Evaluation of water binding, seed coat permeability and germination characteristics of wheat seeds equilibrated at different relative humidities

The relative binding of seed water and seed coat membrane stability were measured in two contrasting wheat (Triticum aestivum L) varieties, HDR 77 (drought-tolerant) and HD 2009 (susceptible) using seed water sorption isotherms, electrical conductivity (EC) of leachates and desorption-absorption isotherms. Analysis of sorption isotherm at 25 degrees C showed that the seeds of HDR 77 had significantly higher number of strong binding sites, with correspondingly greater amount of seed water as strongly bound water, as compared to HD 2009. Total number of binding sites was also higher in HDR 77 than HD 2009, which explained the better desiccation tolerance and higher capacity to bind water in seeds of HDR 77. EC of seed leachate in both varieties did not change with respect to change in equilibrium relative humidity (RII), indicating the general seed coat membrane stability of wheat seeds. However, absolute conductivity values were higher for HD 2009. showing its relatively porous seed coat membrane. Significantly lower area enclosed by the desorption-absorption isotherm loop in HDR 77, as compared to HD 2009 also indicated the greater membrane integrity of HDR 77. Germination and seedling vigour of HD 2009 were reduced when equilibrated over very low and very high RH. In contrast, germination and vigour in HDR 77 were maintained high, except at very high RH, indicating again its desiccation tolerance. Thus, the study demonstrated the relative drought tolerance of HDR 77, on the basis of seed water-binding characteristics and seed membrane stability. Seed membrane stability as measured by seed leachate conductivity or as area under dehydration-rehydration loop may be used as a preliminary screening test for drought tolerance in wheat.     

Glycosylation in the Fc domain of IgG increases resistance to proteolytic cleavage by papain

IgG antibodies (Abs) and fragments of IgG Abs are becoming major biotherapeutics to treat an assortment of human diseases. Commonly prepared fragments of IgGs include Fc, Fab, and F(ab')2 fragments, all of which can be made using the sulfhydryl protease papain, although prolonged digestion times and/or excessive amounts of papain typically result in further cleavage of the Fc domain into smaller fragments. During our attempts to use papain to isolate Fc fragments from different IgG monoclonal Abs, it was observed that prior removal of Fc glycans resulted in a faster rate of papain-mediated degradation of the Fc domain. Subsequent time-course experiments comparing glycosylated and deglycosylated versions of IgG antibodies showed that the majority of molecules in a deglycosylated IgG sample were converted into Fab, Fc, and smaller Fc fragments in less than one hour, whereas the original glycosylated IgG required more than two hours to convert into a comparable amount of Fab and Fc fragments. Furthermore, whereas papain digestion converted almost all of a deglycosylated Fc fragment into smaller fragments of approximately 10 and approximately 12 kDa within 4 h, more than 40% of a glycosylated Fc fragment remained intact even after 24 h of digestion. These results indicate that the presence of CH(2) domain glycans in either IgGs or purified Fc fragments increases resistance to papain digestion. Increased sensitivity of non-glycosylated Fc domains to papain is consistent with the Fc domains lacking a defined structure, as exemplified by their inability to bind Fcgamma receptors, since misfolded proteins are often degraded by proteases because of increased accessibility of their proteolytic cleavage sites. Based on these observations it is possible to use papain sensitivity as a means of assessing proper Fc structure of IgG molecules.     

Characterization of water distribution and activities of enzymes during germination in magnetically-exposed maize (Zea mays L) seeds

Magnetic seed treatment is one of the physical pre-sowing seed treatments to enhance the performance of crop plants. In our earlier experiment, we found significant increase in germination and vigour characteristics of maize (Zea mays L.) seeds subjected to magnetic fields. Among various combinations of magnetic field (MF) strength and duration, best results were obtained with MF of 100 mT for 2 h and 200 mT for 1 h exposure. The quicker germination in magnetically-exposed seeds might be due to greater activities of germination related enzymes, early hydration of membranes as well as greater molecular mobility of bulk and hydration water fractions. Thus, in the present study, changes in water uptake during imbibition and its distribution and activities of germinating enzymes during germination were investigated in maize seeds exposed to static magnetic fields of 100 and 200 mT for 2 and 1 h respectively by nuclear magnetic resonance (NMR) spectroscopy. The magnetically-exposed seed showed higher water uptake in phase II and III than unexposed seed. The longitudinal relaxation time T1 of seed water showed significantly higher values and hence greater molecular mobility of cellular water in magnetically-exposed seeds as compared to unexposed. Component analysis of T2 relaxation times revealed the early appearance of hydration water with least mobility and higher values of relaxation times of cytoplasmic bulk water and hydration water in magnetically-exposed over unexposed seeds. Activities of alpha-amylase, dehydorgenase and protease during germination were higher in magnetically-exposed seeds as compared to unexposed. The quicker germination in magnetically-exposed seeds might be due to greater activities of germination related enzymes, early hydration of membranes as well as greater molecular mobility of bulk and hydration water fractions.     

Monomeric and Dimeric CXCL8 Are Both Essential for In Vivo Neutrophil Recruitment

Rapid mobilization of neutrophils from vasculature to the site of bacterial/viral infections and tissue injury is a critical step in successful resolution of inflammation. The chemokine CXCL8 plays a central role in recruiting neutrophils. A characteristic feature of CXCL8 is its ability to reversibly exist as both monomers and dimers, but whether both forms exist in vivo, and if so, the relevance of each form for in vivo function is not known. In this study, using a ‘trapped’ non-associating monomer and a non-dissociating dimer, we show that (i) wild type (WT) CXCL8 exists as both monomers and dimers, (ii) the in vivo recruitment profiles of the monomer, dimer, and WT are distinctly different, and (iii) the dimer is essential for initial robust recruitment and the WT is most active for sustained recruitment. Using a microfluidic device, we also observe that recruitment is not only dependent on the total amount of CXCL8 but also on the steepness of the gradient, and the gradients created by different CXCL8 variants elicit different neutrophil migratory responses. CXCL8 mediates its function by binding to CXCR2 receptor on neutrophils and glycosaminoglycans (GAGs) on endothelial cells. On the basis of our data, we propose that dynamic equilibrium between CXCL8 monomers and dimers and their differential binding to CXCR2 and GAGs mediates and regulates in vivo neutrophil recruitment. Our finding that both CXCL8 monomer and dimer are functional in vivo is novel, and indicates that the CXCL8 monomer-dimer equilibrium and neutrophil recruitment are intimately linked in health and disease.     

Exposure of seeds to static magnetic field enhances germination and early growth characteristics in chickpea (Cicer arietinum L.)

Seeds of chickpea (Cicer arietinum L.) were exposed in batches to static magnetic fields of strength from 0 to 250 mT in steps of 50 mT for 1-4 h in steps of 1 h for all fields. Results showed that magnetic field application enhanced seed performance in terms of laboratory germination, speed of germination, seedling length and seedling dry weight significantly compared to unexposed control. However, the response varied with field strength and duration of exposure without any particular trend. Among the various combinations of field strength and duration, 50 mT for 2 h, 100 mT for 1 h and 150 mT for 2 h exposures gave best results. Exposure of seeds to these three magnetic fields improved seed coat membrane integrity as it reduced the electrical conductivity of seed leachate. In soil, seeds exposed to these three treatments produced significantly increased seedling dry weights of 1-month-old plants. The root characteristics of the plants showed dramatic increase in root length, root surface area and root volume. The improved functional root parameters suggest that magnetically treated chickpea seeds may perform better under rainfed (un-irrigated) conditions where there is a restrictive soil moisture regime.     

Corona discharge influences ozone concentrations near rats

Ozone can be produced by corona discharge either in dry air or when one electrode is submerged in water. Since ozone is toxic, we examined whether ozone production by corona near laboratory animals could reach levels of concern. Male rats were exposed to a corona discharge and the concentration of ozone produced was measured. The resulting concentration of ozone ranged from ambient levels to 250 ppb when animals were located 1 cm from a 10 kV source. Similar ozone concentrations were observed when a grounded water source was present. Possible explanations for, as well as concerns regarding, ozone production under these conditions are discussed.     

A protein encoded by a member of the multicopy Ssty gene family located on the long arm of the mouse Y chromosome is expressed during sperm development

Multicopy Y-chromosomal genes in human and mouse have been postulated to play a role in spermatogenesis. The mouse Y long arm (Yq) carries hundreds of supposedly intronless copies of Ssty, for which no protein has hitherto been identified; mice lacking Yq are sterile with grossly abnormal sperm. We have now identified an Ssty-encoded protein (Ssty1) that is expressed in spermatids. The protein is absent from spermatids of mice that lack Yq, but is not reduced in mice with a two-thirds reduction of Ssty copies, implying that most do not produce this protein. Furthermore, no protein was produced by a strongly transcribed intronless Ssty transgene, raising doubts as to the protein-encoding potential of these intronless genes. We have now identified an intron-containing copy that is also present in multiple copies on Yq. One or more intron-containing copies are retained in the Ssty-deficient mice and may be the source of the Ssty1 protein.     

An efficient synthesis of a heterobifunctional coupling agent

An efficient synthesis of 4-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl]-N-(6-[[6-([6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl]amino)-6-oxohexyl]amino]-6-oxohexyl)cyclohexanecarboxamide (12), a heterobifunctional coupling agent, was developed, which is critical for chemoselective conjugation of proteins and enzymes. The synthesis involved seven steps starting from 6-{[(benzyloxy)carbonyl]amino}hexanoic acid (1), and multigram quantities of coupling agent (12) were prepared using this protocol in excellent overall yield and 99.6% purity by reversed phase HPLC. The new method is suitable for the synthesis of coupling agent 12, consistently with purity >99%, and is useful for the preparation of other analogous coupling agents.     

13C NMR chemical shifts can predict disulfide bond formation

The presence of disulfide bonds can be detected unambiguously only by X-ray crystallography, and otherwise must be inferred by chemical methods. In this study we demonstrate that ¹³C NMR chemical shifts are diagnostic of disulfide bond formation, and can discriminate between cysteine in the reduced (free) and oxidized (disulfide bonded) state. A database of cysteine ¹³C C and C chemical shifts was constructed from the BMRB and Sheffield databases, and published journals. Statistical analysis indicated that the C shift is extremely sensitive to the redox state, and can predict the disulfide-bonded state. Further, chemical shifts in both states occupy distinct clusters as a function of secondary structure in the C/C chemical shift map. On the basis of these results, we provide simple ground rules for predicting the redox state of cysteines; these rules could be used effectively in NMR structure determination, predicting new folds, and in protein folding studies.     

Novel use of an osmolyte to dissect multiple thermodynamic linkages in a chemokine ligand-receptor system

We have used trimethylamine N-oxide (TMAO), a protecting osmolyte, to dissect the complex thermodynamic linkages involved in the interaction between the chemokine interleukin-8 (IL-8) and the N-domain of its receptor CXCR1. Our results show that TMAO induces folding in the CXCR1 receptor N-domain and that the N-domain upon folding binds ligand with higher affinity. This represents, to our knowledge, the smallest domain that has been shown to be folded in osmolyte. Using the phase diagram method to analyze this thermodynamic relationship graphically, we also observe that TMAO favors ligand dimerization and that the dimeric ligand binds the receptor domain with lower affinity. We have thus been able to dissect coupling among three distinct processes, receptor domain folding, ligand dimerization, and ligand-receptor domain binding in this chemokine-receptor system. We also observe that the affinity of the related chemokine, melanoma growth stimulatory activity (MGSA), increases concurrent with N-domain folding similar to IL-8 but shows more profound differences on ligand dimerization. These studies establish a novel and innovative use of osmolytes to dissect linkages among different processes and exploit the phase diagram as a tool to graphically represent and dissect complex thermodynamic relationships in biological systems. On the basis of our observations and earlier work, we discuss the relevance of ligand dimerization in chemokine regulation.