Zea mays L.) ribosome-inactivating protein (RIP) had varying resistance to the insect species tested. A subset of R(2) plants from the two lines appeared to be more resistant to larvae of the cigarette beetle, Lasioderma serricorne (F.), and the tobacco hornworm, Manduca sexta (L.) than the wild type plants. Progeny (R(3)) of the more resistant R(2) plants were tested more extensively for insect resistance. Resistance to the corn earworm, Helicoverpa zea (Boddie), was most consistent, with significantly decreased feeding often accompanied by increased mortality and reduced weights of survivors fed on leaf disks of the two transgenic lines compared to the wild type. The amount of damage by H. zea was significantly inversely correlated with levels of RIP. Resistance of RIP-producing plants to H. zea was greater than expected on the basis of prior in vitro results using diet-incorporated maize RIP. The R(3) transgenic plant leaf disks were also often more resistant to feeding by larvae of L. serricorne compared to wild type plants. Although reduced feeding by M. sexta was noted when they were fed leaf disks from transgenic compared to wild type plants the first day of exposure, differences were not significant. This information provides further support for maize RIP having a role in resistance to maize-feeding insects.

RIBOSOME-INACTIVATING PROTEINS: A Plant Perspective.

Ribosome-inactivating proteins (RIPs) are toxic N-glycosidases that depurinate the universally conserved alpha-sarcin loop of large rRNAs. This depurination inactivates the ribosome, thereby blocking its further participation in protein synthesis. RIPs are widely distributed among different plant genera and within a variety of different tissues. Recent work has shown that enzymatic activity of at least some RIPs is not limited to site-specific action on the large rRNAs of ribosomes but extends to depurination and even nucleic acid scission of other targets. Characterization of the physiological effects of RIPs on mammalian cells has implicated apoptotic pathways. For plants, RIPs have been linked to defense by antiviral, antifungal, and insecticidal properties demonstrated in vitro and in transgenic plants. How these effects are brought about, however, remains unresolved. At the least, these results, together with others summarized here, point to a complex biological role. With genetic, genomic, molecular, and structural tools now available for integrating different experimental approaches, we should further our understanding of these multifunctional proteins and their physiological functions in plants.