Magnesium-Dependent Enhancement of Endogenous Agonist Binding to A1 Adenosine Receptors: A Complicating Factor in Quantitative Autoradiography

Quantitative autoradiography was used to investigate the effects of Mg2+ on agonist and antagonist binding to A1 receptors in rat striatum. A1 receptors were labelled with the selective agonist N6-[3H]cyclohexyladenosine ([3H]CHA) or the selective antagonist 1,3-[3H]dipropyl-8-cyclopentylxanthine ([3H]DPCPX). Mg2+ had no significant effect on equilibrium binding constants for [3H]CHA [control: KD (95% confidence interval) of 0.34 (0.15-0.80) nM and Bmax of 267 +/- 8 fmol/mg of gray matter; with 10 mM Mg2+: KD of 0.8 (0.13-4.9) nM and Bmax of 313 +/- 8.9 fmol/mg of gray matter] or [3H]DPCPX [control: KD of 0.54 (0.30-0.99) nM and Bmax of 256 +/- 2.3 fmol/mg of gray matter; with 10 mM Mg2+: KD of 1.54 (0.2-11.0) nM and Bmax of 269 +/- 35.7 fmol/mg of gray matter]. In contrast, Mg2+ slowed the apparent association rate for both ligands; this was observed as a shift from a one-component to a two-component model for [3H]DPCPX. Mg2+ also affected the dissociation rates of both ligands; for [3H]CHA, dissociation in the presence of Mg2+ was not detected. Mg2+ produced a concentration-dependent inhibition of [3H]CHA binding only prior to equilibrium. HPLC was performed on untreated sections, sections preincubated with adenosine deaminase (ADA), and sections preincubated with ADA and incubated with ADA in the absence or presence of Mg2+. Adenosine was found in measurable quantities under all conditions, and the concentration was not influenced by Mg2+ or by the inclusion of GTP in the preincubation medium. From these data, we conclude the following: (a) adenosine is present and may be produced continuously in brain sections; (b) ADA is not capable of completely eliminating the produced adenosine; (c) Mg2+ apparently does not influence adenosine production or elimination; (d) A1 receptor-guanine nucleotide binding protein coupling is maximal in this preparation; and (e) Mg2+ decreases the dissociation rate of bound endogenous adenosine from A1 receptors, thus limiting the access of [3H]CHA and [3H]DPCPX to the receptors. Thus, enhancement of endogenous adenosine binding to A1 receptors by Mg2+ is a complicating factor in receptor autoradiography and may be so in other preparations as well.     

Characterization of the basic charge variants of a human IgG1: Effect of copper concentration in cell culture media

We report a case study of an IgG1 with a unique basic charge variant profile caused by C-terminal proline amidation on either one or two heavy chains. The proline amidation was sensitive to copper ion concentration in the production media during cell culture: the higher the Cu²⁺ ion concentration, the higher the level of proline amidation detected. This conclusion was supported by the analysis of samples that revealed direct correlation between the proline amidation level observed from peptide maps and the level of basic peaks measured by imaged capillary isoelectric focusing and a pH gradient ion-exchange chromatography method. The importance of these observations to therapeutic antibody production is discussed.Key words: antibody, basic charge variants, proline amidation, copper, mass spectrometry     

Rain Forest Islands in the Chilean Semiarid Region: Fog-dependency, Ecosystem Persistence and Tree Regeneration

Tree presence in semiarid ecosystems is generally constrained by insufficient annual rainfall. However, in semiarid Chile, rainforest patches dominated by Aextoxicon punctatum are unexpectedly found on coastal mountaintops (450–600 m) at 30°S, surrounded by a xerophytic vegetation matrix that receives only 147 mm of annual precipitation. It has been proposed that these forests persist as a result of fog-water inputs. If so, then because fog-water deposition is spatially heterogeneous and shows strong edge effects, the potential environmental gradient created by the direction of fog input should determine forest structure and tree regeneration patterns. To investigate this hypothesis, we measured fog inputs, forest structural attributes (age and size distribution, basal area, and coarse woody debris), and tree regeneration in three different habitats: the windward edges (WE), leeward edges (LE), and the interior (center) of rainforest patches varying in area from 0.2 to 22 ha. Mean fog-water input was estimated from passive collectors over 1 year in WE and LE of patches. Tree regeneration was greater in the WE and forest interior (FI) and decreased toward the LE of patches, following a marked pattern of decline in fog inputs. Older trees and coarse woody debris were concentrated in the FI and LE of patches. Tree regeneration and patch structure appear to be largely controlled by fog-input direction and edge effects. We propose that forest patches may be slowly growing toward the incoming fog edge, while dying at the opposite edge.     

Epidermal stem cells are retained in vivo throughout skin aging

In healthy individuals, skin integrity is maintained by epidermal stem cells which self-renew and generate daughter cells that undergo terminal differentiation. It is currently unknown whether epidermal stem cells influence or are affected by skin aging. We therefore compared young and aged skin stem cell abundance, organization, and proliferation. We discovered that despite age-associated differences in epidermal proliferation, dermal thickness, follicle patterning, and immune cell abundance, epidermal stem cells were maintained at normal levels throughout life. These findings, coupled with observed dermal gene expression changes, suggest that epidermal stem cells themselves are intrinsically aging resistant and that local environmental or systemic factors modulate skin aging.     

Agrobacterium tumefaciens VirB9, an outer-membrane-associated component of a type IV secretion system, regulates substrate selection and T-pilus biogenesis

Agrobacterium tumefaciens translocates DNA and protein substrates between cells via a type IV secretion system (T4SS) whose channel subunits include the VirD4 coupling protein, VirB11 ATPase, VirB6, VirB8, VirB2, and VirB9. In this study, we used linker insertion mutagenesis to characterize the contribution of the outer-membrane-associated VirB9 to assembly and function of the VirB/D4 T4SS. Twenty-five dipeptide insertion mutations were classified as permissive for intercellular substrate transfer (Tra+), completely transfer defective (Tra-), or substrate discriminating, e.g., selectively permissive for transfer only of the oncogenic transfer DNA and the VirE2 protein substrates or of a mobilizable IncQ plasmid substrate. Mutations inhibiting transfer of DNA substrates did not affect formation of close contacts of the substrate with inner membrane channel subunits but blocked formation of contacts with the VirB2 and VirB9 channel subunits, which is indicative of a defect in assembly or function of the distal portion of the secretion channel. Several mutations in the N- and C-terminal regions disrupted VirB9 complex formation with the outer-membrane-associated lipoprotein VirB7 or the inner membrane energy sensor VirB10. Several VirB9.i2-producing Tra+ strains failed to elaborate T pilus at detectable levels (Pil-), and three such Tra+ Pil- mutant strains were rendered Tra- upon deletion of virB2, indicating that the cellular form of pilin protein is essential for substrate translocation. Our findings, together with computer-based analyses, support a model in which distinct domains of VirB9 contribute to substrate selection and translocation, establishment of channel subunit contacts, and T-pilus biogenesis.     

The application of CRISPR‐Cas for single species identification from environmental DNA

We report the first application of CRISPR‐Cas technology to single species detection from environmental DNA (eDNA). Organisms shed and excrete DNA into their environment such as in skin cells and faeces, referred to as environmental DNA (eDNA). Utilising eDNA allows noninvasive monitoring with increased specificity and sensitivity. Current methods primarily employ PCR‐based techniques to detect a given species from eDNA samples, posing a logistical challenge for on‐site monitoring and potential adaptation to biosensor devices. We have developed an alternative method; coupling isothermal amplification to a CRISPR‐Cas12a detection system. This utilises the collateral cleavage activity of Cas12a, a ribonuclease guided by a highly specific single CRISPR RNA. We used the target species Salmo salar as a proof‐of‐concept test of the specificity of the assay among closely related species and to show the assay is successful at a single temperature of 37°C with signal detection at 535 nM. The specific assay, detects at attomolar sensitivity with rapid detection rates (<2.5 hr). This approach simplifies the challenge of building a biosensor device for rapid target species detection in the field and can be easily adapted to detect any species from eDNA samples from a variety of sources enhancing the capabilities of eDNA as a tool for monitoring biodiversity.     

Effects of storage type and time on DNA amplification success in tropical ungulate faeces

The present study compares the effect of three storage media (silica, RNAlater®, ethanol) and time to extraction (1 week, 1 month and 3 months) on mitochondrial and nuclear marker amplification success in faecal DNA extracts from a sympatric community of small to medium‐sized Central African forest ungulates (genera Cephalophus, Tragelaphus, Hyemoschus). The effect of storage type and time on nuclear DNA concentrations, genotyping errors and percentage recovery of consensus genotypes was also examined. Regardless of storage method, mitochondrial and nuclear amplification success was high in DNA extracted within the first week after collection. Over longer storage periods, RNAlater yielded better amplification success rates in the mitochondrial assay. However, samples stored on silica showed (i) highest nuclear DNA concentrations, (ii) best microsatellite genotyping success, (iii) lowest genotyping errors, and (iv) greatest percentage recovery of the consensus genotype. The quantity of nuclear DNA was generally a good predictor of microsatellite performance with 83% amplification success or greater achieved with sample DNA concentrations of ≥ 50 pg/µL. If faecal DNA samples are to be used for nuclear microsatellite analyses, we recommend silica as the best storage method. However, for maximum mitochondrial amplification success, RNAlater appears to be the best storage medium. In contrast, ethanol appeared inferior to the other two methods examined here and should not be used to store tropical ungulate faeces. Regardless of storage method, samples should be extracted as soon as possible after collection to ensure optimal recovery of DNA.     

Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosis

The lethal genetic disease cystic fibrosis is caused predominantly by in‐frame deletion of phenylalanine 508 in the cystic fibrosis transmembrane conductance regulator (CFTR). F508 is located in the first nucleotide‐binding domain (NBD1) of CFTR, which functions as an ATP‐gated chloride channel on the cell surface. The F508del mutation blocks CFTR export to the surface due to aberrant retention in the endoplasmic reticulum. While it was assumed that F508del interferes with NBD1 folding, biophysical studies of purified NBD1 have given conflicting results concerning the mutation's influence on domain folding and stability. We have conducted isothermal (this paper) and thermal (accompanying paper) denaturation studies of human NBD1 using a variety of biophysical techniques, including simultaneous circular dichroism, intrinsic fluorescence, and static light‐scattering measurements. These studies show that, in the absence of ATP, NBD1 unfolds via two sequential conformational transitions. The first, which is strongly influenced by F508del, involves partial unfolding and leads to aggregation accompanied by an increase in tryptophan fluorescence. The second, which is not significantly influenced by F508del, involves full unfolding of NBD1. Mg‐ATP binding delays the first transition, thereby offsetting the effect of F508del on domain stability. Evidence suggests that the initial partial unfolding transition is partially responsible for the poor in vitro solubility of human NBD1. Second‐site mutations that increase the solubility of isolated F508del‐NBD1 in vitro and suppress the trafficking defect of intact F508del‐CFTR in vivo also stabilize the protein against this transition, supporting the hypothesize that it is responsible for the pathological trafficking of F508del‐CFTR.     

Activation of nuclear factor kappa B in mammary epithelium promotes milk loss during mammary development and infection

We investigated whether nuclear factor kappa B (NF‐κB), which exhibits a regulated pattern of activity during murine mammary gland development, plays an important role during lactation and involution, when milk production ceases and the gland undergoes apoptosis and re‐modeling. We generated a doxycycline inducible transgenic mouse model to activate NF‐κB specifically in the mammary epithelium through expression of a constitutively active form of IKK2, the upstream kinase in the classical NF‐κB signaling cascade. We found that activation of NF‐κB during involution resulted in a more rapid reduction in milk levels and increased cleavage of caspase‐3, an indicator of apoptosis. We also found that activation of NF‐κB during lactation with no additional involution signals had a similar effect. The observation that NF‐κB is a key regulator of milk production led us to investigate the role of NF‐κB during mastitis, an infection of the mammary gland in which milk loss is observed. Mammary gland injection of E. coli LPS resulted in activation of NF‐κB and milk loss during lactation. This milk loss was decreased by selective inhibition of NF‐κB in mammary epithelium. Together, our data reveal that activation of NF‐κB leads to milk clearance in the lactating mammary gland. Therefore, targeting of NF‐κB signaling may prove therapeutic during mastitis in humans and could be beneficial for the dairy industry, where such infections have a major economic impact. J. Cell. Physiol. 222:73–81, 2010. © 2009 Wiley‐Liss, Inc.