ABNORMAL GUARD CELL DEVELOPMENT IN AN OLIVE NECROTIC MUTANT OF MAIZE

A study of a mutant variety of Zea mays (ON8147) revealed that the mutant plants, in contrast with normal maize plants, do not exhibit a light-induced increase in the rate of transpiration, and that the ontogeny of the stomatal complex is abnormal. In later stages of differentiation, the guard cells of mutant plants deteriorate, leaving the mature stomata with only the two subsidiary cells. The subsidiary cells in stomata of mutant leaves are similar to those of normal leaves with respect to their capacity to accumulate K⁺ in the dark, but they do not lose K⁺ in the light, as do subsidiary cells of stomata of nonmutant plants. It is suggested that impairment of guard cell function causes death of the mutant plant seedlings primarily by restricting CO₂ entry into the leaf.     

Microinjection of Intact 200- to 500-kb Fragments of YAC DNA into Mammalian Cells

DNA of yeast artificial chromosomes (YACs) was prepared for microinjection by separation from most of the natural yeast chromosomes on a pulsed-field gel, treatment with agarase, and centrifugation. A salt concentration of 100 mM NaCl was necessary to protect the DNA from shear during these procedures. Injection of a 590-kb YAC, yGART2, into Chinese hamster ovary cells gave rise to cells expressing the 40-kb human GART gene carried on the YAC. Nine of 12 cell lines analyzed contained an intact stretch of at least 110 kb of YAC DNA surrounding the GART gene, and one cell line contained at least 480 kb, but not the entire 590 kb, intact. Mouse L A-9 cells were similarly injected with DNA of a 230-kb YAC containing the human β-globin gene cluster and a mammalian selectable marker. Seven of 10 of the resulting cell lines contained both YAC vector arms plus the intact 140-kb SfiI fragment spanning the β-globin gene. Three cell lines were analyzed by Rec A-assisted restriction endonuclease (RARE) cleavage and found to contain the entire intact 210-kb YAC insert. Introduction of similarly prepared DNA into mammalian cells by lipofection gave rise to cell lines with multiple YAC fragments that were generally shorter than the YAC fragments found in microinjected cell lines. The results show that microinjection of gel-purified YAC DNA into mammalian cells is an efficient method of transferring DNA fragments several hundred kilobase pairs in size into mammalian cells.     

Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.     

Impacts and Recovery from Severe Tropical Cyclone Yasi on the Great Barrier Reef

Full recovery of coral reefs from tropical cyclone (TC) damage can take decades, making cyclones a major driver of habitat condition where they occur regularly. Since 1985, 44 TCs generated gale force winds (≥17 metres/second) within the Great Barrier Reef Marine Park (GBRMP). Of the hurricane strength TCs (≥H1—Saffir Simpson scale; ≥ category 3 Australian scale), TC Yasi (February, 2011) was the largest. In the weeks after TC Yasi crossed the GBRMP, participating researchers, managers and rangers assessed the extent and severity of reef damage via 841 Reef Health and Impact Surveys at 70 reefs. Records were scaled into five damage levels representing increasingly widespread colony-level damage (1, 2, 3) and reef structural damage (4, 5). Average damage severity was significantly affected by direction (north vs south of the cyclone track), reef shelf position (mid-shelf vs outer-shelf) and habitat type. More outer-shelf reefs suffered structural damage than mid-shelf reefs within 150 km of the track. Structural damage spanned a greater latitudinal range for mid-shelf reefs than outer-shelf reefs (400 vs 300 km). Structural damage was patchily distributed at all distances, but more so as distance from the track increased. Damage extended much further from the track than during other recent intense cyclones that had smaller circulation sizes. Just over 15% (3,834 km²) of the total reef area of the GBRMP is estimated to have sustained some level of coral damage, with ~4% (949 km²) sustaining a degree of structural damage. TC Yasi likely caused the greatest loss of coral cover on the GBR in a 24-hour period since 1985. Severely impacted reefs have started to recover; coral cover increased an average of 4% between 2011 and 2013 at re-surveyed reefs. The in situ assessment of impacts described here is the largest in scale ever conducted on the Great Barrier Reef following a reef health disturbance.     

Causes of nondiffusing lipid in the plasma membrane of mammalian spermatozoa

In the plasma membranes of most mammalian somatic cells, lipid is nearly completely free to diffuse laterally in the plane of the membrane. In mammalian spermatozoa and certain other highly polarized mammalian cells, a significant fraction of the plasma membrane lipid is not free to diffuse laterally. Using the technique of fluorescence recovery after photobleaching, we have demonstrated that a variety of fluorescent lipid analogues exhibit a nondiffusing fraction in the plasma membrane of the anterior region of the ram sperm head. The possible causes of this nondiffusing fraction were investigated. The nondiffusing lipid fraction is not the result of lipid oxidation during handling, and it is not released by extensive enzymatic digestion of the membrane surface proteins or the "bleeding" of the membrane by hypoosmotic shock. When lipid bilayers were prepared from protein-free lipid extracts of the plasma membranes of spermatozoa, most of the nondiffusing fraction was retained. These results suggest that the nondiffusing lipid fraction results from lipid factors such as lateral phase separations, which can cause such a nondiffusing fraction in model systems.     

Health-related quality of life and weight loss among overweight and obese U.S. adults, 2001 to 2002

Objective: To examine the prevalence and association of health‐related quality of life (HRQOL) with trying to lose weight and with weight loss practices (eating fewer calories, physical activity, and both) among overweight and obese U.S. adults ≥ 20 years of age. Research Methods and Procedures: This study used data from the 2001 to 2002 National Health and Nutrition Examination Survey, a continuous annual survey of the civilian non‐institutionalized U.S. population. This analysis included those ≥ 20 years of age with BMI ≥ 25 (n = 2578) who responded to four standard HRQOL measures that assessed general health status and recent physical health, mental health, and activity limitation. Results: Among obese men, but not women, there were significant increasing linear trends in the adjusted prevalence of trying to lose weight as physically unhealthy and activity limitation days increased. Regardless of BMI or HRQOL, reducing calories was a common weight loss practice (66% to 86%). Except for recent activity limitation, respondents with BMI ≥ 35 did not generally differ by HRQOL level in the attainment of recommended physical activity either alone or in combination with reduced calories, whereas those in the BMI 25 to 34.9 groups often differed significantly by HRQOL level. Specifically, increased unhealthy or activity limitation days were associated with reduced prevalence of attained physical activity. Discussion: Our findings indicate an association between trying to lose weight and a greater number of unhealthy days reported by obese men, suggesting that these men may be influenced by traditional clinical weight‐loss counseling that is prompted by weight and comorbidity, whereas women had a high prevalence of trying to lose weight irrespective of weight and HRQOL. Assessment of HRQOL, especially measures that evaluate physical domains, could provide subjective information to assist with weight counseling.     

Crystal Structure of the Engineered Neutralizing Antibody M18 Complexed to Domain 4 of the Anthrax Protective Antigen

The virulence of Bacillus anthracis is critically dependent on the cytotoxic components of the anthrax toxin, lethal factor (LF) and edema factor (EF). LF and EF gain entry into host cells through interactions with the protective antigen (PA), which binds to host cellular receptors such as CMG2. Antibodies that neutralize PA have been shown to confer protection in animal models and are undergoing intense clinical development. A murine monoclonal antibody, 14B7, has been reported to interact with domain 4 of PA (PAD4) and block its binding to CMG2. More recently, the 14B7 antibody was used as the platform for the selection of very high affinity, single-chain antibodies that have tremendous potential as a combination anthrax prophylactic and treatment. Here, we report the high-resolution X-ray structures of three high-affinity, single-chain antibodies in the 14B7 family; 14B7 and two high-affinity variants 1H and M18. In addition, we present the first neutralizing antibody-PA structure, M18 in complex with PAD4 at 3.8 Å resolution. These structures provide insights into the mechanism of neutralization, and the effect of various mutations on antibody affinity, and enable a comparison between the binding of the M18 antibody and CMG2 with PAD4.     

Restriction enzyme-free construction of random gene mutagenesis libraries in Escherichia coli

Directed evolution relies on both random and site-directed mutagenesis of individual genes and regulatory elements to create variants with altered activity profiles for engineering applications. Central to these experiments is the construction of large libraries of related variants. However, a number of technical hurdles continue to limit routine construction of random mutagenesis libraries in Escherichia coli, in particular, inefficiencies during digestion and ligation steps. Here, we report a restriction enzyme-free approach to library generation using megaprimers termed MegAnneal. Target DNA is first exponentially amplified using error-prone polymerase chain reaction (PCR) and then linearly amplified with a single 3′ primer to generate long, randomly mutated, single-stranded megaprimers. These are annealed to single-stranded dUTP-containing template plasmid and extended with T7 polymerase to create a complementary strand, and the resulting termini are ligated with T4 DNA ligase. Using this approach, we are able to reliably generate libraries of approximately 10⁷ colony-forming units (cfu)/μg DNA/transformation in a single day. We have created MegAnneal libraries based on three different single-chain antibodies and identified variants with enhanced expression and ligand-binding affinity. The key advantages of this approach include facile amplification, restriction enzyme-free library generation, and a significantly reduced risk of mutations outside the targeted region and wild-type contamination as compared with current methods.