Nicotiana benthamiana Methanol-Inducible Gene (MIG) 21 Encodes a Nucleolus-Localized Protein That Stimulates Viral Intercellular Transport and Downregulates Nuclear Import.

The mechanical damage of plant tissues leads to the activation of methanol production and its release into the atmosphere. The gaseous methanol or vapors emitted by the damaged plant induce resistance in neighboring intact plants to bacterial pathogens but create favorable conditions for viral infection spread. Among the Nicotiana benthamiana methanol-inducible genes (MIGs), most are associated with plant defense and intercellular transport. Here, we characterize NbMIG21, which encodes a 209 aa protein (NbMIG21p) that does not share any homology with annotated proteins. NbMIG21p was demonstrated to contain a nucleolus localization signal (NoLS). Colocalization studies with fibrillarin and coilin, nucleolus and Cajal body marker proteins, revealed that NbMIG21p is distributed among these subnuclear structures. Our results show that recombinant NbMIG21 possesses DNA-binding properties. Similar to a gaseous methanol effect, an increased NbMIG21 expression leads to downregulation of the nuclear import of proteins with nuclear localization signals (NLSs), as was demonstrated with the GFP-NLS model protein. Moreover, upregulated NbMIG21 expression facilitates tobacco mosaic virus (TMV) intercellular transport and reproduction. We identified an NbMIG21 promoter (PrMIG21) and showed that it is methanol sensitive; thus, the induction of NbMIG21 mRNA accumulation occurs at the level of transcription. Our findings suggest that methanol-activated NbMIG21 might participate in creating favorable conditions for viral reproduction and spread.

Nicotiana benthamiana Class 1 Reversibly Glycosylated Polypeptides Suppress Tobacco Mosaic Virus Infection.

Reversibly glycosylated polypeptides (RGPs) have been identified in many plant species and play an important role in cell wall formation, intercellular transport regulation, and plant-virus interactions. Most plants have several RGP genes with different expression patterns depending on the organ and developmental stage. Here, we report on four members of the RGP family in N. benthamiana. Based on a homology search, NbRGP1-3 and NbRGP5 were assigned to the class 1 and class 2 RGPs, respectively. We demonstrated that NbRGP1-3 and 5 mRNA accumulation increases significantly in response to tobacco mosaic virus (TMV) infection. Moreover, all identified class 1 NbRGPs (as distinct from NbRGP5) suppress TMV intercellular transport and replication in N. benthamiana. Elevated expression of NbRGP1-2 led to the stimulation of callose deposition at plasmodesmata, indicating that RGP-mediated TMV local spread could be affected via a callose-dependent mechanism. It was also demonstrated that NbRGP1 interacts with TMV movement protein (MP) in vitro and in vivo. Therefore, class 1 NbRGP1-2 play an antiviral role by impeding intercellular transport of the virus by affecting plasmodesmata callose and directly interacting with TMV MP, resulting in the reduced viral spread and replication.

A novel cellular factor of Nicotiana benthamiana susceptibility to tobamovirus infection.

Viral infection, which entails synthesis of viral proteins and active reproduction of the viral genome, effects significant changes in the functions of many intracellular systems in plants. Along with these processes, a virus has to suppress cellular defense to create favorable conditions for its successful systemic spread in a plant. The virus exploits various cellular factors of a permissive host modulating its metabolism as well as local and systemic transport of macromolecules and photoassimilates. The Nicotiana benthamiana stress-induced gene encoding Kunitz peptidase inhibitor-like protein (KPILP) has recently been shown to be involved in chloroplast retrograde signaling regulation and stimulation of intercellular transport of macromolecules. In this paper we demonstrate the key role of KPILP in the development of tobamovius infection. Systemic infection of N. benthamiana plants with tobacco mosaic virus (TMV) or the closely related crucifer-infecting tobamovirus (crTMV) induces a drastic increase in KPILP mRNA accumulation. KPILP knockdown significantly reduces the efficiency of TMV and crTMV intercellular transport and reproduction. Plants with KPILP silencing become partially resistant to tobamovirus infection. Therefore, KPILP could be regarded as a novel proviral factor in the development of TMV and crTMV infection in N. benthamiana plants.

Nicotiana benthamiana Kunitz peptidase inhibitor-like protein involved in chloroplast-to-nucleus regulatory pathway in plant-virus interaction.

Plant viruses use a variety of strategies to infect their host. During infection, viruses cause symptoms of varying severity, which are often associated with altered leaf pigmentation due to structural and functional damage to chloroplasts that are affected by viral proteins. Here we demonstrate that Nicotiana benthamiana Kunitz peptidase inhibitor-like protein (KPILP) gene is induced in response to potato virus X (PVX) infection. Using reverse genetic approach, we have demonstrated that KPILP downregulates expression of LHCB1 and LHCB2 genes of antenna light-harvesting complex proteins, HEMA1 gene encoding glutamyl-tRNA reductase, which participates in tetrapyrrole biosynthesis, and RBCS1A gene encoding RuBisCO small subunit isoform involved in the antiviral immune response. Thus, KPILP is a regulator of chloroplast retrograde signaling system during developing PVX infection. Moreover, KPILP was demonstrated to affect carbon partitioning: reduced glucose levels during PVX infection were associated with KPILP upregulation. Another KPILP function is associated with plasmodesmata permeability control. Its ability to stimulate intercellular transport of reporter 2xGFP molecules indicates that KPILP is a positive plasmodesmata regulator. Moreover, natural KPILP glycosylation is indispensable for manifestation of this function. During PVX infection KPILP increased expression leads to the reduction of plasmodesmata callose deposition. These results could indicate that KPILP affects plasmodesmata permeability via callose-dependent mechanism. Thus, virus entering a cell and starting reproduction triggers KPILP expression, which leads to downregulation of nuclear-encoded chloroplast genes associated with retrograde signaling, reduction in photoassimilates accumulation and increase in intercellular transport, creating favorable conditions for reproduction and spread of viral infection.

Enhanced Synthesis of Foreign Nuclear Protein Stimulates Viral Reproduction via the Induction of γ-Thionin Expression.

Plants are a promising platform for recombinant protein production. Here we propose a novel approach to increase the level of viral vector-mediated recombinant protein synthesis. This approach is based on the hypothesis that antiviral protection is weakened during the antibacterial cellular response. We suggested that introduced to the cell foreign nuclear localized proteins, including effectors such as bacterial nucleomodulins, can interfere with the import of cellular nuclear proteins and launch antibacterial defense reactions, creating favorable conditions for cytoplasmic virus reproduction. Here, we performed synthesis of an artificial nuclear protein-red fluorescent protein (mRFP) fused with a nuclear localization sequence (NLS)-in plant cells as a mimetic of a bacterial effector. Superproduction of mRFP:NLS induced Nicotiana benthamiana γ-thionin (NbγThio) mRNA accumulation. Both NLS-containing protein synthesis and increased NbγThio expression stimulated reproduction of the viral vector based on the genome of crucifer-infecting tobacco mosaic virus (crTMV) in N. benthamiana leaves. We isolated the NbγThio gene promoter (PrγThio) and showed that PrγThio activity sharply increased in response to massive synthesis of GFP fused with NLS. We conclude that NLS-induced PrγThio activation and increased accumulation of Nbγthio mRNA led to the stimulation of GFP expression from crTMV: GFP vector in the transient expression system.

Approaches to Formaldehyde Measurement: From Liquid Biological Samples to Cells and Organisms.

Formaldehyde (FA) is the simplest aldehyde present both in the environment and in living organisms. FA is an extremely reactive compound capable of protein crosslinking and DNA damage. For a long time, FA was considered a "biochemical waste" and a by-product of normal cellular metabolism, but in recent decades the picture has changed. As a result, the need arose for novel instruments and approaches to monitor and measure not only environmental FA in water, cosmetics, and household products, but also in food, beverages and biological samples including cells and even organisms. Despite numerous protocols being developed for in vitro and in cellulo FA assessment, many of them have remained at the "proof-of-concept" stage. We analyze the suitability of different methods developed for non-biological objects, and present an overview of the recently developed approaches, including chemically-synthesized probes and genetically encoded FA-sensors for in cellulo and in vivo FA monitoring. We also discuss the prospects of classical methods such as chromatography and spectrophotometry, and how they have been adapted in response to the demand for precise, selective and highly sensitive evaluation of FA concentration fluctuations in biological samples. The main objectives of this review is to summarize data on the main approaches for FA content measurement in liquid biological samples, pointing out the advantages and disadvantages of each method; to report the progress in development of novel molecules suitable for application in living systems; and, finally, to discuss genetically encoded FA-sensors based on existing natural biological FA-responsive elements.

The Tobamoviral Movement Protein: A "Conditioner" to Create a Favorable Environment for Intercellular Spread of Infection.

During their evolution, viruses acquired genes encoding movement protein(s) (MPs) that mediate the intracellular transport of viral genetic material to plasmodesmata (Pd) and initiate the mechanisms leading to the increase in plasmodesmal permeability. Although the current view on the role of the viral MPs was primarily formed through studies on tobacco mosaic virus (TMV), the function of its MP has not been fully elucidated. Given the intercellular movement of MPs independent of genomic viral RNA (vRNA), this characteristic may induce favorable conditions ahead of the infection front for the accelerated movement of the vRNA (i.e. the MP plays a role as a "conditioner" of viral intercellular spread). This idea is supported by (a) the synthesis of MP from genomic vRNA early in infection, (b) the Pd opening and the MP transfer to neighboring cells without formation of the viral replication complex (VRC), and (c) the MP-mediated movement of VRCs beyond the primary infected cell. Here, we will consider findings that favor the TMV MP as a "conditioner" of enhanced intercellular virus movement. In addition, we will discuss the mechanism by which TMV MP opens Pd for extraordinary transport of macromolecules. Although there is no evidence showing direct effects of TMV MP on Pd leading to their dilatation, recent findings indicate that MPs exert their influence indirectly by modulating Pd external and structural macromolecules such as callose and Pd-associated proteins. In explaining this phenomenon, we will propose a mechanism for TMV MP functioning as a conditioner for virus movement.

Plasmodesmata Conductivity Regulation: A Mechanistic Model.

Plant cells form a multicellular symplast via cytoplasmic bridges called plasmodesmata (Pd) and the endoplasmic reticulum (ER) that crosses almost all plant tissues. The Pd proteome is mainly represented by secreted Pd-associated proteins (PdAPs), the repertoire of which quickly adapts to environmental conditions and responds to biotic and abiotic stresses. Although the important role of Pd in stress-induced reactions is universally recognized, the mechanisms of Pd control are still not fully understood. The negative role of callose in Pd permeability has been convincingly confirmed experimentally, yet the roles of cytoskeletal elements and many PdAPs remain unclear. Here, we discuss the contribution of each protein component to Pd control. Based on known data, we offer mechanistic models of mature leaf Pd regulation in response to stressful effects.

The biological activity of bispecific trastuzumab/pertuzumab plant biosimilars may be drastically boosted by disulfiram increasing formaldehyde accumulation in cancer cells.

Studies of breast cancer therapy have examined the improvement of bispecific trastuzumab/pertuzumab antibodies interacting simultaneously with two different epitopes of the human epidermal growth factor receptor 2 (HER2). Here, we describe the creation and production of plant-made bispecific antibodies based on trastuzumab and pertuzumab plant biosimilars (bi-TPB-PPB). Using surface plasmon resonance analysis of bi-TPB-PPB antibodies binding with the HER2 extracellular domain, we showed that the obtained Kd values were within the limits accepted for modified trastuzumab and pertuzumab. Despite the ability of bi-TPB-PPB antibodies to bind to Fcγ receptor IIIa and HER2 oncoprotein on the cell surface, a proliferation inhibition assay did not reveal any effect until α1,3-fucose and ß1,2-xylose in the Asn297-linked glycan were removed. Another approach to activating bi-TPB-PPB may be associated with the use of disulfiram (DSF) a known aldehyde dehydrogenase 2 (ALDH2) inhibitor. We found that disulfiram is capable of killing breast cancer cells with simultaneous formaldehyde accumulation. Furthermore, we investigated the capacity of DSF to act as an adjuvant for bi-TPB-PPB antibodies. Although the content of ALDH2 mRNA was decreased after BT-474 cell treatment with antibodies, we only observed cell proliferation inhibiting activity of bi-TPB-PPB in the presence of disulfiram. We concluded that disulfiram can serve as a booster and adjuvant for anticancer immunotherapy.

Plant-Made Antibodies: Properties and Therapeutic Applications.

BACKGROUND: A cost-effective plant platform for therapeutic monoclonal antibody production is both flexible and scalable. Plant cells have mechanisms for protein synthesis and posttranslational modification, including glycosylation, similar to those in animal cells. However, plants produce less complex and diverse Asn-attached glycans compared to animal cells and contain plant-specific residues. Nevertheless, plant-made antibodies (PMAbs) could be advantageous compared to those produced in animal cells due to the absence of a risk of contamination from nucleic acids or proteins of animal origin. OBJECTIVE: In this review, the various platforms of PMAbs production are described, and the widely used transient expression system based on Agrobacterium-mediated delivery of genetic material into plant cells is discussed in detail. RESULTS: We examined the features of and approaches to humanizing the Asn-linked glycan of PMAbs. The prospects for PMAbs in the prevention and treatment of human infectious diseases have been illustrated by promising results with PMAbs against human immunodeficiency virus, rotavirus infection, human respiratory syncytial virus, rabies, anthrax and Ebola virus. The pre-clinical and clinical trials of PMAbs against different types of cancer, including lymphoma and breast cancer, are addressed. CONCLUSION: PMAb biosafety assessments in patients suggest that it has no side effects, although this does not completely remove concerns about the potential immunogenicity of some plant glycans in humans. Several PMAbs at various developmental stages have been proposed. Promise for the clinical use of PMAbs is aimed at the treatment of viral and bacterial infections as well as in anti-cancer treatment.

Methanol in Plant Life.

Until recently, plant-emitted methanol was considered a biochemical by-product, but studies in the last decade have revealed its role as a signal molecule in plant-plant and plant-animal communication. Moreover, methanol participates in metabolic biochemical processes during growth and development. The purpose of this review is to determine the impact of methanol on the growth and immunity of plants. Plants generate methanol in the reaction of the demethylation of macromolecules including DNA and proteins, but the main source of plant-derived methanol is cell wall pectins, which are demethylesterified by pectin methylesterases (PMEs). Methanol emissions increase in response to mechanical wounding or other stresses due to damage of the cell wall, which is the main source of methanol production. Gaseous methanol from the wounded plant induces defense reactions in intact leaves of the same and neighboring plants, activating so-called methanol-inducible genes (MIGs) that regulate plant resistance to biotic and abiotic factors. Since PMEs are the key enzymes in methanol production, their expression increases in response to wounding, but after elimination of the stress factor effects, the plant cell should return to the original state. The amount of functional PMEs in the cell is strictly regulated at both the gene and protein levels. There is negative feedback between one of the MIGs, aldose epimerase-like protein, and PME gene transcription; moreover, the enzymatic activity of PMEs is modulated and controlled by PME inhibitors (PMEIs), which are also induced in response to pathogenic attack.

Human Endogenous Formaldehyde as an Anticancer Metabolite: Its Oxidation Downregulation May Be a Means of Improving Therapy.

Malignant cells are characterized by an increased content of endogenous formaldehyde formed as a by-product of biosynthetic processes. Accumulation of formaldehyde in cancer cells is combined with activation of the processes of cellular formaldehyde clearance. These mechanisms include increased ALDH and suppressed ADH5/FDH activity, which oncologists consider poor and favorable prognostic markers, respectively. Here, the sources and regulation of formaldehyde metabolism in cancer cells are reviewed. The authors also analyze the participation of oncoproteins such as fibulins, FGFR1, HER2/neu, FBI-1, and MUC1-C in the control of genes related to formaldehyde metabolism, suggesting the existence of two mutually exclusive processes in cancer cells: 1) production and 2) oxidation and elimination of formaldehyde from the cell. The authors hypothesize that the study of the anticancer properties of disulfiram and alpha lipoic acid - which affect the balance of formaldehyde in the body - may serve as the basis of future anticancer therapy.

The Antioxidant Cofactor Alpha-Lipoic Acid May Control Endogenous Formaldehyde Metabolism in Mammals.

The healthy human body contains small amounts of metabolic formaldehyde (FA) that mainly results from methanol oxidation by pectin methylesterase, which is active in a vegetable diet and in the gastrointestinal microbiome. With age, the ability to maintain a low level of FA decreases, which increases the risk of Alzheimer's disease and dementia. It has been shown that 1,2-dithiolane-3-pentanoic acid or alpha lipoic acid (ALA), a naturally occurring dithiol and antioxidant cofactor of mitochondrial α-ketoacid dehydrogenases, increases glutathione (GSH) content and FA metabolism by mitochondrial aldehyde dehydrogenase 2 (ALDH2) thus manifests a therapeutic potential beyond its antioxidant property. We suggested that ALA can contribute to a decrease in the FA content of mammals by acting on ALDH2 expression. To test this assumption, we administered ALA in mice in order to examine the effect on FA metabolism and collected blood samples for the measurement of FA. Our data revealed that ALA efficiently eliminated FA in mice. Without affecting the specific activity of FA-metabolizing enzymes (ADH1, ALDH2, and ADH5), ALA increased the GSH content in the brain and up-regulated the expression of the FA-metabolizing ALDH2 gene in the brain, particularly in the hippocampus, but did not impact its expression in the liver in vivo or in rat liver isolated from the rest of the body. After ALA administration in mice and in accordance with the increased content of brain ALDH2 mRNA, we detected increased ALDH2 activity in brain homogenates. We hypothesized that the beneficial effects of ALA on patients with Alzheimer's disease may be associated with accelerated ALDH2-mediated FA detoxification and clearance.

The Intergenic Interplay between Aldose 1-Epimerase-Like Protein and Pectin Methylesterase in Abiotic and Biotic Stress Control.

The mechanical damage that often precedes the penetration of a leaf by a pathogen promotes the activation of pectin methylesterase (PME); the activation of PME leads to the emission of methanol, resulting in a "priming" effect on intact leaves, which is accompanied by an increased sensitivity to Tobacco mosaic virus (TMV) and resistance to bacteria. In this study, we revealed that mRNA levels of the methanol-inducible gene encoding Nicotiana benthamiana aldose 1-epimerase-like protein (NbAELP) in the leaves of intact plants are very low compared with roots. However, stress and pathogen attack increased the accumulation of the NbAELP mRNA in the leaves. Using transiently transformed plants, we obtained data to support the mechanism underlying AELP/PME-related negative feedback The insertion of the NbAELP promoter sequence (proNbAELP) into the N. benthamiana genome resulted in the co-suppression of the natural NbAELP gene expression, accompanied by a reduction in the NbAELP mRNA content and increased PME synthesis. Knockdown of NbAELP resulted in high activity of PME in the cell wall and a decrease in the leaf glucose level, creating unfavorable conditions for Agrobacterium tumefaciens reproduction in injected leaves. Our results showed that NbAELP is capable of binding the TMV movement protein (MPTMV) in vitro and is likely to affect the cellular nucleocytoplasmic transport, which may explain the sensitivity of NbAELP knockdown plants to TMV. Although NbAELP was primarily detected in the cell wall, the influence of this protein on cellular PME mRNA levels might be associated with reduced transcriptional activity of the PME gene in the nucleus. To confirm this hypothesis, we isolated the N. tabacum PME gene promoter (proNtPME) and showed the inhibition of proNtPME-directed GFP and GUS expression in leaves when co-agroinjected with the NbAELP-encoding plasmid. We hypothesized that plant wounding and/or pathogen attack lead to PME activation and increased methanol emission, followed by increased NbAELP expression, which results in reversion of PME mRNA level and methanol emission to levels found in the intact plant.

Tobamovirus 3'-Terminal Gene Overlap May be a Mechanism for within-Host Fitness Improvement.

Overlapping genes (OGs) are a universal phenomenon in all kingdoms, and viruses display a high content of OGs combined with a high rate of evolution. It is believed that the mechanism of gene overlap is based on overprinting of an existing gene. OGs help virus genes compress a maximum amount of information into short sequences, conferring viral proteins with novel features and thereby increasing their within-host fitness. Analysis of tobamovirus 3'-terminal genes reveals at least two modes of OG organization and mechanisms of interaction with the host. Originally isolated from Solanaceae species, viruses (referred to as Solanaceae-infecting) such as tobacco mosaic virus do not show 3'-terminal overlap between movement protein (MP) and coat protein (CP) genes but do contain open reading frame 6 (ORF6), which overlaps with both genes. Conversely, tobamoviruses, originally isolated from Brassicaceae species (referred to as Brassicaceae-infecting) and also able to infect Solanaceae plants, have no ORF6 but are characterized by overlapping MP and CP genes. Our analysis showed that the MP/CP overlap of Brassicaceae-infecting tobamoviruses results in the following: (i) genome compression and strengthening of subgenomic promoters; (ii) CP gene early expression directly from genomic and dicistronic MP subgenomic mRNA using an internal ribosome entry site (IRES) and a stable hairpin structure in the overlapping region; (iii) loss of ORF6, which influences the symptomatology of Solanaceae-infecting tobamoviruses; and (iv) acquisition of an IRES polypurine-rich region encoding an MP nuclear localization signal. We believe that MP/CP gene overlap may constitute a mechanism for host range expansion and virus adjustment to Brassicaceae plants.

An Alternative Nested Reading Frame May Participate in the Stress-Dependent Expression of a Plant Gene.

Although plants as sessile organisms are affected by a variety of stressors in the field, the stress factors for the above-ground and underground parts of the plant and their gene expression profiles are not the same. Here, we investigated NbKPILP, a gene encoding a new member of the ubiquitous, pathogenesis-related Kunitz peptidase inhibitor (KPI)-like protein family, that we discovered in the genome of Nicotiana benthamiana and other representatives of the Solanaceae family. The NbKPILP gene encodes a protein that has all the structural elements characteristic of KPI but in contrast to the proven A. thaliana KPI (AtKPI), it does not inhibit serine peptidases. Unlike roots, NbKPILP mRNA and its corresponding protein were not detected in intact leaves, but abiotic and biotic stressors drastically affected NbKPILP mRNA accumulation. In search of the causes of suppressed NbKPILP mRNA accumulation in leaves, we found that the NbKPILP gene is "matryoshka," containing an alternative nested reading frame (ANRF) encoding a 53-amino acid (aa) polypeptide (53aa-ANRF) which has an amphipathic helix (AH). We confirmed ANRF expression experimentally. A vector containing a GFP-encoding sequence was inserted into the NbKPILP gene in frame with 53aa-ANRF, resulting in a 53aa-GFP fused protein that localized in the membrane fraction of cells. Using the 5'-RACE approach, we have shown that the expression of ANRF was not explained by the existence of a cryptic promoter within the NbKPILP gene but was controlled by the maternal NbKPILP mRNA. We found that insertion of mutations destroying the 53aa-ANRF AH resulted in more than a two-fold increase of the NbKPILP mRNA level. The NbKPILP gene represents the first example of ANRF functioning as a repressor of a maternal gene in an intact plant. We proposed a model where the stress influencing the translation initiation promotes the accumulation of NbKPILP and its mRNA in leaves.

Metabolic methanol: molecular pathways and physiological roles.

Methanol has been historically considered an exogenous product that leads only to pathological changes in the human body when consumed. However, in normal, healthy individuals, methanol and its short-lived oxidized product, formaldehyde, are naturally occurring compounds whose functions and origins have received limited attention. There are several sources of human physiological methanol. Fruits, vegetables, and alcoholic beverages are likely the main sources of exogenous methanol in the healthy human body. Metabolic methanol may occur as a result of fermentation by gut bacteria and metabolic processes involving S-adenosyl methionine. Regardless of its source, low levels of methanol in the body are maintained by physiological and metabolic clearance mechanisms. Although human blood contains small amounts of methanol and formaldehyde, the content of these molecules increases sharply after receiving even methanol-free ethanol, indicating an endogenous source of the metabolic methanol present at low levels in the blood regulated by a cluster of genes. Recent studies of the pathogenesis of neurological disorders indicate metabolic formaldehyde as a putative causative agent. The detection of increased formaldehyde content in the blood of both neurological patients and the elderly indicates the important role of genetic and biochemical mechanisms of maintaining low levels of methanol and formaldehyde.

Dietary methanol regulates human gene activity.

Methanol (MeOH) is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of MeOH to formaldehyde (FA), which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC) from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimer's disease (AD). There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, GAPDH and SNX27, and genes revealed in this study, including MME, SORL1, DDIT4, HBA and HBB. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling.

Cell wall methanol as a signal in plant immunity.

Cell wall pectin forms a matrix around the cellulose-xyloglucan network that is composed of rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan (HG), a major pectic polymer consisting of α-1,4-linked galacturonic acids. HG is secreted in a highly methyl-esterified form and selectively de-methyl-esterified by pectin methylesterases (PMEs) during cell growth and pathogen attack. The mechanical damage that often precedes the penetration of the leaf by a pathogen promotes the activation of PME, which in turn leads to the emission of methanol (MeOH), an abundant volatile organic compound, which is quickly perceived by the intact leaves of the damaged plant, and the neighboring plants. The exposure to MeOH may result in a "priming" effect on intact leaves, setting the stage for the within-plant, and neighboring plant immunity. The emission of MeOH by a wounded plant enhances the resistance of the non-wounded, neighboring "receiver" plants to bacterial pathogens and promotes cell-to-cell communication that facilitates the spread of viruses in neighboring plants.

Endogenous methanol regulates mammalian gene activity.

We recently showed that methanol emitted by wounded plants might function as a signaling molecule for plant-to-plant and plant-to-animal communications. In mammals, methanol is considered a poison because the enzyme alcohol dehydrogenase (ADH) converts methanol into toxic formaldehyde. However, the detection of methanol in the blood and exhaled air of healthy volunteers suggests that methanol may be a chemical with specific functions rather than a metabolic waste product. Using a genome-wide analysis of the mouse brain, we demonstrated that an increase in blood methanol concentration led to a change in the accumulation of mRNAs from genes primarily involved in detoxification processes and regulation of the alcohol/aldehyde dehydrogenases gene cluster. To test the role of ADH in the maintenance of low methanol concentration in the plasma, we used the specific ADH inhibitor 4-methylpyrazole (4-MP) and showed that intraperitoneal administration of 4-MP resulted in a significant increase in the plasma methanol, ethanol and formaldehyde concentrations. Removal of the intestine significantly decreased the rate of methanol addition to the plasma and suggested that the gut flora may be involved in the endogenous production of methanol. ADH in the liver was identified as the main enzyme for metabolizing methanol because an increase in the methanol and ethanol contents in the liver homogenate was observed after 4-MP administration into the portal vein. Liver mRNA quantification showed changes in the accumulation of mRNAs from genes involved in cell signalling and detoxification processes. We hypothesized that endogenous methanol acts as a regulator of homeostasis by controlling the mRNA synthesis.