Hydrologic regulation of gross methyl chloride and methyl bromide uptake from Alaskan Arctic tundra

The Arctic tundra has been shown to be a potentially significant regional sink for methyl chloride (CH₃Cl) and methyl bromide (CH₃Br), although prior field studies were spatially and temporally limited, and did not include gross flux measurements. Here we compare net and gross CH₃Cl and CH₃Br fluxes in the northern coastal plain and continental interior. As expected, both regions were net sinks for CH₃Cl and CH₃Br. Gross uptake rates (−793 nmol CH₃Cl m⁻² day⁻¹ and −20.3 nmol CH₃Br m⁻² day⁻¹) were 20–240% greater than net fluxes, suggesting that the Arctic is an even greater sink than previously believed. Hydrology was the principal regulator of methyl halide flux, with an overall trend towards increasing methyl halide uptake with decreasing soil moisture. Water table depth was one of the best predictors of net and gross uptake, with uptake increasing proportionately with water table depth. In drier areas, gross uptake was very high, averaging −1201 nmol CH₃Cl m⁻² day⁻¹ and −34.9 nmol CH₃Br m⁻² day⁻¹; in flooded areas, gross uptake was significantly lower, averaging −61 nmol CH₃Cl m⁻² day⁻¹ and −2.3 nmol CH₃Br m⁻² day⁻¹. Net and gross uptake was greater in the continental interior than in the northern coastal plain, presumably due to drier inland conditions. Within certain microtopographic features (low‐ and high‐centered polygons), uptake rates were positively correlated with soil temperature, indicating that temperature played a secondary role in methyl halide uptake. Incubations suggested that the inverse relationship between water content and methyl halide uptake was the result of mass transfer limitation in saturated soils, rather than because of reduced microbial activity under anaerobic conditions. These findings have potential regional significance, as the Arctic is expected to become warmer and drier due to anthropogenic climate forcing, potentially enhancing the Arctic sink for CH₃Cl and CH₃Br.     

Monolithic microfluidic mixing–spraying devices for time-resolved cryo-electron microscopy

The goal of time-resolved cryo-electron microscopy is to determine structural models for transient functional states of large macromolecular complexes such as ribosomes and viruses. The challenge of time-resolved cryo-electron microscopy is to rapidly mix reactants, and then, following a defined time interval, to rapidly deposit them as a thin film and freeze the sample to the vitreous state. Here we describe a methodology in which reaction components are mixed and allowed to react, and are then sprayed onto an EM grid as it is being plunged into cryogen. All steps are accomplished by a monolithic, microfabricated silicon device that incorporates a mixer, reaction channel, and pneumatic sprayer in a single chip. We have found that microdroplets produced by air atomization spread to sufficiently thin films on a millisecond time scale provided that the carbon supporting film is made suitably hydrophilic. The device incorporates two T-mixers flowing into a single channel of four butterfly-shaped mixing elements that ensure effective mixing, followed by a microfluidic reaction channel whose length can be varied to achieve the desired reaction time. The reaction channel is flanked by two ports connected to compressed humidified nitrogen gas (at 50 psi) to generate the spray. The monolithic mixer-sprayer is incorporated into a computer-controlled plunging apparatus. To test the mixing performance and the suitability of the device for preparation of biological macromolecules for cryo-EM, ribosomes and ferritin were mixed in the device and sprayed onto grids. Three-dimensional reconstructions of the ribosomes demonstrated retention of native structure, and 30S and 50S subunits were shown to be capable of reassociation into ribosomes after passage through the device.     

DOG-SPOT database for comprehensive management of dog genetic research data

Research laboratories studying the genetics of companion animals have no database tools specifically designed to aid in the management of the many kinds of data that are generated, stored and analyzed. We have developed a relational database, "DOG-SPOT," to provide such a tool. Implemented in MS-Access, the database is easy to extend or customize to suit a lab's particular needs. With DOG-SPOT a lab can manage data relating to dogs, breeds, samples, biomaterials, phenotypes, owners, communications, amplicons, sequences, markers, genotypes and personnel. Such an integrated data structure helps ensure high quality data entry and makes it easy to track physical stocks of biomaterials and oligonucleotides.     

The C. elegans ric-3 gene is required for maturation of nicotinic acetylcholine receptors

Mutations in ric-3 (resistant to inhibitors of cholinesterase) suppress the neuronal degenerations caused by a gain of function mutation in the Caenorhabditis elegans DEG-3 acetylcholine receptor. RIC-3 is a novel protein with two transmembrane domains and extensive coiled-coil domains. It is expressed in both muscles and neurons, and the protein is concentrated within the cell bodies. We demonstrate that RIC-3 is required for the function of at least four nicotinic acetylcholine receptors. However, GABA and glutamate receptors expressed in the same cells are unaffected. In ric-3 mutants, the DEG-3 receptor accumulates in the cell body instead of in the cell processes. Moreover, co-expression of ric-3 in Xenopus laevis oocytes enhances the activity of the C.elegans DEG-3/DES-2 and of the rat alpha-7 acetylcholine receptors. Together, these data suggest that RIC-3 is specifically required for the maturation of acetylcholine receptors.     

Mutations in the extracellular domain and in the membrane-spanning domains interfere with nicotinic acetylcholine receptor maturation

The deg-3(u662) mutation is a degeneration-causing mutation in a Caenorhabditis elegans nicotinic acetylcholine receptor. In a large screen for mutations that suppress the deleterious effects of this mutation we identified 32 mutations in the deg-3 gene. Among these, 11 are missense mutations, affecting seven residues within the extracellular domain or the membrane-spanning domains. All of these mutations greatly reduce the degeneration-causing activity of deg-3(u662). All but one of these mutations cause defective localization of the DEG-3 protein, as seen in immunohistochemical analysis. Thus our screen identifies multiple residues within the nicotinic acetylcholine receptor needed for normal folding, assembly, or trafficking of this receptor. Interestingly, these mutations lead to distinct localization defects suggesting differences in their effect on DEG-3's maturation process. Specifically, mutations in the extracellular domain lead to a phenotype more severe than mutations in the membrane-spanning domains. Differences in the effects of the mutations are also predicted by homology-based modeling, showing that some mutations in the extracellular domain are likely to disrupt the native fold of the protein, while others are likely to disrupt trafficking.     

Understanding down-regulation of photosynthesis under water stress: future prospects and searching for physiological tools for irrigation management

Photosynthetic down-regulation and/or inhibition under water stress conditions are determinants for plant growth, survival and yield in drought-prone areas. Current knowledge about the sequence of metabolic events that leads to complete inhibition of photosynthesis under severe water stress is reviewed. An analysis of published data reveals that a key regulatory role for Rubisco in photosynthesis is improbable under water stress conditions. By contrast, the little data available for other Calvin cycle enzymes suggest the possibility of a key regulatory role for some enzymes involved in the regeneration of RuBP. There are insufficient data to determine the role of photophosphorylation. Several important gaps in our knowledge of this field are highlighted. The most important is the remarkable scarcity of data about the regulation/inhibition of photosynthetic enzymes other than Rubisco under water stress. Consequently, new experiments are urgently needed to improve our current understanding of photosynthetic down-regulation under water stress. A second gap is the lack of knowledge of photosynthetic recovery after irrigation of plants which have been subjected to different stages of water stress. This knowledge is necessary in order to match physiological down-regulation by water stress with controlled irrigation programmes.     

STUDY ON THE PROLIFERATIVE STATE OF HAEMOPOIETIC STEM CELLS (CFU)

Hydroxyurea was used to study the proliferation rate of haemopoietic stem cells (CFUJ in normal mice, after irradiation or transplantation into irradiated recipients. It was demonstrated that the proliferation rate of endogenous CFU_S (endo-CFU,) and exogenous CFU_S (exo-CFU_s) are identical. After irradiation (650 R) the surviving endo-CFU_s begin to proliferate immediately. By contrast exo-CFU, transplanted into the irradiated recipient mouse (850 R), begin to proliferate only after about 30 hr. However, injection of isoproterenol (which stimulates adenyl cyclase) or dibutyryl cyclic adenosine 3′,5′-monophosphate shortly after marrow cell graft, triggers the transplanted CFU_S into cell cycle as shown by an almost immediately increased sensitivity to hydroxyurea. Isoproterenol is capable of inducing DNA synthesis also in stem cells of normal mice but it takes about 20 hr before CFU, become to be increasingly sensitive to hydroxyurea.     

Catching the phylogenic history through the ontogenic hourglass: a phylogenomic analysis of Drosophila body segmentation genes

SUMMARY The phylogenetic information content of different developmental stages is a long‐standing issue in the study of development and evolution. We performed phylogenetic analyses of 51 body segmentation genes in 12 species of Drosophila in order to investigate the impact of the mode of evolution of development on phylogeny inference. Previous studies of these genes in Drosophila using pairwise phenetic comparisons at the species group level revealed the presence of an “hourglass model” (HG), wherein mid‐embryonic stages are the most evolutionarily constrained. We utilized two character‐based approaches: taxonomic congruence using the relative consensus fork index (RCFI), in which phylogenies are inferred from each gene separately and compared with a total evidence tree (TET), and partitioned simultaneous analysis using several indices such as branch support (BS) and localized incongruence length difference (LILD) test. We also proposed a new index, the recapitulatory index (R), which divides the number of synapomorphies on the total number of informative characters in a data set. Polynomial adjustment of both BS and R indices showed strong support for the hourglass model regardless of the taxonomic level (species subgroup vs. subgenera), showing less phylogenetic information content for mid‐developmental stages (mainly the zygotic segment polarity stage). Significant LILD scores were randomly distributed among developmental stages revealing the absence of differential selective constraints, but were significantly related to chromosomal location showing physical (linkage) impact on phylogenetic incongruence. RCFI was the most sensitive measure to taxonomic level, having a convex parabola at the species subgroup level in support of the hourglass model and a concave parabola at the subgeneric level in support of the adaptive penetrance model. This time‐dependent discrepancy of best fit developmental model parallels previous conflicting results from the vertebrates. Because of the quasi‐phenetic nature of this index, we argue that the discrepancy is due to the evolutionary rate heterogeneity of developmental genes rather than to fundamental differences among organisms. We suggest that simultaneous character‐based analyses give better macroevolutionary support to the hourglass model of the developmental constraints on genome evolution than pairwise phenetic comparisons.