A Non-Synonymous HMGA2 Variant Decreases Height in Shetland Ponies and Other Small Horses

The identification of quantitative trait loci (QTL) such as height and their underlying causative variants is still challenging and often requires large sample sizes. In humans hundreds of loci with small effects control the heritable portion of height variability. In domestic animals, typically only a few loci with comparatively large effects explain a major fraction of the heritability. We investigated height at withers in Shetland ponies and mapped a QTL to ECA 6 by genome-wide association (GWAS) using a small cohort of only 48 animals and the Illumina equine SNP70 BeadChip. Fine-mapping revealed a shared haplotype block of 793 kb in small Shetland ponies. The HMGA2 gene, known to be associated with height in horses and many other species, was located in the associated haplotype. After closing a gap in the equine reference genome we identified a non-synonymous variant in the first exon of HMGA2 in small Shetland ponies. The variant was predicted to affect the functionally important first AT-hook DNA binding domain of the HMGA2 protein (c.83G>A; p.G28E). We assessed the functional impact and found impaired DNA binding of a peptide with the mutant sequence in an electrophoretic mobility shift assay. This suggests that the HMGA2 variant also affects DNA binding in vivo and thus leads to reduced growth and a smaller stature in Shetland ponies. The identified HMGA2 variant also segregates in several other pony breeds but was not found in regular-sized horse breeds. We therefore conclude that we identified a quantitative trait nucleotide for height in horses.     

Both developmental and metabolic signals activate the promoter of a class I patatin gene

Patatin is one of the major soluble proteins in potato tubers and is encoded by a multigene family. Based on structural considerations two classes of patatin genes are distinguished. The 5′-upstream regulatory region of a class I gene contained within a 1.5 kb sequence is essential and sufficient to direct a high level of tuber-specific gene activity which was on average 100- to 1000-fold higher in tubers as compared to leaf, stem and roots in greenhouse grown transgenic potato plants when fused to the β-glucuronidase reporter gene. Histochemical analysis revealed this activity to be present in parenchymatic tissue but not in the peripheral phellem cells of transgenic tubers. Furthermore the promoter fragment can be activated in leaves under conditions that simulate the need for the accumulation of starch in storage organs, i.e. high levels of sucrose. The expression is restricted to both mesophyll and epidermal cells in contrast to vascular tissue or hair cells.     

Reduced Glycine Stimulation of [3H]MK-801 Binding in Alzheimer''s Disease

The novel N-methyl-D-aspartate receptor channel ligand (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine maleate ([3H]MK-801) has been utilized to label this receptor in human brain tissue. Characteristics of [3H]MK-801 binding to well-washed membranes from 17 control subjects and 16 patients with Alzheimer's disease were determined in frontal, parietal, and temporal cerebral cortex and cerebellar cortex. In control tissue the pharmacological specificity of the binding of this substance is entirely consistent with the profile previously reported for rat brain. Binding could be stimulated by the addition of glutamic acid to the incubation medium; addition of glycine produced further enhancement which was not prevented by strychnine. The specificity of the effects of these and other amino acids on the binding was the same as in the rat. In Alzheimer's disease significantly less binding was observed in the frontal cortex under glutamate- and glycine-stimulated conditions. This appears to be associated with a reduced affinity of the site whereas the pharmacological specificity of the site remained unchanged. The effect did not appear to be due to differences in mode of death between Alzheimer's disease and control subjects and is unlikely to be related to factors for which the groups were matched. In contrast, binding was not altered in the absence of added amino acids and presence of glutamate alone. These results imply that in the cerebral cortex the agonist site and a site in the cation channel of the receptor are not selectively altered, but that their coupling to a strychnine-insensitive glycine recognition site is impaired.     

The unexpected long period of elevated CH4 emissions from an inundated fen meadow ended only with the occurrence of cattail (Typha latifolia)

Drainage and agricultural use transform natural peatlands from a net carbon (C) sink to a net C source. Rewetting of peatlands, despite of high methane (CH₄) emissions, holds the potential to mitigate climate change by greatly reducing CO₂ emissions. However, the time span for this transition is unknown because most studies are limited to a few years. Especially, nonpermanent open water areas often created after rewetting, are highly productive. Here, we present 14 consecutive years of CH₄ flux measurements following rewetting of a formerly long-term drained peatland in the Peene valley. Measurements were made at two rewetted sites (non-inundated vs. inundated) using manual chambers. During the study period, significant differences in measured CH₄ emissions occurred. In general, these differences overlapped with stages of ecosystem transition from a cultivated grassland to a polytrophic lake dominated by emergent helophytes, but could also be additionally explained by other variables. This transition started with a rapid vegetation shift from dying cultivated grasses to open water floating and submerged hydrophytes and significantly increased CH₄ emissions. Since 2008, helophytes have gradually spread from the shoreline into the open water area, especially in drier years. This process was periodically delayed by exceptional inundation and eventually resulted in the inundated site being covered by emergent helophytes. While the period between 2009 and 2015 showed exceptionally high CH₄ emissions, these decreased significantly after cattail and other emergent helophytes became dominant at the inundated site. Therefore, CH₄ emissions declined only after 10 years of transition following rewetting, potentially reaching a new steady state. Overall, this study highlights the importance of an integrative approach to understand the shallow lakes CH₄ biogeochemistry, encompassing the entire area with its mosaic of different vegetation forms. This should be ideally done through a study design including proper measurement site allocation as well as long-term measurements.     

Cholinomimetics Increase Glutamate Outflow via an Action on the Corticostriatal Pathway: Implications for Alzheimer's Disease

Abstract: Physostigmine, the acetylcholinesterase inhibitor (0.3 mg/kg, i.m.), increased extracellular glutamate but not aspartate concentrations in the striatum of anaesthetised rats, determined using microdialysis and HPLC. The rise was both tetrodotoxin and calcium dependent. In contrast, neither physostigmine (10 µ M ) added to the perfusion fluid nor vehicle (injected intramuscularly) affected amino acid concentrations. To obtain evidence that the action of acetylcholine was to modulate positively cortical pyramidal neurone activity via the M₁ receptor, the selective M₁ agonist PD 142505-0028 (10 µ M ) was topically applied to the frontal cortex. Like physostigmine, PD 142505-0028 rapidly increased glutamate but not aspartate concentrations in the striatum. Moreover, the effect of intramuscular physostigmine was blocked by a topically applied M₁ antagonist. These new data add to our hypothesis that cholinomimetics increase pyramidal neurone function.     

Development of a pollen-mediated transformation method forNicotiana glutinosa

The development is described of a new procedure to genetically transform plant species using the male gametophyte as a natural transformation vector. Our system avoids the need for complicated regeneration procedures thus making it broadly applicable. Naked plasmid DNA encoding kanamycin resistance and GUS activity was introduced by particle gun bombardment into mature pollen grains ofNicotiana glutinosa. Bombarded pollen was used for pollinations and the resulting seeds were selected for kanamycin resistance. Two different kanamycin-resistant plants, designated VIP A and VIP B, were obtained in two independent experiments. In VIP A, TR2-driven GUS activity was observed in vascular bundles, trichomes and in a small number of pollen grains. DNA gel blot analysis indicated that the introduced DNA was integrated independently into the genome of VIP A and VIP B. It was shown that male and female gametophyte development and seed set were highly aberrant in both VIP A and VIP B and that the offspring of self- and cross-pollinations did not contain the transgenes. This might be caused by a recombination event during the integration of the naked DNA resulting in a deletion of part of the target chromosome. After meiosis such a deletion is lethal for the gametes. Our observation that the transgenes were detected in DNA isolated from sporophytic tissues but not in DNA from VIP A and VIP B pollen grains is in line with this explanation. Future experiments designed to increase the frequency of transformation and to transfer the transgenes to the offspring are discussed.     

Characterisation of the Glycine Modulatory Site of the N-Methyl-d-Aspartate Receptor-Ionophore Complex in Human Brain

[3H]Glycine binding and glycine modulation of [3H]MK-801 binding have been used to study the glycine allosteric site associated with the N-methyl-D-aspartate receptor complex in postmortem human brain. The effect of glycine on [3H]MK-801 binding appeared sensitive to duration of terminal coma, and possibly postmortem delay. Thirty percent of the binding occurred in a subfraction of brain tissue and did not show enhancement by glycine and glutamic acid. [3H]Glycine binding to a subfraction free from this component was studied and showed high specific binding. KD and Bmax values showed considerable intersubject variability which did not appear to be due to demographic features or to tissue content of amino acids with an affinity for this site. The pharmacological characteristics of binding in this subfraction and a correlation between Bmax values and the maximal enhancement of [3H]MK-801 binding by glycine are consistent with [3H]glycine binding occurring to an N-methyl-D-aspartate receptor complex associated site. Further support for this is provided by a significantly lower Bmax value for [3H]glycine binding in subjects with Alzheimer's disease and reduced glycine enhancement of [3H]MK-801 binding. However, the effect of perimortem factors makes it difficult to confidently attribute this solely to a disease-related change in the receptor. The possible role of the glycine allosteric site in the treatment of neuropsychiatric disorders is discussed.     

Electron probe X-ray microanalysis of calcium and phosphorus distribution in developing hamster tooth germs in vitro and in vivo

The aim of the present study was to investigate the spatial distribution of Ca and P in dentin and enamel of developing first (M1) and second (M2) maxillary hamster molars (age: 3-5 days) in comparison with cultured molars. For culturing the germs were dissected from 3-day-old hamsters and incubated for 1 and 2 days, respectively, in a modified BGJb medium. Electron probe X-ray measurements were carried out on 3 regions extending in a vertical axis from cusp tip over cusp middle to cusp base next to the cervical loop region. Neither the in vivo nor the in vitro group was statistically different in the Ca and P concentration in the regions of dentin. In both groups the measurements in enamel showed a gradient with an increase in Ca and P from enamel surface towards dentin-enamel junction and a gradient with an increase from cusp base towards cusp tip. Direct comparison of the in vivo group with the in vitro group did not demonstrate a statistical difference between the mineral content of the 4-day-old germs and the 1-day culture germs, respectively the 5-day-old germs and the 2-day culture germs. The results indicate a high correspondence between the mineralization process of in vitro and in vivo tooth germ development.