Amylolytic activity and carbohydrate levels in relation to coleoptile anoxic elongation in Oryza sativa genotypes

Among starchy seeds, rice has the unique capacity to germinate successfully under complete anaerobiosis. In this conditions, starch degradation is supported by a complete set of starch-degrading enzymes that are absent or inactive in cereals except rice. A characterization of carbohydrate metabolism and starch-degrading enzyme activity across twenty-nine genotypes of Oryza sativa L. is presented here. The zymogram of amylolytic activities present in rice embryos and endosperms under anaerobic conditions seven days after sowing (DAS) revealed marked differences among cultivars. Coleoptile elongation was positively correlated with total amylolytic activities and α-amylase activity in embryos, and negatively correlated with α-amylase activity in endosperm. Moreover, carbohydrate content in embryos was found to be positively correlated with total amylolytic activities under anaerobic conditions, while a negative relationship was recorded in the endosperm. Carbohydrate status in rice seedlings has a primary importance in sustaining coleoptile elongation towards the surface. The relationship between carbohydrate level in embryo and anoxic germination, as well as with total amylolytic activities present in rice embryo under anaerobic condition 7 DAS, is consistent with the role of sugar metabolism to support rice germination under oxygen-deprived environment.     

Expiration rate drives human airway design

Analyses of human airway architecture based on calculations of airflow resistance or energy dissipation suggest that the branching pattern is not optimized for minimizing energy loss by flow dissipation during respiration. Airway flow dissipates only a few percent of the total body work during normal breathing, so branching patterns deviate from minimum energy loss to also optimize other physiological needs. Studies of airway performance often record some measure of expiration, such as FEV1 (Forced Expiratory Volume in 1 s), because airway constriction during expiration limits the rate of rapid respiration. We posit that lung structure is optimized for the rate of expiration as well as minimum energy loss. By increasing the daughter-to-parent airway diameter ratio (h) from 0.794 (corresponding to the energy minimum for symmetrically branching airways) to 0.85 (the observed value in humans) luminal pressures at airway generations 4-15 were substantially increased during exercise (a 4.5 and 15 cmH₂O increase during moderate and heavy exercise, respectively). Values of h somewhat larger than 0.794 help airways remain open during expiration by increasing both viscous pressure drop and convective acceleration pressure drop. Asymmetric bifurcations also exhibit higher proximal airway pressures than symmetric ones, but the improvement was not large.     

Glutamate 85 and glutamate 228 contribute to the pH-response of the soluble form of chloride intracellular channel 1

The chloride intracellular channel protein, CLIC1, is synthesised as a soluble monomer that can reversibly bind membranes. Soluble CLIC1 is proposed to respond to the low pH found at a membrane surface by partially unfolding and restructuring into a membrane-competent conformation. This transition is proposed to be controlled by strategically located “pH-sensor” residues that become protonated at acidic pH. In this study, we investigate the role of two conserved glutamate residues, Glu85 in the N-domain and Glu228 in the C-domain, as pH-sensors. E85L and E228L CLIC1 variants were created to reduce pH sensitivity by permanently breaking the bonds these residues form. The structure and stability of each variant was compared to the wild type at both pH 7.0 and pH 5.5. Neither substitution significantly altered the structure but both decreased the conformational stability. Furthermore, E85L CLIC1 formed a urea-induced unfolding intermediate state at both pH 7 and pH 5.5 compared to wild-type and E228L CLIC1 which only formed the intermediate at pH 5.5. We conclude that Glu85 and Glu228 are two of the five pH-sensor residues of CLIC1 and contribute to the pH-response in different ways. Glu228 lowers the stability of the native state at pH 5.5, while Glu85 contributes both to the stability of the native state and to the formation of the intermediate state. By putting these interactions into the context of the three previously described CLIC1 pH-sensor residues, we propose a mechanism for the conversion of CLIC1 from the soluble state to the pre-membrane form.     

Epithelial cell distribution and abundance in rhesus monkey airways during postnatal lung growth and development.

Lung development is both a pre- and postnatal process. Although many lung diseases have their origins in early childhood, few quantitative data are available on the normal growth and differentiation of both the conducting airways and the airway epithelium during the postnatal period. We examined rhesus monkey lungs from five postnatal ages: 4-6 days and 1, 2, 3, and 6 mo. Airways increase significantly in both length and circumference as monkeys increase significantly in body weight from 5 days to 6 mo. In this study we asked: as basement membrane surface area increases, does the epithelial cell organization change? To answer this question, we quantified total epithelial cell mass using high-resolution light micrographs and morphometric techniques on sections from defined airway regions: trachea, proximal intrapulmonary bronchus (generations 1 or 2), and distal intrapulmonary bronchus (generations 6-8). Epithelial thickness decreased in the smaller, more distal, airways compared with trachea but did not change with age in the trachea and proximal bronchus. The volume fraction of all cell types measured did not change significantly. Ciliated cells in the distal bronchus and goblet cells in the trachea both decreased in abundance with increasing age. Overall, the epithelial cell populations changed little in terms of mass or relative abundance to each other during this period of active postnatal lung growth. Regarding the proximal conducting airway epithelium, we conclude that 1) the steady-state abundance is tightly regulated to keep the proportion of cell types constant, and 2) establishment of these cell types occurs before 4-6 days postnatal age. We conclude that growth of the proximal airways occurs primarily in length and lags behind that of the lung parenchyma.     

Role of p53/FAK association and p53 phosphorylation in staurosporine-mediated apoptosis: Wild type versus mutant p53-R175H

A novel survival role of focal adhesion kinase (FAK) that involves its nuclear translocation and direct association with p53 has been demonstrated. Here we examined the relationship between the p53/FAK interaction and Ser46 phosphorylation of p53 (p-p53^Ser46) in the apoptotic regulation of human esophageal squamous cell carcinoma (HOSCC) cell lines, expressing either wild type (wt) p53 or mutant (mt) p53-R175H. In contrast to the wt p53 cell lines, the mt p53-R175H cell line was resistant to staurosporine (STS)-mediated detachment and caspase-3 activation. Furthermore, despite the resistance of mt p53-R175H to Ser46 phosphorylation, both wt and mt HOSCC cells translocate FAK into the nucleus and maintain the p53/FAK interaction post STS treatment. These findings provide unique insight into how tumor cells harboring the R175H mutant may resist chemotherapeutic intervention.

Structured summary

MINT-7294020: FAK (uniprotkb:Q05397) physically interacts (MI:0915) with p53 (uniprotkb:P04637) by anti-bait coimmunoprecipitation (MI:0006)     

A conserved interdomain interaction is a determinant of folding cooperativity in the GST fold

The canonical glutathione transferase (GST) fold found in many monomeric and dimeric proteins consists of two domains that differ in structure and conformational dynamics. However, no evidence exists that the two domains unfold/fold independently at equilibrium, indicating the significance of interdomain interactions in governing cooperativity between domains. Bioinformatics analyses indicate the interdomain interface of the GST fold is large, predominantly hydrophobic with a high packing density explaining cooperative interdomain behavior. Structural alignments reveal a topologically conserved lock-and-key interaction across the domain interface in which a bulky hydrophobic residue ("key") protrudes from the surface of the N-domain and inserts into a pocket ("lock") in the C-domain. To better understand the molecular basis for the contribution of interdomain interactions toward cooperativity within the GST fold in the absence of any influence from quaternary interactions, studies were done with two monomeric GST proteins: Escherichia coli Grx2 (EcGrx2) and human CLIC1 (hCLIC1). Replacing the methionine "key" residue with alanine is structurally nondisruptive, whereas it significantly diminishes the folding cooperativity of both proteins. The loss in cooperativity between domains in the mutants is reflected by a change in the equilibrium folding mechanism from a wild-type two-state process to a three-state process, populating a stable folding intermediate.     

Proteomic identification of differentially expressed proteins in the anoxic rice coleoptile

Rice is the staple food for more than fifty percent of the world's population, and is therefore an important crop. However, its production is hindered by several biotic and abiotic stresses. Although rice is the only crop that can germinate even in the complete absence of oxygen (i.e. anoxia), flooding (low oxygen) is one of the major causes of reduced rice production. Rice germination under anoxia is characterized by the elongation of the coleoptile, but leaf growth is hampered. In this work, a comparative proteomic approach was used to detect and identify differentially expressed proteins in the anoxic rice coleoptile compared to the aerobic coleoptile. Thirty-one spots were successfully identified by MALDI-TOF MS analysis. The majority of the identified proteins were related to stress responses and redox metabolism. The expression levels of twenty-three proteins and their respective mRNAs were analyzed in a time course experiment.     

Fluorometric high-performance liquid chromatographic analysis of 10-deazaaminopterin, 10-ethyl-10-deazaaminopterin, and known metabolites

The antifolate compounds 10-deazaaminopterin (10-dAM) and 10-ethyl-10-deazaaminopterin (10-EdAM) are therapeutically superior to methotrexate in transplanted murine tumor systems and in human tumor xenografts growing in immunodeficient "nude" mice. The increased therapeutic index of these analogs correlates with their selective uptake, retention, and polyglutamation within neoplastic cells. We have developed a fluorescence high-performance liquid chromatographic assay applicable to 10-dAM, 10-EdAM, their polyglutamate anabolites, and their 7-hydroxy (7-OH) and deglutamate catabolites. The assay is based upon the high native fluorescence of pteridine-containing compounds which contain carbon in the 10 position. The assay employs a reverse-phase C-18 column and an ascending acetonitrile gradient in 50 mM phosphate, pH 7.0. The compounds are extracted from plasma and urine with 95 +/- 7% and 98 +/- 2% recoveries, respectively, using C-18 Sep-Paks. The linear range of the assay is, for 10-dAM, 2-100 nM, and for 10-EdAM, 1-100 nM. Polyglutamated metabolites of [3H]10-EdAM isolated from L1210 cells have been separated by HPLC with identification of five derivatives (Glu 1-5) confirmed by enzymatic peak shift using serum conjugase and by quantitative correlation of fluorescence intensity, radioactivity, and titration inhibition of dihydrofolate reductase. The assay has been used successfully in pharmacokinetic analyses of plasma and urine samples from patients receiving 10-dAM and 10-EdAM. In patients who had received 10-EdAM, 7-OH-10-EdAM, and the deglutamate catabolite were also detected. This HPLC fluorescence assay is superior to the dihydrofolate reductase inhibition and binding assays with regard to specificity and precision; moreover, it can provide a means for simultaneous assay of the physiologically important anabolites and catabolites of these new antifolates.     

Recent advances of novel targeted therapy in non-small cell lung cancer

Lung cancer is the leading cause of cancer deaths world-wide. Recent advances in cancer biology have led to the identification of new targets in neoplastic cells and the development of novel targeted therapies. At this time, two targeted agents are approved by the FDA in advanced non-small cell lung cancer (NSCLC): the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib, and the anitangiogenic bevacizumab. A third agent, cetuximab, which was recently shown to enhance survival when used with cisplatin and vinorelbine as first line therapy for advanced NSCLC, will likely be approved by regulatory agencies. With more than 500 molecularly targeted agents under development, the prospects of identifying novel therapies that benefit individual patients with lung cancer are bright.     

Stability of the domain interface contributes towards the catalytic function at the H-site of class alpha glutathione transferase A1-1

Cytosolic glutathione transferases (GSTs) are major detoxification enzymes in aerobes. Each subunit has two distinct domains and an active site consisting of a G-site for binding GSH and an H-site for an electrophilic substrate. While the active site is located at the domain interface, the role of the stability of this interface in the catalytic function of GSTs is poorly understood. Domain 1 of class alpha GSTs has a conserved tryptophan (Trp21) in helix 1 that forms a major interdomain contact with helices 6 and 8 in domain 2. Replacing Trp21 with an alanine is structurally non-disruptive but creates a cavity between helices 1, 6 and 8 thus reducing the packing density and van der Waals contacts at the domain interface. This results in destabilization of the protein and a marked reduction in catalytic activity. While functionality at the G-site is not adversely affected by the W21A mutation, the H-site becomes more accessible to solvent and less favorable for the electrophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB). Not only does the mutation result in a reduction in the energy for stabilizing the transition state formed in the S_NAr reaction between the substrates GSH and CDNB, it also compromises the ability of the enzyme to form and stabilize a transition state analogue (Meisenheimer complex) formed between GSH and 1,3,5-trinitrobenzene (TNB). The study demonstrates that the stability of the domain–domain interface plays a role in mediating the catalytic functionality of the active site, particularly the H-site, of class alpha GSTs.