Modulation of TOR complex 2 signaling by the stress-activated MAPK pathway in fission yeast.

Sin1 is a substrate-binding subunit of target of rapamycin complex 2 (TORC2), an evolutionarily conserved protein kinase complex. In fission yeast, Sin1 has also been identified as a protein that interacts with Spc1 (also known as Sty1) in the stress-activated protein kinase (SAPK) pathway. Therefore, this study examined the relationship between TORC2 and Spc1 signaling. We found that the common docking (CD) domain of Spc1 interacts with a cluster of basic amino acid residues in Sin1. Although diminished TORC2 activity in the absence of the functional Spc1 cascade suggests positive regulation of TORC2 by Spc1, such regulation appears to be independent of the Sin1-Spc1 interaction. Hyperosmotic stress transiently inhibits TORC2, and its swift recovery is dependent on Spc1, the transcription factor Atf1, and the glycelrol-3-phosphate dehydrogenase Gpd1, whose expression is induced upon osmostress by the Spc1-Atf1 pathway. Thus, cellular adaptation to osmostress seems important for TORC2 reactivation, though Spc1 and Atf1 contribute to TORC2 activation also in the absence of osmostress. These results indicate coordinated actions of the SAPK and TORC2 pathways, both of which are essential for fission yeast cells to survive environmental stress.

Glycolytic enzyme GAPDH promotes peroxide stress signaling through multistep phosphorelay to a MAPK cascade.

Phosphorelay signaling of environmental stimuli by two-component systems is prevailing in bacteria and also utilized by fungi and plants. In the fission yeast Schizosaccharomyces pombe, peroxide stress signals are transmitted from the Mak2/3 sensor kinases to the Mpr1 histidine-containing phosphotransfer (HPt) protein and finally to the Mcs4 response regulator, which activates a MAP kinase cascade. Here we show that, unexpectedly, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) physically associates with the Mcs4 response regulator and stress-responsive MAP kinase kinase kinases (MAPKKKs). In response to H2O2 stress, Cys-152 of the Tdh1 GAPDH is transiently oxidized, which enhances the association of Tdh1 with Mcs4. Furthermore, Tdh1 is essential for the interaction between the Mpr1 HPt protein and the Mcs4 response regulator and thus for phosphorelay signaling. These results demonstrate that the glycolytic enzyme GAPDH plays an essential role in the phosphorelay signaling, where its redox-sensitive cysteine residue may provide additional input signals.

Roseiterribacter gracilis gen. nov., sp. nov., a novel filterable alphaproteobacterium isolated from soil using a gel-filled microwell array device.

Our previous studies indicate the abundant and diverse presence of yet-to-be-cultured microorganisms in the micropore-filtered fractions of various environmental samples. Here, we isolated a novel bacterium (designated as strain TMPK1T) from a 0.45-µm-filtered soil suspension by using a gel-filled microwell array device comprising 900 microwells and characterized its phylogenetic and physiological features. This strain showed low 16S rRNA gene sequence identities (<91%) and low average nucleotide identity values (<70%) to the closest validly described species, and belonged to a novel-family-level lineage within the order Rhodospirillales of Alphaproteobacteria. Strain TMPK1T exhibited small cell sizes (0.08-0.23 µm3) and had a high cyclopropane fatty acid content (>13%), and these characteristics were differentiated from other Rhodospirillales bacteria. A comprehensive habitability search using amplicon datasets suggested that TMPK1T and its close relatives are mainly distributed in soil and plant-associated environments. Based on these results, we propose that strain TMPK1T represents a novel genus and species named Roseiterribacter gracilis gen. nov., sp. nov. (JCM 34627T = KCTC 82790T). We also propose Roseiterribacteraceae fam. nov. to accommodate the genus Roseiterribacter.

Temporal and fertilizer-dependent dynamics of soil bacterial communities in buckwheat fields under long-term management.

This study integrated bacterial community and soil chemicals to characterize the soil ecosystem in an open upland field managed by six controlled fertilizer programs using the minimum amount of pesticides. Amplicon sequencing the 16S rRNA gene revealed that inorganic nitrogen fertilizer and compost altered the diversity and structure of the soil bacterial community throughout buckwheat (Fagopyrum esculentum Moench 'Hitachiakisoba') cultivation. The bacterial community comprised three clusters that contained bacteria that are prevalent in soils fertilized with nitrogen (cluster 1, 340 taxa), without nitrogen and compost (cluster 2, 234 taxa), and with compost-fertilized (cluster 3, 296 taxa). Cluster 2 contained more taxa in Actinobacteriota and less in Acidobacteriota, and cluster 3 contained more taxa in Gemmatimonadota compared with the other clusters. The most frequent taxa in cluster 1 were within the Chloroflexi phylum. The bacterial community structure correlated with soil chemical properties including pH, total organic carbon, SO42-, soluble Ca2+. A co-occurrence network of bacterial taxa and chemicals identified key bacterial groups comprising the center of a community network that determined topology and dynamics of the network. Temporal dynamics of the bacterial community structure indicated that Burkholderiales were associated with buckwheat ripening, indicating plant-bacteria interaction in the ecosystem.

Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli.

BACKGROUND: Escherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli. RESULTS: Because the redox enzymes can reduce the disulfide that forms on proteins, we first tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coliL-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (ΔcysI and ΔcysJ) and the L-cysteine synthase gene (ΔcysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (ΔcysC or ΔcysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coliL-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell. CONCLUSIONS: In this work, we showed that Grx1 and NrdH reduce SSC to L-cysteine, and the generated sulfite is then utilized as the sulfur source to produce additional L-cysteine molecule through the sulfate pathway in E. coli. We also found that co-overexpression of NrdH, CysI, and CysK increases L-cysteine production. Our results propose that the enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from SSC is a novel method for improvement of L-cysteine production.

The L-cysteine/L-cystine shuttle system provides reducing equivalents to the periplasm in Escherichia coli.

Intracellular thiols like L-cysteine and glutathione play a critical role in the regulation of cellular processes. Escherichia coli has multiple L-cysteine transporters, which export L-cysteine from the cytoplasm into the periplasm. However, the role of L-cysteine in the periplasm remains unknown. Here we show that an L-cysteine transporter, YdeD, is required for the tolerance of E. coli cells to hydrogen peroxide. We also present evidence that L-cystine, a product from the oxidation of L-cysteine by hydrogen peroxide, is imported back into the cytoplasm in a manner dependent on FliY, the periplasmic L-cystine-binding protein. Remarkably, this protein, which is involved in the recycling of the oxidized L-cysteine, is also found to be important for the hydrogen peroxide resistance of this organism. Furthermore, our analysis of the transcription of relevant genes revealed that the transcription of genes encoding FliY and YdeD is highly induced by hydrogen peroxide rather than by L-cysteine. These findings led us to propose that the inducible L-cysteine/L-cystine shuttle system plays an important role in oxidative stress tolerance through providing a reducing equivalent to the periplasm in E. coli.

Defect of RNA pyrophosphohydrolase RppH enhances fermentative production of L-cysteine in Escherichia coli.

Fermentative production of L-cysteine has been established using Escherichia coli. In that procedure, thiosulfate is a beneficial sulfur source, whereas repressing sulfate utilization. We first found that thiosulfate decreased transcript levels of genes related to sulfur assimilation, particularly whose expression is controlled by the transcription factor CysB. Therefore, a novel approach, i.e. increment of expression of genes involved in sulfur-assimilation, was attempted for further improvement of L-cysteine overproduction. Disruption of the rppH gene significantly augmented transcript levels of the cysD, cysJ, cysM and yeeE genes (≥1.5-times) in medium containing sulfate as a sole sulfur source, probably because the rppH gene encodes mRNA pyrophosphohydrolase that triggers degradation of certain mRNAs. In addition, the ΔrppH strain appeared to preferentially uptake thiosulfate rather than sulfate, though thiosulfate dramatically reduced expression of the known sulfate/thiosulfate transporter complexes in both ΔrppH and wild-type cells. We also found that both YeeE and YeeD are required for the strain without the transporters to grow in the presence of thiosulfate as a sole sulfur source. Therefore, yeeE and yeeD are assigned as genes responsible for thiosulfate uptake (tsuA and tsuB, respectively). In final, we applied the ΔrppH strain to the fermentative production of L-cysteine. Disruption of the rppH gene enhanced L-cysteine biosynthesis, as a result, a strain producing approximately twice as much L-cysteine as the control strain was obtained.

Draft Genome Sequence of Novel Filterable Rhodospirillales Bacterium Strain TMPK1, Isolated from Soil.

We report the draft genome sequence of novel Rhodospirillales bacterium strain TMPK1, isolated from a micropore-filtered soil suspension. This strain has a genome of 4,249,070 bp, comprising 4,151 protein-coding sequences. The genome sequence data further suggest that strain TMPK1 is an alphaproteobacterium capable of carotenoid production.

Wsh3/Tea4 is a novel cell-end factor essential for bipolar distribution of Tea1 and protects cell polarity under environmental stress in S. pombe.

BACKGROUND: The fission yeast Schizosaccharomyces pombe has a cylindrical cell shape, for which growth is strictly limited to both ends, and serves as an excellent model system for genetic analysis of cell-polarity determination. Previous studies identified a cell-end marker protein, Tea1, that is transported by cytoplasmic microtubules to cell tips and recruits other cell-end factors, including the Dyrk-family Pom1 kinase. The deltatea1 mutant cells cannot grow in a bipolar fashion and show T-shaped morphology after heat shock. RESULTS: We identified Wsh3/Tea4 as a novel protein that interacts with Win1 MAP kinase kinase kinase (MAPKKK) of the stress-activated MAP kinase cascade. Wsh3 forms a complex with Tea1 and is transported to cell tips by growing microtubules. The deltawsh3 mutant shows monopolar growth with abnormal Tea1 aggregate at the non-growing cell end; this abnormal aggregate fails to recruit Pom1 kinase. Consistent with the observed interaction between Win1 and Wsh3, cells lacking Wsh3 or Tea1 show more severe cell-polarity defects under osmolarity and heat-stress stimuli that are known to activate the stress MAPK cascade. Furthermore, mutants of the stress MAPK also exhibit cell-polarity defects when exposed to the same stress. CONCLUSIONS: Wsh3/Tea4 is an essential component of the Tea1 cell-end complex. In addition to its role in bipolar growth during the normal cell cycle, the Wsh3-Tea1 complex, together with the stress-signaling MAPK cascade, contributes to cell-polarity maintenance under stress conditions.

S-Adenosylmethionine Synthetase Is Required for Cell Growth, Maintenance of G0 Phase, and Termination of Quiescence in Fission Yeast.

S-adenosylmethionine is an important compound, because it serves as the methyl donor in most methyl transfer reactions, including methylation of proteins, nucleic acids, and lipids. However, cellular defects in the genetic disruption of S-adenosylmethionine synthesis are not well understood. Here, we report the isolation and characterization of temperature-sensitive mutants of fission yeast S-adenosylmethionine synthetase (Sam1). Levels of S-adenosylmethionine and methylated histone H3 were greatly diminished in sam1 mutants. sam1 mutants stopped proliferating in vegetative culture and arrested specifically in G2 phase without cell elongation. Furthermore, sam1 mutants lost viability during nitrogen starvation-induced G0 phase quiescence. After release from the G0 state, sam1 mutants could neither increase in cell size nor re-initiate DNA replication in the rich medium. Sam1 is thus required for cell growth and proliferation, and maintenance of and exit from quiescence. sam1 mutants lead to broad cellular and drug response defects, as expected, since S. pombe contains more than 90 S-adenosylmethionine-dependent methyltransferases.

Fission yeast Ryh1 GTPase activates TOR Complex 2 in response to glucose.

The Target Of Rapamycin (TOR) is an evolutionarily conserved protein kinase that forms 2 distinct protein complexes referred to as TOR complex 1 (TORC1) and 2 (TORC2). Recent extensive studies have demonstrated that TORC1 is under the control of the small GTPases Rheb and Rag that funnel multiple input signals including those derived from nutritional sources; however, information is scarce as to the regulation of TORC2. A previous study using the model system provided by the fission yeast Schizosaccharomyces pombe identified Ryh1, a Rab-family GTPase, as an activator of TORC2. Here, we show that the nucleotide-binding state of Ryh1 is regulated in response to glucose, mediating this major nutrient signal to TORC2. In glucose-rich growth media, the GTP-bound form of Ryh1 induces TORC2-dependent phosphorylation of Gad8, a downstream target of TORC2 in fission yeast. Upon glucose deprivation, Ryh1 becomes inactive, which turns off the TORC2-Gad8 pathway. During glucose starvation, however, Gad8 phosphorylation by TORC2 gradually recovers independently of Ryh1, implying an additional TORC2 activator that is regulated negatively by glucose. The paired positive and negative regulatory mechanisms may allow fine-tuning of the TORC2-Gad8 pathway, which is essential for growth under glucose-limited environment.

Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli.

Intracellular thiols like L-cystine and L-cystine play a critical role in the regulation of cellular processes. Here we show that Escherichia coli has two L-cystine transporters, the symporter YdjN and the ATP-binding cassette importer FliY-YecSC. These proteins import L-cystine, an oxidized product of L-cystine from the periplasm to the cytoplasm. The symporter YdjN, which is expected to be a new member of the L-cystine regulon, is a low affinity L-cystine transporter (Km = 1.1 µM) that is mainly involved in L-cystine uptake from outside as a nutrient. E. coli has only two L-cystine importers because ΔydjNΔyecS mutant cells are not capable of growing in the minimal medium containing L-cystine as a sole sulfur source. Another protein YecSC is the FliY-dependent L-cystine transporter that functions cooperatively with the L-cystine transporter YdeD, which exports L-cystine as reducing equivalents from the cytoplasm to the periplasm, to prevent E. coli cells from oxidative stress. The exported L-cystine can reduce the periplasmic hydrogen peroxide to water, and then generated L-cystine is imported back into the cytoplasm via the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (Km = 110 nM) in a manner dependent on FliY, the periplasmic L-cystine-binding protein. The double disruption of ydeD and fliY increased cellular levels of lipid peroxides. From these findings, we propose that the hydrogen peroxide-inducible L-cystine/L-cystine shuttle system plays a role of detoxification of hydrogen peroxide before lipid peroxidation occurs, and then might specific prevent damage to membrane lipids.

Phosphorelay-dependent and -independent regulation of MAPKKK by the Mcs4 response regulator in fission yeast.

In a "two-component system," extracellular stimuli are transmitted by the transfer of a phosphoryl group from a sensor histidine kinase to a response regulator (RR), a mechanism referred to as phosphorelay. In the fission yeast Schizosaccharomyces pombe, peroxide stress signals are transmitted by phosphorelay to the Mcs4 RR, which activates the Spc1 MAP kinase (MAPK) cascade. We previously demonstrated that a glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) physically interacts with Mcs4 and promotes phosphorelay signaling to Mcs4. Independently of the phosphorelay mechanism, Mcs4 also plays a critical role in osmostress signaling, as a part of the stable ternary complex with the Wis4 and Win1 MAPK kinase kinases (MAPKKKs). Interestingly, GAPDH dissociates from Mcs4 upon osmostress, while oxidative stress promotes their association. The Mcs4 RR may serve as a switching hub that mediates activation of the Wis4-Win1 MAPKKK heteromer in response to different forms of stress.

Response regulator-mediated MAPKKK heteromer promotes stress signaling to the Spc1 MAPK in fission yeast.

The Spc1 mitogen-activated protein kinase (MAPK) cascade in fission yeast is activated by two MAPK kinase kinase (MAPKKK) paralogues, Wis4 and Win1, in response to multiple forms of environmental stress. Previous studies identified Mcs4, a "response regulator" protein that associates with the MAPKKKs and receives peroxide stress signals by phosphorelay from the Mak2/Mak3 sensor histidine kinases. Here we show that Mcs4 has an unexpected, phosphorelay-independent function in promoting heteromer association between the Wis4 and Win1 MAPKKKs. Only one of the MAPKKKs in the heteromer complex needs to be catalytically active, but disturbing the integrity of the complex by mutations to Mcs4, Wis4, or Win1 results in reduced MAPKKK-MAPKK interaction and, consequently, compromised MAPK activation. The physical interaction among Mcs4, Wis4, and Win1 is constitutive and not responsive to stress stimuli. Therefore the Mcs4-MAPKKK heteromer complex might serve as a stable platform/scaffold for signaling proteins that convey input and output of different stress signals. The Wis4-Win1 complex discovered in fission yeast demonstrates that heteromer-mediated mechanisms are not limited to mammalian MAPKKKs.

Two-component signaling to the stress MAP kinase cascade in fission yeast.

In the fission yeast Schizosaccharomyces pombe, the Mak2/3 sensor histidine kinases (HKs), the Mpr1 histidine-containing phosphotransfer (HPt) protein, and the Mcs4 response regulator (RR) constitute a multistep phosphorelay, which is connected to a stress-activated mitogen-activated protein kinase (MAPK) cascade. This hybrid signaling pathway senses H2O2 and transmits the stress signal by sequential phosphorylation of the component proteins, whose physical interactions play crucial roles to attain eventual activation of Spc1 MAPK. This chapter describes methodological details of the copurification assays in S. pombe cell lysate to detect the physical interactions between the Mpr1 HPt and Mcs4 RR proteins and between Mcs4 and the MAPK kinase kinases (MAPKKKs) of the Spc1 cascade. Unexpectedly, we found that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by tdh1+ is involved in the H2O2 signaling process, and its association with Mcs4 and MAPKKKs in cell lysate is also detectable by copurification assays. In response to H2O2, the catalytic cysteine residue of Tdh1 GAPDH is subjected to S-thiolation, of which detection protocol is described as well.

Rab-family GTPase regulates TOR complex 2 signaling in fission yeast.

BACKGROUND: From yeast to human, TOR (target of rapamycin) kinase plays pivotal roles in coupling extracellular stimuli to cell growth and metabolism. TOR kinase functions in two distinct protein complexes, TOR complex 1 (TORC1) and 2 (TORC2), which phosphorylate and activate different AGC-family protein kinases. TORC1 is controlled by the small GTPase Rheb, but little is known about TORC2 regulators. RESULTS: We have identified the Ryh1 GTPase, a human Rab6 ortholog, as an activator of TORC2 signaling in the fission yeast Schizosaccharomyces pombe. Mutational inactivation of Ryh1 or its guanine nucleotide exchange factor compromises the TORC2-dependent phosphorylation of the AGC-family Gad8 kinase. In addition, the effector domain of Ryh1 is important for its physical interaction with TORC2 and for stimulation of TORC2 signaling. Thus, GTP-bound Ryh1 is likely to be the active form stimulatory to TORC2-Gad8 signaling. Consistently, expression of the GTP-locked mutant Ryh1 is sufficient to promote interaction between TORC2 and Gad8 and to induce Gad8 hyperphosphorylation. The loss of functional Ryh1, TORC2, or Gad8 brings about similar vacuolar fragmentation and stress sensitivity, further corroborating their involvement in a common cellular process. Human Rab6 can substitute Ryh1 in S. pombe, and therefore Rab6 may be a potential activator of TORC2 in mammals. CONCLUSIONS: In its GTP-bound form, Ryh1, an evolutionarily conserved Rab GTPase, activates TORC2 signaling to the AGC kinase Gad8. The Ryh1 GTPase and the TORC2-Gad8 pathway are required for vacuolar integrity and cellular stress resistance in S. pombe.

The level of expression of thioredoxin is linked to fundamental properties and applications of wheat seeds.

Work with cereals (barley and wheat) and a legume (Medicago truncatula) has established thioredoxin h (Trx h) as a central regulatory protein of seeds. Trx h acts by reducing disulfide (S-S) groups of diverse seed proteins (storage proteins, enzymes, and enzyme inhibitors), thereby facilitating germination. Early in vitro protein studies were complemented with experiments in which barley seeds with Trx h overexpressed in the endosperm showed accelerated germination and early or enhanced expression of associated enzymes (alpha-amylase and pullulanase). The current study extends the transgenic work to wheat. Two approaches were followed to alter the expression of Trx h genes in the endosperm: (1) a hordein promoter and its protein body targeting sequence led to overexpression of Trx h5, and (2) an antisense construct of Trx h9 resulted in cytosolic underexpression of that gene (Arabidopsis designation). Underexpression of Trx h9 led to effects opposite to those observed for overexpression Trx h5 in barley-retardation of germination and delayed or reduced expression of associated enzymes. Similar enzyme changes were observed in developing seeds. The wheat lines with underexpressed Trx showed delayed preharvest sprouting when grown in the greenhouse or field without a decrease in final yield. Wheat with overexpressed Trx h5 showed changes commensurate with earlier in vitro work: increased solubility of disulfide proteins and lower allergenicity of the gliadin fraction. The results are further evidence that the level of Trx h in cereal endosperm determines fundamental properties as well as potential applications of the seed.

Fission yeast TOR complex 2 activates the AGC-family Gad8 kinase essential for stress resistance and cell cycle control.

Members of the mitogen-activated protein kinase (MAPK) subfamily responsive to environmental stress stimuli are known as SAPKs (stress-activated protein kinases), which are conserved from yeast to humans. In the fission yeast Schizosaccharomyces pombe, Spc1/Sty1 SAPK is activated by diverse forms of stress, such as osmostress, oxidative stress and heat shock, and induces gene expression through the Atf1 transcription factor. Sin1 (SAPK interacting protein 1) was originally isolated as a protein that interacts with Spc1, and its orthologs were also found in diverse eukaryotes. Here we report that Sin1 is not required for the stress gene expression regulated by Spc1 and Atf1, and that Sin1 is an essential component of TOR (target of rapamycin) complex 2 (TORC2). TORC2 is not essential for cell viability in S. pombe but plays important roles in cellular survival of stress conditions through phosphorylation and activation of an AGC-family protein kinase, Gad8. In addition, inactivation of Gad8 results in a synthetic growth defect with cdc25-22, a temperature-sensitive mutation of the Cdc25 phosphatase that activates Cdc2 kinase at G(2)/M. Gad8 also positively regulates expression of the CDK inhibitor gene rum1+, which is essential for cell cycle arrest in G(1) after nitrogen starvation. These results strongly suggest that the TORC2-Gad8 pathway has multiple physiological functions in cellular stress resistance and cell cycle progression at both G(1)/S and G(2)/M transitions.

The atopic dog as a model of peanut and tree nut food allergy.

BACKGROUND: Animal models are needed that mimic human IgE-mediated peanut and tree nut allergy. Atopic dogs have been previously used in a model of food allergy to cow's milk, beef, wheat, and soy, with the demonstration of specific IgE production and positive oral challenges similar to those seen in human subjects. OBJECTIVE: We sought to sensitize dogs to peanut, walnut, and Brazil nut and to assess whether sensitization is accompanied by clinical reactions and whether there is cross-reactivity among the different preparations. METHODS: Eleven dogs were sensitized subcutaneously by using an established protocol with 1 microg each of peanut, English walnut, or Brazil nut protein extracts in alum first at birth and then after modified live virus vaccinations at 3, 7, and 11 weeks of age. The dogs were sensitized to other allergens, including soy and either wheat or barley. Intradermal skin tests, IgE immunoblotting to nut proteins, and oral challenges were performed with ground nut preparations. RESULTS: At 6 months of age, the dogs' intradermal skin test responses were positive to the nut extracts. IgE immunoblotting to peanut, walnut, and Brazil nut showed strong recognition of proteins in the aqueous preparations. Each of the 4 peanut- and the 3 Brazil nut-sensitized dogs and 3 of the 4 walnut-sensitized dogs reacted on oral challenge with the corresponding primary immunogen at age 2 years. None of the peanut-sensitized dogs reacted clinically with walnut or Brazil nut challenges. One of the walnut-sensitized dogs had delayed (overnight) vomiting to Brazil nut. CONCLUSIONS: On the basis of measurements of the mean amount of allergen eliciting a skin test response in dogs, the hierarchy of reactivity by skin testing is similar to the clinical experience in human subjects (peanut > tree nuts > wheat > soy > barley). Cross-reactivity, which was not apparent between soy and peanut or tree nuts or between peanut and tree nuts, was slight between walnut and Brazil nut. The results give further support to the dog as a model of human food allergy.