MicroRNA-141-regulated KLK10 and TNFSF-15 gene expression in hepatoblastoma cells as a novel mechanism in liver carcinogenesis.

Liver cancer is one of the most pivotal global health problems, leading hepatocellular carcinoma (HCC) with a significant increase in cases worldwide. The role of non-coding-RNA in cancer proliferation and carcinogenesis has attracted much attention in the last decade; however, microRNAs (miRNAs), as non-coding RNA, are considered master mediators in various cancer progressions. Yet the role of miR-141 as a modulator for specific cellular processes in liver cancer cell proliferation is still unclear. This study identified the role of miR-141 and its potential functions in liver carcinogenesis. The level of miR-141 in HepG2 and HuH7 cells was assessed using quantitative real-time PCR (qRT-PCR) and compared with its expression in normal hepatocytes. A new miR-141 construct has been performed in a CMV promoter vector tagged with GFP. Using microarray analysis, we identified the potentially regulated genes by miR-141 in transfected HepG2 cells. The protein profile of the kallikrein-related peptidase 10 (KLK10) and tumor necrosis factor TNFSF-15 was investigated in HepG2 cells transfected with either an inhibitor, antagonist miR-141, or miR-141 overexpression vector using immunoblotting and flow cytometry assay. Finally, ELISA assay has been used to monitor the produced inflammatory cytokines from transfected HepG2 cells. Our findings showed that the expression of miR-141 significantly increased in HepG2 and HuH7 cells compared to the normal hepatocytes. Transfection of HepG2 cells with an inhibitor, antagonist miR-141, showed a significant reduction of HepG2 cell viability, unlike the transfection of miR-141 overexpression vector. The microarray data of HepG2 cells overexpressed miR-141 provided a hundred downregulated genes, including KLK10 and TNFSF-15. Furthermore, the expression profile of KLK10 and TNFSF-15 markedly depleted in HepG2 cells transfected with miR-141 overexpression accompanied by a decreasing level of interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α), indicating the role of miR-141 in HepG2 cell proliferation and programmed cell death. Interestingly, the experimental rats with liver cancer induced by Diethylnitrosamine injection further confirmed the upregulation of miR-141 level, IL-10, and TNF-α and the disturbance in KLK10 and TNFSF-15 gene expression compared with their expression in normal rats. The in-silico online tools, IntaRNA and miRWalk were used to confirm the direct interaction and potential binding sites between miR-141 and identified genes. Thus, the seeding regions of potential targeted sequences was cloned upstream of luciferase reporter gene in pGL3 control vector. Interestingly, the luciferase activities of constructed vectors were significantly decreased in HepG2 cells pre-transfected with miR-141 overexpression vector, while increasing in cells pre-transfected with miR-141 specific inhibitor. In summary, these data suggest the crucial role of miR-141 in liver cancer development via targeting KLK10 and TNFSF-15 and provide miR-141 as an attractive candidate in liver cancer treatment and protection.

Supplying rats with halfa-bar and liquorice extracts ameliorate doxorubicin-induced nephrotic syndrome.

In the current work, we aimed to evaluate the protective effects of liquorice and halfa-bar extract against doxorubicin (DOX)-induced nephritic syndrome (NS) in rats. Twenty albino male rats were intraperitoneally injected with 50 mg/kg of DOX. The injected rats were supplied daily with 400 mg/kg of liquorice, halfa-bar extract, or their combination for 2 weeks. Our findings confirmed the induction of NS in rats indicated by alteration in Bowman's space, damaged in glomerular capsules, and tubules. Moreover, the levels of produced tumour necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8) were increased, accompanied by decreasing levels of IL-4 and IL-10. Supplement NS-rats with liquorice and halfa-bar extracts restored the glomerular and tubules damage and adjusted the level of produced TNF-α and IL-8. Interestingly, both extracts can stimulate the expression profile of small proline-rich protein 2 F (sprr2f) and metalloproteinase-10 (MMP-10), which are responsible for repairing and regeneration mechanisms of renal syndromes.

LGR6 is a prognostic biomarker for less differentiated tumors in lymph nodes of colon cancer patients.

Introduction: The aim was to investigate whether the stem cell marker LGR6 has prognostic value in colon cancer, alone or in combination with the prognostic biomarkers CEA and CXCL16. Methods: LGR6 mRNA levels were determined in 370 half lymph nodes of 121 colon cancer patients. Ability to predict relapse after curative surgery was estimated by Kaplan-Meier survival model and Cox regression analyses. Results: Patients with high LGR6 levels [LGR6(+)] had a decreased mean survival time of 11 months at 5-year follow-up and 47 months at 12-year follow-up, respectively, with hazard ratios of 3.2 and 2.8. LGR6 mRNA analysis added prognostic value to CEA and CXCL16 mRNA analysis. In the poor prognosis groups CEA(+) and CXCL16(+), further division was achieved by LGR6 analysis. LGR6(+) patients had a very poor prognosis. LGR6 also identified a small number of CEA(-), TNM stage I patients who relapsed suggesting stem cell origin of these tumors. LGR6 and LGR5 levels correlated strongly in lymph nodes of stage I and IV patients but not in stage II patients, suggesting that these stem cell markers are differentially regulated. Conclusion: This study highlights LGR6 as a useful prognostic biomarker independently and in combination with CEA, CXCL16 or LGR5 identifying different risk groups.

Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication.

The antiviral response against influenza A virus (IAV) infection includes the induction of the interferon (IFN) signaling pathway, including activation of the STATs protein family. Subsequently, antiviral myxovirus resistance (MxA) protein and other interferon-stimulated genes control virus replication; however, the molecular interaction of viral-mediated IFN signaling needs more investigation. Host microRNAs (miRNAs) are small non-coding molecules that posttranscriptionally regulate gene expression. Here, we sought to investigate the possible involvement of miR-141 in IAV-mediated IFN signaling. Accordingly, the microarray analysis of A549 cells transfected with precursor miR-141 (pre-miR-141) was used to capture the potentially regulated genes in response to miR-141 overexpression independent of IAV infection. The downregulation of targeted genes by miR-141, in addition to viral gene expression, was investigated by quantitative real-time PCR, western blot analysis, and flow cytometric assay. Our findings showed a significant upregulation of miR-141 in infected A549 cells with different strains of IAV. Notably, IAV replication was firmly interrupted in cells transfected with the miR-141 inhibitor. While its replication significantly increased in cells transfected with pre-miR-141 confirming the crucial role of miRNA-141 in supporting virus replication. Interestingly, the microarray data of miR-141 transduced A549 cells showed many downregulated genes, including MxA, STAT3, IFI27, and LAMP3. The expression profile of MxA and STAT3 was significantly depleted in infected cells transfected with the pre-miR-141, while their expression was restored in infected cells transfected with the miR-141 inhibitor. Unlike interleukin 6 (IL-6), the production of IFN-ß markedly decreased in infected cells that transfected with pre-miR-141, while it significantly elevated in infected cells transfected with miR-141 inhibitor. These data provide evidence for the crucial role of miR-141 in regulating the antiviral gene expression induced by IFN and IL-6 signaling during IAV infection to ensure virus replication.

Purification, biochemical characterization, and molecular cloning of cellulase from Bacillus licheniformis strain Z9 isolated from soil.

BACKGROUND: Cellulose is the most prevalent biomass and renewable energy source in nature. The hydrolysis of cellulosic biomass to glucose units is essential for the economic exploitation of this natural resource. Cellulase enzyme, which is largely generated by bacteria and fungus, is commonly used to degrade cellulose. Cellulases are used in a variety of industries, including bioethanol manufacturing, textiles, detergents, drugs, food, and paper. As part of our quest to find an efficient biocatalyst for the hydrolysis of cellulosic biomass, we describe the amplification, cloning, and sequencing of cellulase (cel9z) from Bacillus licheniformis strain Z9, as well as the characterization of the resulting enzyme. RESULTS: Cellulase was partially purified from B. licheniformis strain Z9 using (NH4)2SO4 precipitation and Sephadex G-100 gel column chromatography with 356.5 U/mg specific activity, 2.1-purification fold, and 3.07 % yield. The nucleotide sequence of the cellulase gene was deposited to the GenBank, B. licheniformis strain Z9 cellulase (cel9z) gene, under accession number MK814929. This corresponds to 1453 nucleotides gene and encodes for a protein composed of 484 amino acids. Comparison of deduced amino acids sequence to other related cellulases showed that the enzyme cel9z can be classified as a glycoside hydrolase family 9. SDS-PAGE analysis of the purified enzyme revealed that the molecular mass was 54.5 kDa. The optimal enzyme activity was observed at pH 7.4 and 30 °C. The enzyme was found to be strongly inhibited by Mg2+ and Na+, whereas strongly activated by Fe3+, Cu2+, and Ca2+. CONCLUSIONS: B. licheniformis strain Z9 and its cellulase gene can be further utilized for recombinant production of cellulases for industrial application.

Metabolomic Profiling of Nicotiana Spp. Nectars Indicate That Pollinator Feeding Preference Is a Stronger Determinant Than Plant Phylogenetics in Shaping Nectar Diversity.

Floral nectar is a rich secretion produced by the nectary gland and is offered as reward to attract pollinators leading to improved seed set. Nectars are composed of a complex mixture of sugars, amino acids, proteins, vitamins, lipids, organic and inorganic acids. This composition is influenced by several factors, including floral morphology, mechanism of nectar secretion, time of flowering, and visitation by pollinators. The objective of this study was to determine the contributions of flowering time, plant phylogeny, and pollinator selection on nectar composition in Nicotiana. The main classes of nectar metabolites (sugars and amino acids) were quantified using gas chromatography/mass spectrometric analytical platforms to identify differences among fifteen Nicotiana species representing day- and night-flowering plants from ten sections of the genus that are visited by five different primary pollinators. The nectar metabolomes of different Nicotiana species can predict the feeding preferences of the target pollinator(s) of each species, and the nectar sugars (i.e., glucose, fructose, and sucrose) are a distinguishing feature of Nicotiana species phylogeny. Moreover, comparative statistical analysis indicate that pollinators are a stronger determinant of nectar composition than plant phylogeny.

Interruption of Autophagosome Formation in Cardiovascular Disease, an Evidence for Protective Response of Autophagy.

BACKGROUND: A heart attack occurs when coronary artery blockage interrupts the blood supply to the heart such as is seen in cardiovascular disease (CVD). Importantly, autophagy is commonly regarded as a host defense mechanism against microbial invaders. METHODS: A total of 50 blood samples were obtained from cardiovascular (CV) patients in addition to 30 samples that were obtained from healthy individuals and served as controls. Macrophages were isolated in vitro and propagated from the blood samples. Autophagosome formation, cytokine secretion, and apolipoprotein B (ApoB) gene expression were monitored in patient samples and their derived macrophages. RESULTS: The results showed that autophagy-related (Atg) LC3 and Atg5 genes were significantly down-regulated in all samples obtained from CV patients. Furthermore, the relative gene expression of ApoB, which plays the major role in lipoprotein metabolism, was significantly increased in CV patients. Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) levels were increased in these blood samples. Interestingly, targeting of ApoB by small interference RNA (siRNA) reduced the production levels of low-density lipoprotein (LDL), IL-6 and TNF-α in patient-derived macrophages. Further, treatment of patient-derived macrophages with rapamycin, an autophagy inducer agent, successfully regulated the production of LDL, IL-6, TNF-α, and ApoB expression via activation of autophagosome formation. CONCLUSION: The current data reveal the potential disturbance of autophagy in CV patients that accompanied ApoB over-expression. Furthermore, our findings provide evidence for the protective role of autophagy in accumulation of pro-inflammatory cytokines and intracellular LDL degradation in CV patient-derived macrophages.

LC-MS/MS based comparative proteomics of floral nectars reveal different mechanisms involved in floral defense of Nicotiana spp., Petunia hybrida and Datura stramonium.

Tobacco floral nectar (FN) is a biological fluid produced by nectaries composed of sugars, amino acids and proteins called nectarins, involved in the floral defense. FN provides an ideal source of nutrients for microorganisms. Understanding the role of nectar proteins is essential to predict impacts in microbial growth, composition and plants-pollinators interactions. Using LC-MS/MS-based comparative proteomic analysis we identified 22 proteins from P. hybrida, 35 proteins from D. stramonium, and 144 proteins from 23 species of Nicotiana. The data are available at ProteomeXchance (PXD014760). GO analysis and secretory signal prediction demonstrated that defense/stress was the largest group of proteins in the genus Nicotiana. The Nicotiana spp. proteome consisted of 105 exclusive proteins such as lipid transfer proteins (LTPs), Nectar Redox Cycle proteins, proteases inhibitors, and PR-proteins. Analysis by taxonomic sections demonstrated that LTPs were most abundant in Undulatae and Noctiflora, while nectarins were more abundant in Rusticae, Suaveolens, Polydicliae, and Alata sections. Peroxidases (Pox) and chitinases (Chit) were exclusive to P. hybrida, while D. stramonium had only seven unique proteins. Biochemical analysis confirmed these differences. These findings support the hypothesis that, although conserved, there is differential abundance of proteins related to defense/stress which may impact the mechanisms of floral defense. SIGNIFICANCE: This study represents a comparative proteomic analysis of floral nectars of the Nicotiana spp. with two correlated Solanaceous species. Significant differences were identified between the proteome of taxonomic sections providing relevant insights into the group of proteins related to defense/stress associated with Nectar Redox Cycle, antimicrobial proteins and signaling pathways. The activity of FNs proteins is suggested impact the microbial growth. The knowledge about these proteomes provides significant insights into the diversity of proteins secreted in the nectars and the array of mechanisms used by Nicotiana spp. in its floral defense.

Association of C3435T, C1236T and C4125A Polymorphisms of the MDR-1 Gene in Egyptian Children with Acute Lymphoblastic Leukaemia

Background: P-glycoprotein (P-gp), a membrane transporter encoded by the multidrug resistance-1 (MDR1) gene, influences pharmacokinetics and metabolism of anticancer drugs and contributes to multidrug resistance phenotype in acute lymphoblastic leukemia (ALL). Genetic variation ofMDR1 in ALL patients is increasingly recognized as a factor influencing response to treatment. Aim: To investigate the possible role of MDR-1 gene polymorphisms (C3435T, C1236T and C4125A) as risk factors for the development and clinical outcome of ALL in Egyptian children. Materials and Methods: Genotyping of MDR-1 C3435T, C1236T and C4125A single nucleotide polymorphisms (SNPs) was accomplished using a polymerase chain reaction­restriction fragment length polymorphism (RFLP-PCR) assay with 120 childhood ALL patients and 100 healthy controls. Results: Homozygous T with the C3435T SNP showed a protective effect as compared to homozygous C (OR=0.748) while heterozygous CT correlated with a poor outcome (high risk, drug unresponsiveness, relapse and high percentage of death). Additionally, the T allele of the C1236T SNP showed a significant relation with ALL risk (OR=1.6). However, there were no significant differences in the genotype and allele frequencies of MDR-1 SNPs between patients and controls. Only one genotype (CC) and one allele of MDR-1 (C4125A) were seen. Neither CA/AA genotypes nor A alleles were present in ALL patients and normal controls. TC was the predominant haplotype in both groups, while CT proved to be minor. The cumulative incidence of relapse was higher with the CC genotype of C1236T as compared with TT. Conclusion: From our preliminary data, the CT genotype of C3435T is associated with a poor ALL outcome while the CC genotype of C1236T is related with an increased incidence of relapse. Although our results provide assistance for oncologist choice of individual therapeutic strategy taking the patient genetic repertoire into consideration, further investigations with larger sample size should be conducted to validate our results.

Nectar Analysis Throughout the Genus Nicotiana Suggests Conserved Mechanisms of Nectar Production and Biochemical Action.

We have evaluated the floral nectars of nine species from different sections of the genus Nicotiana. These nine species effectively cover the genus. We found that the nectary glands from these different species showed similar developmental regulation with swelling of nectaries during the first half of development and a distinct color change in the nectary gland as development approaches anthesis. When we examined the composition of the nectar from these nine different species we found that they were similar in content. Carbohydrate compositions of these various nectars varied between these species with N. bonariensis showing the highest and N. sylvestris lowest level of sugars. Based upon the amount of carbohydrates, the nectars fell into two groups. We found that hydrogen peroxide accumulated in the nectars of each of these species. While all species showed the presence of hydrogen peroxide in nectar, the quantitative amounts of hydrogen peroxide which was very high in N. rustica and N. bonariensis, suggesting be a common characteristic in short flower Nicotiana species. We further found that the antioxidant ascorbate accumulated in nectar and ß-carotene accumulated in nectaries. ß-carotene was most high in nectaries of N. bonariensis. We also examined the presence of proteins in the nectars of these species. The protein profile and quantities varied significantly between species, although all species have showed the presence of proteins in their nectars. We performed a limited proteomic analysis of several proteins from these nectars and determined that each of the five abundant proteins examined were identified as Nectarin 1, Nectarin 3, or Nectarin 5. Thus, based upon the results found in numerous species across the genus Nicotiana, we conclude that the mechanisms identified are similar to those mechanisms found in previous studies on ornamental tobacco nectars. Further, these similarities are remarkably conserved, throughout the genus Nicotiana.

Interleukin 10 (- 1082 G/A) and (- 819 C/T) gene polymorphisms in Egyptian women with polycystic ovary syndrome (PCOS).

Cytokines play critical roles in the pathogenesis of Polycystic Ovarian Syndrome (PCOS). This work was designed to study the implication of IL10 gene polymorphisms (- 1082 G/A and - 819 C/T) on the susceptibility of Egyptian women to have PCOS. Rotterdam consensus criteria were used to diagnose PCOS patients. Genotyping was performed by single-stranded polymorphism-polymerase chain reaction (SSP-PCR) in 61 PCOS patients and 80 healthy controls, and IL-10 serum levels were measured using Enzyme linked immunosorbent assay (ELISA). The frequency of IL10 - 1082 G/G (46%) genotype was significantly increased (p < 0.001) while the frequency of - 1082 A/A (16%) genotype was significantly decreased (p < 0.05) in PCOS patients compared to controls (14% and 35% for G/G and A/A genotypes; respectively). G allele (65%) is significantly increased (p < 0.01( in PCOS patients while A allele (61%) is significantly increased (p < 0.001( in control subjects. The distribution of IL10 -819 T/T genotype was significantly increased (p < 0.05) in PCOS group. G/G genotype (odd ratio (OR = 5.322) with confidence interval (CI = 2.364-11.982) and the G allele (OR = 2.828 with CI = 1.73-4.61) of - 1082 G/A and T/T genotype of - 819 C/T (OR = 4.18 with CI = 1.26-13.86) could be considered as risk factors for PCOS. IL-10 levels were significantly lower among PCOS patients (313.42 ± 30.10) compared to normal controls (4914.36 ± 303.72). Depending on our preliminary work, IL10 - 1082 G/G might be considered as a host genetic factor for PCOS susceptibility in Egyptian women. Studies concerning other cytokine gene polymorphisms are required to get a better understanding of the pathogenesis of PCOS disease.

An alternative process to treat boiler feed water for reuse.

A bench-scale process to treat boiler feed water for reuse in steam generation was developed. Industrial water samples from a steam-assisted gravity drainage plant in northern Alberta, Canada, were obtained and samples characterized. The technology, which consists of coagulation-settling to remove oil/grease and particulates followed by an advanced oxidative treatment, led to clean water samples with negligible organic carbon. Coagulation followed by settling removed most particulates and some insoluble organics. The advanced oxidative treatment removed any remaining color in the samples, decreased the organic content to near-zero, and provided water ready for reuse.

Effect of thalidomide dithiocarbamate analogs on the intercellular adhesion molecule-1 expression.

Thalidomide has been reported to have anti-angiogenic and antimetastatic effects. Intercellular adhesion molecule-1 (ICAM-1) was shown to be involved in monocyte adherence to epithelial cells and cancer cell invasion. Novel thalidomide dithiocarbamate analogs (containing 2 sulfur atoms) were designed and synthesized as potential anti-tumor agents. The aim of this work is to investigate their anti-tumor effect against transplantable experimental tumor, Ehrlich ascites carcinoma (EAC), in mice by studying the changes in cell's biochemical profile, the expression of ICAM-1 and nitric oxide (NO) and their association with tumor burden. As shown in our results, treatment of solid tumor-bearing mice with thalidomide 1 resulted in a significant reduction in tumor volume with 75.4% inhibition, a significant decrease in lactate dehydrogenase (LDH), ICAM-1 expression and NO. Thalidomide dithiocarbamate analogs 2 and 3 exhibited a potent effect to reduce the volume of solid tumor with 96.7% and 96.5% inhibition, respectively, a significant ability to increase the albumin, alanine aminotransferase (ALT) and glucose levels and to diminish LDH, ICAM-1 expression and NO. Thalidomide dithiocarbamate analog 3 has more potent anti-tumor activity as compared with thalidomide 1 or its dithiocarbamate analog 2. Taken together, our study improved that the dithiocarbamate analogs 2 and 3 are more potent anti-tumor agents with more pronounced effect than thalidomide 1 itself.

The MYB305 transcription factor regulates expression of nectarin genes in the ornamental tobacco floral nectary.

We have isolated and characterized the cDNA encoding the ornamental tobacco (Nicotiana langsdorffii X N. sanderae) homolog of the antirrhinum (Antirrhinum majus) MYB305. This transcription factor was robustly expressed at Stage 12 of nectary development but was only weakly expressed in the earlier Stage 6 nectaries. The ornamental tobacco MYB305 contains a conserved R2R3 MYB DNA binding domain with 76 amino acids in the activation domain. A green fluorescent protein-MYB305 fusion localized to nucleus of tobacco protoplasts and yeast one-hybrid assays demonstrated that it functions as a transcription activator. A conserved 23-amino acid C-terminal domain is required to activate gene expression. The coding region of the myb305 cDNA was expressed in Escherichia coli as a glutathione S-transferase fusion protein and was purified to homogeneity. This protein shows binding to two consensus MYB binding sites on the ornamental tobacco Nectarin I (nec1) promoter as well as to the single site located on the Nectarin V (nec5) promoter. Deletions of either of the binding sites from the nec1 promoter significantly reduced expression in nectary tissues. Temporally, MYB305 expression precedes that of nec1 and nec5, as would be expected if the MYB305 factor regulates expression of the nec1 and nec5 genes. Ectopic expression of MYB305 in foliage was able to induce expression of both nec1 and nec5, as well as two flavonoid biosynthetic genes in the foliage. Finally, RNA interference knockdown of MYB305 resulted in reduced expression of both nectarins and flavonoid biosynthetic genes. We conclude that expression of MYB305 regulates expression of the major nectarin genes in the floral nectary.

Nectarin IV, a potent endoglucanase inhibitor secreted into the nectar of ornamental tobacco plants. Isolation, cloning, and characterization.

We have isolated and characterized the Nectarin IV (NEC4) protein that accumulates in the nectar of ornamental tobacco plants (Nicotiana langsdorffii x Nicotiana sanderae var LxS8). This 60-kD protein has a blocked N terminus. Three tryptic peptides of the protein were isolated and sequenced using tandem mass spectroscopy. These unique peptides were found to be similar to the xyloglucan-specific fungal endoglucanase inhibitor protein (XEGIP) precursor in tomato (Lycopersicon esculentum) and its homolog in potato (Solanum tuberosum). A pair of oligonucleotide primers was designed based on the potato and tomato sequences that were used to clone a 1,018-bp internal piece of nec4 cDNA from a stage 6 nectary cDNA library. The remaining portions of the cDNA were subsequently captured by 5' and 3' rapid amplification of cDNA ends. Complete sequencing of the nec4 cDNA demonstrated that it belonged to a large family of homologous proteins from a wide variety of angiosperms. Related proteins include foliage proteins and seed storage proteins. Based upon conserved identity with the wheat (Triticum aestivum) xylanase inhibitor TAXI-1, we were able to develop a protein model that showed that NEC4 contains additional amino acid loops that are not found in TAXI-1 and that glycosylation sites are surface exposed. Both these loops and sites of glycosylation are on the opposite face of the NEC4 molecule from the site that interacts with fungal hemicellulases, as indicated by homology to TAXI-I. NEC4 also contains a region homologous to the TAXI-1 knottin domain; however, a deletion in this domain restructures the disulfide bridges of this domain, resulting in a pseudoknottin domain. Inhibition assays were performed to determine whether purified NEC4 was able to inhibit fungal endoglucanases and xylanases. These studies showed that NEC4 was a very effective inhibitor of a family GH12 xyloglucan-specific endoglucanase with a K(i) of 0.35 nm. However, no inhibitory activity was observed against other family GH10 or GH11 xylanases. The patterns of expression of the NEC4 protein indicate that, while expressed in nectar at anthesis, it is most strongly expressed in the nectary gland after fertilization, indicating that inhibition of fungal cell wall-degrading enzymes may be more important after fertilization than before.

Regulation of pyrimidine metabolism in plants.

Pyrimidine nucleotides represent one of the most fundamental of cellular components. They are the building blocks for the direct synthesis of DNA and RNA that function in information storage and retrieval within the cell, but they also participate in the metabolism of a large number of other cellular components from sugar interconversion to cellular polysaccharides to glycoproteins and phospholipids. Thus, the metabolism of pyrimidine nucleotides and their intracellular pool sizes influence vast areas of normal cellular metabolism. The first pyrimidine, UMP, is synthesized by a de novo pathway that appears to be mechanistically invariant in all organisms. UMP is then further modified to form other pyrimidines. Breakdown of deoxyribo- and ribonucleic acids, the main sink for pyrimidine nucleotides, allows pyrimidines to be reutilized for resynthesis of these important cellular components. Pyrimidines are salvaged by converting the modified components into the free base, uracil for reutilization. Finally, pyrimidines are degraded into simple cellular metabolites permitting reutilization of nitrogen and carbon from pyrimidine ring systems into cellular metabolic pools. The regulation of pyrimidine metabolism is tightly controlled in plants. Additionally, plants produce toxic secondary metabolites derived from pyrimidines for use as defense compounds.