A Three-Dimensional Skeletal Reconstruction of the Stem Amniote Orobates pabsti (Diadectidae): Analyses of Body Mass,Centre of Mass Position,and Joint Mobility

Orobates pabsti, a basal diadectid from the lower Permian, is a key fossil for the understanding of early amniote evolution. Quantitative analysis of anatomical information suffers from fragmentation of fossil bones, plastic deformation due to diagenetic processes and fragile preservation within surrounding rock matrix, preventing further biomechanical investigation. Here we describe the steps taken to digitally reconstruct MNG 10181, the holotype specimen of Orobates pabsti, and subsequently use the digital reconstruction to assess body mass, position of the centre of mass in individual segments as well as the whole animal, and study joint mobility in the shoulder and hip joints. The shape of most fossil bone fragments could be recovered from micro-focus computed tomography scans. This also revealed structures that were hitherto hidden within the rock matrix. However, parts of the axial skeleton had to be modelled using relevant isolated bones from the same locality as templates. Based on the digital fossil, mass of MNG 10181 was estimated using a model of body shape that was varied within a plausible range to account for uncertainties of the dimension. In the mean estimate model the specimen had an estimated mass of circa 4 kg. Varying of the mass distribution amongst body segments further revealed that Orobates carried most of its weight on the hind limbs. Mostly unrestricted joint morphology further suggested that MNG 10181 was able to effectively generate propulsion with the pelvic limbs. The digital reconstruction is made available for future biomechanical studies.     

High-resolution SNP scan of chromosome 6p21 in pooled samples from patients with complex diseases

We apply a high-throughput protocol of chip-based mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight; MALDI-TOF) as a method of screening for differences in single-nucleotide polymorphism (SNP) allele frequencies. Using pooled DNA from individuals with asthma, Crohn's disease (CD), schizophrenia, type 1 diabetes (T1D), and controls, we selected 534 SNPs from an initial set of 1435 SNPs spanning a 25-Mb region on chromosome 6p21. The standard deviations of measurements of time of flight at different dots, from different PCRs, and from different pools indicate reliable results on each analysis step. In 90% of the disease-control comparisons we found allelic differences of <10%. Of the T1D samples, which served as a positive control, 10 SNPs with significant differences were observed after taking into account multiple testing. Of these 10 SNPs, 5 are located between DQB1 and DRB1, confirming the known association with the DR3 and DR4 haplotypes whereas two additional SNPs also reproduced known associations of T1D with DOB and LTA. In the CD pool also, two earlier described associations were found with SNPs close to DRB1 and MICA. Additional associations were found in the schizophrenia and asthma pools. They should be confirmed in individual samples or can be used to develop further quality criteria for accepting true differences between pools. The determination of SNP allele frequencies in pooled DNA appears to be of value in assigning further genotyping priorities also in large linkage regions.     

Disoriented growth of pollen tubes of Lilium longiflorum Thunb. induced by prolonged treatment with the calcium-chelating antibiotic,chlorotetracycline

Pollen of Lilium longiflorum Thunb. was germinated for 12 h in growth medium containing 1·10^-4 M chlorotetracycline (CTC), or growing tubes were treated with 1·10^-4 M CTC for up to 2 h. These treatments have drastic effects: In the CTC-containing medium, out-growing tubes form only short tubes. Irregular wall thickenings are visible. Thirty minutes CTC-treatment cause growing tubes to bend and grow back toward the grain. Electron micrographs of CTC-treated tubes show that CTC affects the organelle distribution: The polar zonation of organelles is disturbed. Vesicle-and endoplasmic reticulum-accumulations are found in the wrong places, together with extensive wall thickenings and a very irregular plasma membrane. The structural details of most cell organelles look normal after CTC treatment, but the mitochondria possess unusual cristae, and microtubules are absent. The disoriented growth is interpreted as an effect of the ability of CTC to chelate intracellular calcium ions, to bind them to membranes, and thus to disturb the dynamics of the delicate Ca²⁺-equilibria thought to regulate oriented exocytosis.Abbreviations CTC chlorotetracycline - ER endoplasmic reticulum     

Exome sequences and multi‐environment field trials elucidate the genetic basis of adaptation in barley

Broadening the genetic base of crops is crucial for developing varieties to respond to global agricultural challenges such as climate change. Here, we analysed a diverse panel of 371 domesticated lines of the model crop barley to explore the genetics of crop adaptation. We first collected exome sequence data and phenotypes of key life history traits from contrasting multi‐environment common garden trials. Then we applied refined statistical methods, including some based on exomic haplotype states, for genotype‐by‐environment (G×E) modelling. Sub‐populations defined from exomic profiles were coincident with barley's biology, geography and history, and explained a high proportion of trial phenotypic variance. Clear G×E interactions indicated adaptation profiles that varied for landraces and cultivars. Exploration of circadian clock‐related genes, associated with the environmentally adaptive days to heading trait (crucial for the crop's spread from the Fertile Crescent), illustrated complexities in G×E effect directions, and the importance of latitudinally based genic context in the expression of large‐effect alleles. Our analysis supports a gene‐level scientific understanding of crop adaption and leads to practical opportunities for crop improvement, allowing the prioritisation of genomic regions and particular sets of lines for breeding efforts seeking to cope with climate change and other stresses.     

Effects of heat and drought stress on post‐illumination bursts of volatile organic compounds in isoprene‐emitting and non‐emitting poplar

Over the last decades, post‐illumination bursts (PIBs) of isoprene, acetaldehyde and green leaf volatiles (GLVs) following rapid light‐to‐dark transitions have been reported for a variety of different plant species. However, the mechanisms triggering their release still remain unclear. Here we measured PIBs of isoprene‐emitting (IE) and isoprene non‐emitting (NE) grey poplar plants grown under different climate scenarios (ambient control and three scenarios with elevated CO₂ concentrations: elevated control, periodic heat and temperature stress, chronic heat and temperature stress, followed by recovery periods). PIBs of isoprene were unaffected by elevated CO₂ and heat and drought stress in IE, while they were absent in NE plants. On the other hand, PIBs of acetaldehyde and also GLVs were strongly reduced in stress‐affected plants of all genotypes. After recovery from stress, distinct differences in PIB emissions in both genotypes confirmed different precursor pools for acetaldehyde and GLV emissions. Changes in PIBs of GLVs, almost absent in stressed plants and enhanced after recovery, could be mainly attributed to changes in lipoxygenase activity. Our results indicate that acetaldehyde PIBs, which recovered only partly, derive from a new mechanism in which acetaldehyde is produced from methylerythritol phosphate pathway intermediates, driven by deoxyxylulose phosphate synthase activity.     

Patterns of expression of trichocytic and epithelial cytokeratins in mammalian tissues II. Concomitant and mutually exclusive synthesis of trichocytic and epithelial cytokeratins in diverse human and bovine tissues (hair follicle, nail bed and matrix, lingual papilla, thymic reticulum)

The hair-forming cells (trichocytes) and the mature hair contain four major trichocytic cytokeratins from each of the subfamilies, basic (Hb1-4) and acidic (Ha1-4); these are related - but not identical - to the epithelial cytokeratins. Here we show, by biochemical methods and immunofluorescence microscopy using antibodies specific for either epithelial or trichocyte cytokeratins, that the same set of hair-type cytokeratins, including two newly identified minor components, designated Hax (type I) and Hbx (type II), are also expressed in cells forming nails, in the filiform papillae of the dorsal surface of human and bovine tongue, and, most surprisingly, in some cells of the epithelial reticulum of bovine and human thymus. By double-label immunofluorescence microscopy, we also show that the expression of the two subsets of cytokeratins, i.e., the epithelial and the trichocytic ones, is not necessarily mutually exclusive, but that certain cells of hair follicles, nail matrix and bed, lingual papillae, and the nonlymphoid cell system of the thymus contain both trichocytic and certain epithelial cytokeratins. This indicates that these cells coexpress representatives of both kinds of cytokeratin. Implications of these findings with respect to problems of regulatory control of cytokeratin synthesis in tissue development and differentiation, and the possible functional meaning of the occurrence of trichocytic cytokeratins in such histologically diverse tissues, are discussed.     

Identification of Merkel cells in human skin by specific cytokeratin antibodies:

Merkel cells are special neurosecretory cells which, in adult human skin, are usually very scarce. By immunofluorescence microscopy using antibodies to human cytokeratin polypeptide no. 18, we localized distinct non-keratinocyte cells in the glandular ridges of human fetal and adult plantar epidermis. Using electron and immunofluorescence microscopy, these cells were identified as Merkel cells containing typical neurosecretory granules as well as bundles of intermediate-sized filaments and desmosomes. Two-dimensional gel electrophoresis of the cytoskeletal fractions of microdissected epidermal preparations highly enriched in Merkel cells indicated the presence of cytokeratin polypeptides nos. 8, 18 and 19 which are typical of diverse simple epithelia of the human body. Double immunofluorescence microscopy showed that these human Merkel cells contain neither neurofilaments nor vimentin filaments. In human fetuses of 18-24 weeks of age, conspicuously high concentrations of Merkel cells, reaching a density of approximately 1,700 Merkel cells/mm2 skin, were found in the glandular ridges of plantar skin. The concentration decreased considerably at newborn and adult stages. Thin cell processes (up to 20 microns long) were observed in many fetal epidermal Merkel cells. In addition, we detected isolated Merkel cells deeper in the dermis (i.e. at distances of, at most, 100 microns from the epidermis) in fetal and newborn plantar skin. Our results show that Merkel cells are true epithelial cells which, however, differ profoundly from epidermal keratinocytes in their cytokeratin expression. The findings are discussed in relation to the much disputed question of the origin of Merkel cells. The present data speak against the immigration of Merkel cells from the neural crest, but rather suggest that they originate from epithelial cells of the skin, although most probably not from differentiated keratinocytes.     

A TEA-insensitive flickering potassium channel active around the resting potential in myelinated nerve

A novel potassium-selective channel which is active at membrane potentials between -100 mV and +40 mV has been identified in peripheral myelinated axons of Xenopus laevis using the patch-clamp technique. At negative potentials with 105 mM-K on both sides of the membrane, the channel at 1 kHz resolution showed a series of brief openings and closings interrupted by longer closings, resulting in a flickery bursting activity. Measurements with resolution up to 10 kHz revealed a single-channel conductance of 49 pS with 105 mM-K and 17 pS with 2.5 mM-K on the outer side of the membrane. The channel was selective for K ions over Na ions (PNa/PK = 0.033). The probability of being within a burst in outside-out patches varied from patch to patch (> 0.2, but often > 0.9), and was independent of membrane potential. Open-time histograms were satisfactorily described with a single exponential (tau o = 0.09 msec), closed times with the sum of three exponentials (tau c = 0.13, 5.9, and 36.6 msec). Sensitivity to external tetraethylammonium was comparatively low (IC50 = 19.0 mM). External Cs ions reduced the apparent unitary conductance for inward currents at Em = -90 mV (IC50 = 1.1 mM). Ba and, more potently, Zn ions lowered not only the apparent single-channel conductance but also open probability. The local anesthetic bupivacaine with high potency reduced probability of being within a burst (IC50 = 165 nM). The flickering K channel is clearly different from the other five types of K channels identified so far in the same preparation. We suggest that this channel may form the molecular basis of the resting potential in vertebrate myelinated axons.     

Histological examination of spruce needles from a long-term gas exchange experiment in pure and polluted air in the field

Summary At the end of a 4-year period of gas exchange measurements in a natural stand in the Lower Bavarian Forest, needles of an adult spruce [Picea abies (L.) Karst.] were harvested from two chambers, one with pure air and the other with ambient air. The needles were examined as to their histological properties in the stomatal apparatus and in the bundle sheath. In needles from the polluted air UV absorbance at 280 nm was decreased in the walls of the stomatal apparatus. Simultaneously, the deposition of compounds with an absorption maximum at 310 nm increased within the encrusted plate-like thickenings of the subsidiary cells. The contents of the lumina of hypodermal cells and of the bundle sheath exhibited a greater degree of autofluorescence in ambient-air material than in pure-air leaf organs. Differences between needles exposed to pure and polluted air are gradual. The damaged condition is rare in pure air, common in polluted air. The needles from outside the chambers occupied an intermediate position between pure-air and ambient-air needles. This fact is traced to an unnaturally high pollutant load in the liquid phase of the needle surfaces within the ambient-air chamber because in order to compensate pollutant losses within the system, SO₂ and O₃ were added even during periods of irrigation. The reduction of absorption capacity at 280 nm in the walls of the stomatal apparatus is attributed to destruction of lignin due to the high reactivity of the pollutants in the liquid phase on the damp needle surface. The importance of delignification with regard to hydroregulation is discussed.