Comparison of the capacity of enamel matrix derivative gel and enamel matrix derivative in liquid formulation to adsorb to bone grafting materials.

BACKGROUND The use of an enamel matrix derivative (EMD) has been shown to enhance periodontal regeneration (e.g., formation of root cementum, periodontal ligament, and alveolar bone). However, in certain clinical situations, the use of EMD alone may not be sufficient to prevent flap collapse or provide sufficient stability of the blood clot. Data from clinical and preclinical studies have demonstrated controversial results after application of EMD combined with different types of bone grafting materials in periodontal regenerative procedures. The aim of the present study is to investigate the adsorption properties of enamel matrix proteins to bone grafts after surface coating with either EMD (as a liquid formulation) or EMD (as a gel formulation). METHODS Three different types of grafting materials, including a natural bone mineral (NBM), demineralized freeze-dried bone allograft (DFDBA), or a calcium phosphate (CaP), were coated with either EMD liquid or EMD gel. Samples were analyzed by scanning electron microscopy or transmission electron microscopy (TEM) using an immunostaining assay with gold-conjugated anti-EMD antibody. Total protein adsorption to bone grafting material was quantified using an enzyme-linked immunosorbent assay (ELISA) kit for amelogenin. RESULTS The adsorption of amelogenin to the surface of grafting material varied substantially based on the carrier system used. EMD gel adsorbed less protein to the surface of grafting particles, which easily dissociated from the graft surface after phosphate-buffered saline rinsing. Analyses by TEM revealed that adsorption of amelogenin proteins were significantly farther from the grafting material surface, likely a result of the thick polyglycolic acid gel carrier. ELISA protein quantification assay demonstrated that the combination of EMD liquid + NBM and EMD liquid + DFDBA adsorbed higher amounts of amelogenin than all other treatment modalities. Furthermore, amelogenin proteins delivered by EMD liquid were able to penetrate the porous surface structure of NBM and DFDBA and adsorb to the interior of bone grafting particles. Grafting materials coated with EMD gel adsorbed more frequently to the exterior of grafting particles with little interior penetration. CONCLUSIONS The present study demonstrates a large variability of adsorbed amelogenin to the surface of bone grafting materials when enamel matrix proteins were delivered in either a liquid formulation or gel carrier. Furthermore, differences in amelogenin adsorption were observed among NBM, DFDBA, and biphasic CaP particles. Thus, the potential for a liquid carrier system for EMD, used to coat EMD, may be advantageous for better surface coating.

Mimicking respiratory phosphorylation using purified enzymes

The enzymes of oxidative phosphorylation are a striking example of the functional association of multiple enzyme complexes, working together to form ATP from cellular reducing equivalents. These complexes, such as cytochrome c oxidase or the ATP synthase, are typically investigated individually and therefore, their functional interplay is not well understood. Here, we present methodology that allows the co-reconstitution of purified terminal oxidases and ATP synthases in synthetic liposomes. The enzymes are functionally coupled via proton translocation where upon addition of reducing equivalents the oxidase creates and maintains a transmembrane electrochemical proton gradient that energizes the synthesis of ATP by the F1F0 ATP synthase. The method has been tested with the ATP synthases from Escherichia coli and spinach chloroplasts, and with the quinol and cytochrome c oxidases from E. coli and Rhodobacter sphaeroides, respectively. Unlike in experiments with the ATP synthase reconstituted alone, the setup allows in vitro ATP synthesis under steady state conditions, with rates up to 90 ATP×s(-1)×enzyme(-1). We have also used the novel system to study the phenomenon of "mild uncoupling" as observed in mitochondria upon addition of low concentrations of ionophores (e.g. FCCP, SF6847) and the recoupling effect of 6-ketocholestanol. While we could reproduce the described effects, our data with the in vitro system does not support the idea of a direct interaction between a mitochondrial protein and the uncoupling agents as proposed earlier.

Effect of Enamel Matrix Derivative Liquid on Osteoblast and Periodontal Ligament Cell Proliferation and Differentiation.

BACKGROUND Enamel matrix derivatives (EMDs) have been used clinically for more than a decade for the regeneration of periodontal tissues. The aim of the present study is to analyze the effect on cell growth of EMDs in a gel carrier in comparison to EMDs in a liquid carrier. EMDs in a liquid carrier have been shown to adsorb better to bone graft materials. METHODS Primary human osteoblasts and periodontal ligament (PDL) cells were exposed to EMDs in both gel and liquid carriers and compared for their ability to induce cell proliferation and differentiation. Alizarin red staining and real-time polymerase chain reaction for expression of genes encoding collagen 1, osteocalcin, and runt-related transcription factor 2, as well as bone morphogenetic protein 2 (BMP2), transforming growth factor (TGF)-β1, and interleukin (IL)-1β, were assessed. RESULTS EMDs in both carriers significantly increased cell proliferation of both osteoblasts and PDL cells in a similar manner. Both formulations also significantly upregulated the expression of genes encoding BMP2 and TGF-β1 as well as decreased the expression of IL-1β. EMDs in the liquid carrier further retained similar differentiation potential of both osteoblasts and PDL cells by demonstrating increased collagen and osteocalcin gene expression and significantly higher alizarin red staining. CONCLUSIONS The results from the present study indicate that the new formulation of EMDs in a liquid carrier is equally as potent as EMDs in a gel carrier in inducing osteoblast and PDL activity. Future study combining EMDs in a liquid carrier with bone grafting materials is required to further evaluate its potential for combination therapies.

Plant Adenosine 5′-phosphosulfate Reductase Is a Novel Iron-Sulfur Protein

Adenosine 5′-phosphosulfate reductase (APR) catalyzes the two-electron reduction of adenosine 5′-phosphosulfate to sulfite and AMP, which represents the key step of sulfate assimilation in higher plants. Recombinant APRs from both Lemna minorand Arabidopsis thaliana were overexpressed inEscherichia coli and isolated as yellow-brown proteins. UV-visible spectra of these recombinant proteins indicated the presence of iron-sulfur centers, whereas flavin was absent. This result was confirmed by quantitative analysis of iron and acid-labile sulfide, suggesting a 4Fe-4S cluster as the cofactor. EPR spectroscopy of freshly purified enzyme showed, however, only a minor signal at g = 2.01. Therefore, Mössbauer spectra of 57Fe-enriched APR were obtained at 4.2 K in magnetic fields of up to 7 tesla, which were assigned to a diamagnetic 4Fe-4S2+ cluster. This cluster was unusual because only three of the iron sites exhibited the same Mössbauer parameters. The fourth iron site gave, because of the bistability of the fit, a significantly smaller isomer shift or larger quadrupole splitting than the other three sites. Thus, plant assimilatory APR represents a novel type of adenosine 5′-phosphosulfate reductase with a 4Fe-4S center as the sole cofactor, which is clearly different from the dissimilatory adenosine 5′-phosphosulfate reductases found in sulfate reducing bacteria.

VERIFICATION OF DNA PREDICTED PROTEIN SEQUENCES BY ENZYME HYDROLYSIS AND MASS SPECTROMETRIC ANALYSIS

The focus of this thesis lies in the development of a sensitive method for the analysis of protein primary structure which can be easily used to confirm the DNA sequence of a protein's gene and determine the modifications which are made after translation. This technique involves the use of dipeptidyl aminopeptidase (DAP) and dipeptidyl carboxypeptidase (DCP) to hydrolyze the protein and the mass spectrometric analysis of the dipeptide products.^ Dipeptidyl carboxypeptidase was purified from human lung tissue and characterized with respect to its proteolytic activity. The results showed that the enzyme has a relatively unrestricted specificity, making it useful for the analysis of the C-terminal of proteins. Most of the dipeptide products were identified using gas chromatography/mass spectrometry (GC/MS). In order to analyze the peptides not hydrolyzed by DCP and DAP, as well as the dipeptides not identified by GC/MS, a FAB ion source was installed on a quadrupole mass spectrometer and its performance evaluated with a variety of compounds.^ Using these techniques, the sequences of the N-terminal and C-terminal regions and seven fragments of bacteriophage P22 tail protein have been verified. All of the dipeptides identified in these analysis were in the same DNA reading frame, thus ruling out the possibility of a single base being inserted or deleted from the DNA sequence. The verification of small sequences throughout the protein sequence also indicates that no large portions of the protein have been removed after translation. ^

CHARACTERIZATION OF HIGH MOBILITY GROUP AND HISTONE H1 PROTEIN LEVELS AND SYNTHESIS DURING SPERMATOGENESIS IN THE RAT

In order to propose a role for internucleosomal high mobility group proteins (HMGs), and HI histone variants study of their levels and synthesis in a system of development and differentiation--rat spermatogenesis--was undertaken. HMG1, 2, 14, and 17 were isolated from rat testes and found to be very similar to calf thymus HMGs. Testis levels of HMGs, relative to DNA, were equivalent to other rat tissues for HMG1 (13 ug/mg DNA), HMG14 (2 ug/mg DNA), and HMG17 (5 ug/mg DNA). HMG2 levels were different among rat tissues, with three groups observed: (1) nonproliferating tissues (1-5 ug/mg DNA); (2) proliferating tissues (8-13 ug/mg DNA); and (3) the testis (32 ug/mg DNA). Other species (toad, opposum, mouse, dog, and monkey) showed the same testis-specific increase of HMG2. Populations of purified testis cell types were separated by centrifugal elutriation and density gradient centrifugation from adult and immature rat testes. Pachytene spermatocytes and early spermatids (56 and 47 ug/mg DNA, respectively) caused the testis-specific increase of HMG2 levels. Cell types preceding pachytenes (types A and B spermatogonia, mixtures of spermatogonia and early primary spermatocytes, and early pachytenes contained HMG2 levels similar to proliferating tissues (12 ug/mg DNA). Late spermatids did not contain HMGs. Somatic Sertoli and Leydig cells (2 ug/mg DNA) exhibited HMG2 levels similar to nonproliferating tissues. HMGs synthesized in spermatogonia and spermatocytes had similar specific activities, but early spermatids did not synthesize HMGs. Germ cells also contained an HMG2 species (on acid-urea gels) not found in somatic tissues. Other investigators have shown that HMGs may be associated with transcriptional or replicative processes. Thus, it is proposed that HMG2 plays a role in modulatable gene expression, while HMG1 is associated with housekeeping functions.^ HI histone variants were also studied throughout spermatogenesis. The minor somatic variant, HIa, is the predominant variant in spermatogonia and early primary spermatocytes. In early pachytenes, the testis-specific variant, HIt, is first synthesized and appears, largely replacing somatic variants HIbcd and e by late pachytene stage. Early spermatids contain the same HI composition as pachytenes, but do not synthesize HI histones. HI('0) is present in low amounts in all germ cells. These results suggest that expression of HI variants is developmentally controlled.^

The regulation of protein kinase C by thiol oxidation

The Ser/Thr protein kinase C (PKC) isozyme family plays an important role in cell growth and differentiation and also contributes to key events in the development and progression of cancer. PKC isozymes are activated by phospholipid-dependent mechanisms, and they are also subject to oxidative activation and inactivation. Oxidative regulatory mechanisms are important in the governance of PKC isozyme action. While oxidative PKC activation involves phospho-tyrosine (P-Y) stabilization, the molecular mechanism(s) for oxidative PKC inactivation have not been defined. We previously reported that Thr → Cys peptide-substrate analogs inactivate several PKC isozymes including PKC-α via S-thiolation, i.e., by forming disulfides with PKC thiols. This inactivation mechanism is chemically analogous to protein S-glutathiolation, a post-translational modification that has been shown to oxidatively regulate several enzymes. To determine if PKC-α could be inactivated by S-glutathiolation, we employed the thiol-specific oxidant diamide (0.01–10mM) and 100μM glutathione (GSH). Diamide alone (0.1–5.0 mM) weakly inactivated PKC-α (<20%), and GSH alone had no effect on the isozyme activity. Marked potentiation of diamide-induced PKC-α inactivation (>90%) was achieved by 100μM GSH, resulting in full inactivation of the isozyme. Inactivation was reversed by DTT, consistent with a mechanism involving PKC-α S-glutathiolation. S-glutathiolation was demonstrated as DTT-reversible incorporation of [35S] GSH into PKC-α isozyme structure. These results indicate that a mild oxidative stimulus can inactivate purified PKC-α via S-glutathiolation. In addition, diamide treatment of metabolically labeled NIH3T3 cells induced potent PKC-α inactivation via isozyme [35S] S-thiolation. These results indicate that cellular PKC-α can be regulated via S-glutathiolation. ^

Characterization of hLodestar: A CDC5L interacting protein

Lodestar, a Drosophila maternal-effect gene, is essential for proper chromosome segregation during embryonic mitosis. Mutations in lodestar cause chromatin bridging in anaphase, preventing the sister chromatids from fully separating and leaving chromatin tangled at the metaphase plate. Drosophila lodestar protein was originally identified, in purified fractions of Drosophila Kc cell nuclear extracts, by its ability to suppress the generation of long RNA polymerase II transcripts. The human homolog of this protein (hLodestar) was cloned and studied in comparison to the Drosophila lodestar activities. The results of these studies show, similar to the Drosophila protein, hLodestar has dsDNA-dependent ATPase and transcription termination activity in vitro. hLodestar has also been shown to release RNA polymerase I and II stalled at a cyclobutane thymine dimer. Lodestar belongs to the SNF2 family of proteins, which are members of the DExH/D helicase super-family. The SNF2 family of proteins are believed to play a critical role in altering protein-DNA interactions in a variety of cellular contexts. We have recently isolated a human cDNA (hLodestar) that shares significant homology to the Drosophila lodestar gene. The 4.6 kb clone contains an open reading frame of 1162 amino acids, and shares 55% similarity and 46% identity to the Drosophila Lodestar protein sequence. Our studies looking for hLodestar interacting proteins revealed an association with CDC5L in the yeast two-hybrid system and co-immunoprecipitation experiments. CDC5L has been well documented to be a component of the spliceosome. Our data suggests hLodestar is involved in splicing through in vitro assembly and splicing reactions, in addition to its association with spliceosomes purified from HeLa nuclear extract. Although many other members of the DExH/D helicase super-family have been linked to splicing, this is the first SNF2 family member to be implicated in the splicing reaction. ^

CCTeta: A novel soluble guanylyl cyclase-interacting protein

Nitric oxide (NO) transduces most of its biological effects through activation of the heterodimeric enzyme, soluble guanylyl cyclase (sGC). Activation of sGC results in the production of 3′,5 ′-cyclic guanosine monophosphate (cGMP) from 5′ -guanosine triphosphate (GTP). In this thesis, we demonstrate a novel protein interaction between CCT (chaperonin containing t-complex polypeptide) subunit η and the α1β1 isoform of sGC. Using the yeast-two-hybrid system, CCTη was found to interact with the N-terminal portion of β1 subunit of sGC. This interaction was then confirmed in vitro with a co-immunoprecipitation from mouse brain. The interaction between these two proteins was further supported by a co-localization of the proteins within rat brain. Using the yeast-two-hybrid system, CCTη was found to bind to the N-terminal portion of sGC. In vitro assays with purified CCTη and Sf9 lysate expressing sGC resulted in a 33% inhibition of sodium nitroprusside (SNP)-stimulated sGC activity. The same assays were then performed using BAY41-2272, an NO-independent allosteric sGC activator, and CCTη had no effect on this activity. Furthermore, CCTη had no effect on the activity of αβCys105 sGC a constitutively active mutant that lacks a heme group. Of note is the fact that the full-length CCTη-expressing bacterial lysate inhibited the activity of sGC-expressing Sf9 lysate by 48% compared with GST alone. This indicates that the amino terminal 94 amino acids of CCTη are important to the inhibition of sGC activity. Lastly, a 45% inhibition of sGC activity by CCTη was seen in vivo in BE2 cells stably transfected with CCTη and treated with SNP. The fact that the inhibition of sGC was more pronounced with bacterial lysate expressing CCTη versus the purified CCTη implies that some factor in the bacterial lysate enhances the inhibitory effect of CCTη. Because the level of inhibition seen in bacterial lysate and in vivo experiments is similar, might imply that the factor that aids in CCTη effect on sGC is conserved. Together, these data suggest that CCTη is a novel type of sGC inhibitor that inhibits sGC by modifying the binding of NO to the heme group or the subsequent conformational changes induced by NO binding. ^

Contribution of transmembrane and cytoplasmic domains to membrane protein association

Membrane proteins are critical to every aspect of cell physiology, with their association mediating important biological functions. The transmembrane and cytoplasmic domains are known to be important for their association. In order to characterize their role in detail, we have applied different biophysical techniques in detergent micelles to two model systems. The first study involves FcRγ, a single transmembrane domain protein existing as a disulfide linked homodimer. We investigated the role of a conserved transmembrane polar residue and the cytoplasmic tail in FcRγ homo-interactions. Our results by various biophysical techniques including SDS-PAGE, circular dichroism and sedimentation equilibrium in detergent micelles indicate importance of both the transmembrane polar residue and cytoplasmic tail in maintaining proper conformation for FcRγ homo-interactions. A contrasting second study was on L-selectin, another single transmembrane domain protein with a large extracellular domain and a short cytoplasmic tail. Previous cross-linking experiments indicate its possible dimerization. However, the purified fragment of L-selectin and corresponding mutants did not dimerize when analyzed by TOXCAT assay, sedimentation equilibrium and fluorescence resonance energy transfer. It was likely that the presence of juxtamembrane positively charged residues led to decreased migrational rates in SDS PAGE. In conclusion, complementary biophysical techniques should be used with care when studying membrane protein association in detergent micelles. As an extension to our study on L-selectin, we also investigated its interaction with Calmodulin (CaM) in detergent micelles. CaM was found to interact with different detergents. We applied fluorescence and NMR spectroscopy to characterize the interaction of both the apo and Ca 2+ bound form of CaM, with commonly used detergents, below and above their respective critical micelle concentrations. The interaction of apo-CaM with detergents was found to vary with the nature of the detergent head group, whereas Ca2+-CaM interacted with individual detergent molecules irrespective of the nature of their head group. NMR titration experiments of CaM with detergents indicated involvement of specific residues from the N-lobe, linker and C-lobe of CaM. ^

GMZ27 is a new organic arsenic derivative with anti-leukemic activity, via the induction of reactive oxygen species through NADPH oxidase and mitochondrial pathway that lead to leukemia cell apoptosis

Arsenic trioxide (ATO) is an inorganic arsenic derivative that is very effective against relapsed acute promyelocytic leukemia. It is being investigated as therapy for other cancers, but the risk/benefit ratio is questionable due to significant side effects. In contrast, organic arsenic derivatives (OAD) are known to be much less toxic than ATO. Based on high activity, we selected GMZ27 (dipropil-s-glycerol arsenic) for further study and have confirmed its potent activity against human acute leukemia cell lines. This anti-leukemic activity is significantly higher than that of ATO. Both in vivo and in vitro tests have shown that GMZ27 is significantly less toxic to normal bone marrow mononuclear cells and normal mice. Therefore, further study of the biological activity of GMZ27 was undertaken. ^ GMZ27, in contrast to ATO, can only marginally induce maturation of leukemic cells. GMZ27 has no effect on cell cycle. The anti-leukemic activity of GMZ27 against acute myeolocytic leukemia cells is not dependent upon degradation of PML-RARα fusion protein. GMZ27 causes dissipation of mitochondrial transmembrane potential, cleavage of caspase 9, caspase 3 activation. Further studies indicated that GMZ27 induces intracellular reactive oxygen species (ROS) production, and modification of intracellular ROS levels had profound effect on its potential to inhibit proliferation of leukemic cells. Therefore ROS production plays a major role in the anti-leukemic activity of GMZ27. ^ To identify how GMZ27 induces ROS, our studies focused on mitochondria and NADPH oxidase. The results indicated that the source of ROS generation induced by GMZ27 is dose dependent. At the low dose (0.3 uM) GMZ27 induces NADPH oxidase activity that leads to late ROS production, while at the high dose (2.0 uM) mitochondria function is disrupted and early ROS production is induced leading to dramatic cell apoptosis. Therefore, late, ROS production can be detected in mitochondria are depleted Rho-0 cells. Our work not only delineates a major biologic pathway for the anti-leukemic activity of GMZ27, but also discusses possible ways of enhancing the effect by the co-application of NADPH oxidase activator. Further study of this interaction may lead to achieving better therapeutic index.^

Pigment, carbohydrate and protein content of microbial mats from northern Victoria Land lake sediments

The composition of algal pigments and extracellular polymeric substances (EPS) was determined in microbial mats from two lakes in Victoria Land (Continental Antarctica) with different lithology and environmental features. The aim was to expand knowledge of benthic autotrophic communities in Antarctic lacustrine ecosystems, providing reference data for future assessment of possible changes in environmental conditions and freshwater communities. The results of chemical analyses were supported by microscopy observations. Pigment profiles showed that filamentous cyanobacteria are dominant in both lakes. Samples from the water body at Edmonson Point had greater biodiversity, fewer pigments and lower EPS ratios than those from the lake at Kar Plateau. Differences in mat composition and in pigment and EPS profile between the two lakes are discussed in terms of local environmental conditions such as lithology, ice-cover and UV radiation. The present study suggests that a chemical approach could be useful in the study of benthic communities in Antarctic lakes and their variations in space and time.

Heterologous Expression of a Plant Small Heat-Shock Protein Enhances Escherichia Coli Viability under Heat And Cold Stress Ref.: 1

A small heat-shock protein (sHSP) that shows molecular chaperone activity in vitro was recently purified from mature chestnut (Castanea sativa) cotyledons. This protein, renamed here as CsHSP17.5, belongs to cytosolic class I, as revealed by cDNA sequencing and immunoelectron microscopy. Recombinant CsHSP17.5 was overexpressed in Escherichia coli to study its possible function under stress conditions. Upon transfer from 37°C to 50°C, a temperature known to cause cell autolysis, those cells that accumulated CsHSP17.5 showed improved viability compared with control cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cell lysates suggested that such a protective effect in vivo is due to the ability of recombinant sHSP to maintain soluble cytosolic proteins in their native conformation, with little substrate specificity. To test the recent hypothesis that sHSPs may be involved in protection against cold stress, we also studied the viability of recombinant cells at 4°C. Unlike the major heat-induced chaperone, GroEL/ES, the chestnut sHSP significantly enhanced cell survivability at this temperature. CsHSP17.5 thus represents an example of a HSP capable of protecting cells against both thermal extremes. Consistent with these findings, high-level induction of homologous transcripts was observed in vegetative tissues of chestnut plantlets exposed to either type of thermal stress but not salt stress

A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses

The bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non-host tobacco, was found to also suppress the production of defence-associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His 6 -tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1 D299A non-catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death.

The involvement of thaumatin-like proteins in plant food cross-reactivity: a multicenter study using a specific protein microarray.

Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs) in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG) and another against pollens but tolerant to food-plant allergens (PAG), were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%), chestnut TLP (24%) and plane pollen TLP (22%) proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited mayor que50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy.