Natural and anthropogenic variations in methane sources during the past two millennia.

Methane is an important greenhouse gas that is emitted from multiple natural and anthropogenic sources. Atmospheric methane concentrations have varied on a number of timescales in the past, but what has caused these variations is not always well understood. The different sources and sinks of methane have specific isotopic signatures, and the isotopic composition of methane can therefore help to identify the environmental drivers of variations in atmospheric methane concentrations. Here we present high-resolution carbon isotope data (δ(13)C content) for methane from two ice cores from Greenland for the past two millennia. We find that the δ(13)C content underwent pronounced centennial-scale variations between 100 BC and AD 1600. With the help of two-box model calculations, we show that the centennial-scale variations in isotope ratios can be attributed to changes in pyrogenic and biogenic sources. We find correlations between these source changes and both natural climate variability--such as the Medieval Climate Anomaly and the Little Ice Age--and changes in human population and land use, such as the decline of the Roman empire and the Han dynasty, and the population expansion during the medieval period.

Significant contribution of small icebergs to the freshwater budget in Greenland fjords.

Icebergs represent nearly half of the mass loss from the Greenland Ice Sheet and provide a distributed source of freshwater along fjords which can alter fjord circulation, nutrient levels, and ultimately the Meridional Overturning Circulation. Here we present analyses of high resolution optical satellite imagery using convolutional neural networks to accurately delineate iceberg edges in two East Greenland fjords. We find that a significant portion of icebergs in fjords are comprised of small icebergs that were not detected in previously-available coarser resolution satellite images. We show that the preponderance of small icebergs results in high freshwater delivery, as well as a short life span of icebergs in fjords. We conclude that an inability to identify small icebergs leads to inaccurate frequency-size distribution of icebergs in Greenland fjords, an underestimation of iceberg area (specifically for small icebergs), and an overestimation of iceberg life span.

Friction at the bed does not control fast glacier flow.

The largest uncertainty in the ice sheet models used to predict future sea level rise originates from our limited understanding of processes at the ice/bed interface. Near glacier termini, where basal sliding controls ice flow, most predictive ice sheet models use a parameterization of sliding that has been theoretically derived for glacier flow over a hard bed. We find that this sliding relation does not apply to the 140 Greenland glaciers that we analyzed. There is no relationship between basal sliding and frictional stress at the glacier bed, contrary to theoretical predictions. There is a strong relationship between sliding speed and net pressure at the glacier bed. This latter finding is in agreement with earlier observations of mountain glaciers that have been largely overlooked by the glaciological community.

Inhibition of mitochondrial protein synthesis influences the glucocorticoid sensitivity of lymphoid cells.

Inhibition of mitochondrial protein synthesis impairs the formation of the 13 polypeptides encoded on the mitochondrial genome. These polypeptides are part of enzyme complexes involved in oxidative phosphorylation. Prolonged inhibition of mitochondrial protein synthesis thus reduces the oxidative phosphorylation capacity which ultimately results in impairment of energy-requiring processes. Via a different mechanism glucocorticoid hormones also decrease the oxidative phosphorylation capacity of, e.g., lymphoid cells. The present study shows that inhibition of mitochondrial protein synthesis influences glucocorticoid-induced responses of lymphoid cells in two opposing manners. (a) It is enhanced after induction in cells with a reduced oxidative phosphorylation capacity resulting from preceding inhibition of mitochondrial protein synthesis. This can be explained by the synergistic effects of glucocorticoids and prolonged inhibition of mitochondrial protein synthesis on energy-producing processes. (b) It is counteracted when mitochondrial protein synthesis is impaired during induction of the response. The latter observation suggests that mitochondrial protein synthesis is involved in the generation of glucocorticoid-induced effects on lymphoid cells.

Lipids and oxidised lipids in human atherosclerotic lesions at different stages of development.

Lipids and oxidised lipids were analysed by GC and GC-MS in human necropsy samples of normal artery and individual atherosclerotic lesions, from aorta and common carotid artery, including fatty streaks, intermediate lesions and advanced lesions. Age-related increases were seen for linoleate, oleate and cholesterol in normal artery, but not in lesions. Each category of lesion was much richer than normal artery in all the lipids measured and in oxidised lipids (oxysterols and hydroxyoctadecadienoic acids), although a degree of overlap existed between the compositions of the various categories of lesion. 26-Hydroxycholesterol and 7 beta-hydroxycholesterol levels were extremely low or undetectable in normal artery, but significantly higher in each of the categories of lesions. The generally wide variation in lipid composition of individual lesions within each category, and the fact that a few individual lesions showed no detectable 26-hydroxycholesterol or 7 beta-hydroxycholesterol, suggested that the lipid oxidation in lesions and therefore perhaps the progression of lesions may be intermittent. Fatty streaks showed the highest concentration of 7 beta-hydroxycholesterol relative to cholesterol, and the lowest ratio of linoleate to oleate, suggesting that this type of lesion experiences the greatest concentration of free radical activity. Levels of the enzymatic product 26-hydroxycholesterol were approximately proportional to cholesterol in all the categories of lesions. 26-Hydroxycholesterol was significantly more abundant in advanced lesions than in intermediate lesions or fatty streaks. 26-Hydroxycholesterol levels were higher in macrophage-rich intermediate and advanced lesions than in their fibrous counterparts. This distinction between macrophage-rich and fibrous lesions was also true for most of the other lipid components, consistent with the involvement of macrophages in lipid accumulation, lipid oxidation and lesion development.

MHC class I expression in murine skin: developmentally controlled and strikingly restricted intraepithelial expression during hair follicle morphogenesis and cycling, and response to cytokine treatment in vivo.

Hair bulb keratinocytes generate one of the few "immune privileged" tissue compartments of the mammalian organism by suppressing classical MHC class I (MHC Ia) antigens. Expression of non-classical MHC class I (MHC Ib) antigens in the follicle has been found, but only in its distal epithelium. Here, we have defined when during murine hair follicle morphogenesis these peculiar MHC Ia and Ib expression patterns are established, how they change during the murine hair cycle, and how different MHC I modulatory agents alter follicular MHC Ia and Ib expression in vivo. During neonatal hair follicle morphogenesis in C57BL/6 mice, distal follicle keratinocytes began to express MHC Ia (H2b) only late in development. The MHC Ib antigens, Qa-1 and Qa-2, did not become visible until the initiation of follicle cycling, with Qa-1 expression being more widespread than that of Qa-2. H2b, Qa-1, and TAP-1 immunoreactivity on previously negative keratinocytes of the proximal anagen hair bulb was upregulated by intradermal injection of the proinflammatory cytokine interferon-gamma, but not by tumor necrosis factor-alpha or interleukin-1beta. Injection of the reportedly MHC class I downregulating agents interleukin-10, insulin-like growth factor-1, transforming growth factor-beta, alpha-melanocyte stimulating hormone, or dexamethasone, however, all failed to downregulate constitutive or interferon-gamma-induced follicular MHC Ia expression. This shows that the hair follicle is a previously unrecognized site of Qa-1 expression and that interferon-gamma is a key regulator of follicular MHC I expression in vivo. It also suggests that the developmental and immunologic controls of MHC I expression by follicle keratinocytes differ from those of other epithelial cells.

A comprehensive guide for the recognition and classification of distinct stages of hair follicle morphogenesis.

Numerous spontaneous and experimentally induced mouse mutations develop a hair phenotype, which is often associated with more or less discrete abnormalities in hair follicle development. In order to recognize these, it is critically important to be able to determine and to classify accurately the major stages of normal murine hair follicle morphogenesis. As an aid, we propose a pragmatic and comprehensive guide, modified after previous suggestions by Hardy, and provide a list of easily recognizable classification criteria, illustrated by representative micrographs. Basic and more advanced criteria are distinguished, the former being applicable to all mouse strains and requiring only simple histologic stains (hematoxylin and eosin, Giemsa, periodic acid Schiff, alkaline phosphatase activity), the latter serving as auxiliary criteria, which require a pigmented mouse strain (like C57BL/6J) or immunohistochemistry (interleukin-1 receptor type I, transforming growth factor-beta receptor type II). In addition, we present simplified, computer-generated schematic drawings for the standardized recording and reporting of gene and antigen expression patterns during hair follicle development. This classification aid serves as a basic introduction into the field of hair follicle morphogenesis, aims at standardizing the presentation of related hair research data, and should become a useful tool when screening new mouse mutants for discrete abnormalities of hair follicle morphogenesis (compared with the respective wild type) in a highly reproducible, easily applicable, and quantifiable manner.

Neurotrophin-3 involvement in the regulation of hair follicle morphogenesis.

Hair follicle epithelium and nervous system share a common ectodermal origin, and some neurotrophins can modulate keratinocyte proliferation and apoptosis. It is therefore reasonable to ask whether growth factors that control neural development are also involved in the regulation of hair follicle morphogenesis. Focusing on neurotrophin-3 (NT-3) and its high-affinity-receptor [tyrosine kinase C (TrkC)], we show that hair placode keratinocytes express TrkC mRNA and immunoreactivity early during murine hair follicle morphogenesis. In later stages of hair follicle development, TrkC mRNA, TrkC-, and NT-3-immunoreactivity are seen in keratinocytes of the proximal hair bulb as well as in dermal papilla fibroblasts. Compared with the corresponding wild-type animals, early stages of hair follicle morphogenesis are significantly accelerated in newborn NT-3 overexpressing mice, whereas these are retarded in newborn heterozygous NT-3 knockout (+/-) mice. These observations suggest that NT-3 is an important growth modulator during morphogenesis and remodeling of neuroectodermal-mesenchymal interaction systems like the hair follicle.

Towards defining the pathogenesis of the hairless phenotype.

Mutation of the hairless (hr) gene in mice causes severe abnormalities during the first hair follicle regression (catagen), resulting in complete baldness. Here, we further characterize how hairlessness develops in HRS/J hairless mouse skin (hr) by histology, histochemistry, immunohistology, and in situ hybridization. We show that, in hr skin, only two defined epithelial cell populations in the distal outer root sheath (ORS) retain their integrity, whereas the rest of the ORS disintegrates. The surviving distal ORS forms the characteristic utriculi, whereas the remnants of the bulge get isolated from other epithelial compartments, but retain the capacity to proliferate and to produce either columnar epithelial outgrowths or selected dermal cysts. Normal dermal papilla structures get lost during the development of hairlessness. Based on the patterns of keratin 17 mRNA and neural cell adhesion molecule antigen expression, and on the distribution of alkaline phosphatase activity, we propose that dermal cysts in hr skin arise from (i) the central ORS, (ii) bulge-derived cells, or (iii) the disintegrating proximal ORS under the influence of dermal papilla remnants. The hr mutation seems to disrupt the integrity of key functional tissue units in the hair follicle, possibly due to a dysregulation of normal, catagen-associated apoptosis and/or an impairment of cell adhesion, whereas the distal follicle epithelium (including its stem cell region) seems to be largely protected from this. Thus, hairless mice offer a unique model for dissecting the as yet obscure functional properties of the hr gene product in maintaining follicle integrity during normal catagen.

Generation and cyclic remodeling of the hair follicle immune system in mice.

In this immunohistomorphometric study, we have defined basic characteristics of the hair follicle (HF) immune system during follicle morphogenesis and cycling in C57BL/6 mice, in relation to the skin immune system. Langerhans cells and gammadelta T cell receptor immunoreactive lymphocytes were the predominant intraepithelial hematopoietic cells in neonatal mouse skin. After their numeric increase in the epidermis, these cells migrated into the HF, although only when follicle morphogenesis was almost completed. In contrast to Langerhans cells, gammadelta T cell receptor immunoreactive lymphocytes entered the HF only via the epidermis. Throughout HF morphogenesis and cycling, both cell types remained strikingly restricted to the distal outer root sheath. On extremely rare occasions, CD4+ or CD8+ alphabetaTC were detected within the HF epithelium or the sebaceous gland. Major histocompatibility complex class II+, MAC-1+ cells of macrophage phenotype and numerous mast cells appeared very early on during HF development in the perifollicular dermis, and the percentage of degranulated mast cells significantly increased during the initiation of synchronized HF cycling (first catagen). During both depilation- and cyclosporine A-induced HF cycling, the numbers of intrafollicular Langerhans cells, gammadelta T cell receptor immunoreactive lymphocytes, and perifollicular dermal macrophages fluctuated significantly. Yet, no numeric increase of perifollicular macrophages was detectable during HF regression, questioning their proposed role in catagen induction. In summary, the HF immune system is generated fairly late during follicle development, shows striking differences to the extrafollicular skin immune system, and undergoes substantial hair cycle-associated remodeling. In addition, synchronized HF cycling is accompanied by profound alterations of the skin immune system.

A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.

Numerous strains of mice with defined mutations display pronounced abnormalities of hair follicle cycling, even in the absence of overt alterations of the skin and hair phenotype; however, in order to recognize even subtle, hair cycle-related abnormalities, it is critically important to be able to determine accurately and classify the major stages of the normal murine hair cycle. In this comprehensive guide, we present pragmatic basic and auxiliary criteria for recognizing key stages of hair follicle growth (anagen), regression (catagen) and quiescence (telogen) in C57BL/6NCrlBR mice, which are largely based on previous work from other authors. For each stage, a schematic drawing and representative micrographs are provided in order to illustrate these criteria. The basic criteria can be employed for all mouse strains and require only routine histochemical techniques. The auxiliary criteria depend on the immunohistochemical analysis of three markers (interleukin-1 receptor type I, transforming growth factor-beta receptor type II, and neural cell-adhesion molecule), which allow a refined analysis of anatomical hair follicle compartments during all hair cycle stages. In contrast to prior staging systems, we suggest dividing anagen III into three distinct substages, based on morphologic differences, onset and progression of melanogenesis, and the position of the dermal papilla in the subcutis. The computer-generated schematic representations of each stage are presented with the aim of standardizing reports on follicular gene and protein expression patterns. This guide should become a useful tool when screening new mouse mutants or mice treated with pharmaceuticals for discrete morphologic abnormalities of hair follicle cycling in a highly reproducible, easily applicable, and quantifiable manner.

Oxidized low-density lipoprotein is cytotoxic to human monocyte-macrophages: protection with lipophilic antioxidants.

Human monocyte-macrophages were incubated for 24 h with low-density lipoprotein (LDL) which had been previously oxidized for varying periods up to 24 h with copper ions, in the presence or absence of DL-alpha-tocopherol or probucol. The release of radioactivity from cells preloaded with tritiated adenine was used as an assay of toxicity. Toxicity of oxidized LDL increased with duration of copper oxidation and with increasing evidence of lipid oxidation, measured by assay of thiobarbituric acid-reactive substances and by gas chromatography. Oxidation and toxicity were inhibited by DL-alpha-tocopherol (200 microM) and probucol (50 microM).

The carotenoids beta-carotene, canthaxanthin and zeaxanthin inhibit macrophage-mediated LDL oxidation.

Human monocyte-macrophages were incubated for 24 h in Ham's F-10 medium with human low-density lipoprotein (LDL) in the presence or absence of beta-carotene, canthaxanthin or zeaxanthin, at final concentrations of 2.5, 12.5 and 25 mg/l. LDL oxidation, measured by agarose gel electrophoresis, the thiobarbituric acid assay and gas chromatography, was inhibited by each of the carotenoids in a concentration-dependent manner. Canthaxanthin was more effective when incorporated into LDL before addition to the cultures whereas beta-carotene and zeaxanthin were more effective when added simultaneously with LDL. The results suggest that dietary carotenoids might help slow atherosclerosis progression.

Clusters of perifollicular macrophages in normal murine skin: physiological degeneration of selected hair follicles by programmed organ deletion.

In back skin sections from adolescent C57BL/6 mice, regularly distributed, perifollicular inflammatory cell clusters (PICC) were found located around the distal noncycling portion of about 2% of all hair follicles examined. The PICC and the affected hair follicles were characterized during spontaneously developed or induced hair cycle stages, using antibodies against MHC Class II, F4/80, ER-MP23, NLDC 145, CD4, CD8, gammadeltaTCR, IL-1 receptor, and ICAM-1. PICC consisted predominantly of macrophages (MAC), accompanied by a few CD4+ cells, whereas gammadeltaTCR+ and CD8+ cells were absent. During anagen and catagen, some of the PICC+ hair follicles showed variable degenerative phenomena reminiscent of scarring alopecia: thickened basement membrane, ectopic MHC II expression, MAC infiltration into the follicle epithelium, and signs of keratinocyte apoptosis. Loss of distal outer root sheath keratinocytes was detected in 10% of PICC+ hair follicles (0.2% of all hair follicles). Because PICC were located in the vicinity of the bulge region, MAC-dependent damage to follicle stem cells might eventually lead to follicle degeneration. These perifollicular MAC clusters around selected hair follicles may indicate the existence of a physiological program of MAC-dependent controlled follicle degeneration by which damaged or malfunctioning follicles are removed by programmed organ deletion (POD).

Factors affecting events during oxidation of low density lipoprotein: correlation of multiple parameters of oxidation.

The present study shows that copper oxidation of LDL is a tightly-ordered process which can be finely controlled by appropriate selection of duration of oxidation and of concentrations of LDL and copper. Oxidation of LDL (0.1-2.0 mg LDL protein/ml) was carried out by copper catalysis (in the ratio of 2.5 microM Cu2+ to 0.1 mg LDL protein/ml) in phosphate-buffered saline, and was monitored by agarose gel electro-phoresis, gas chromatography (GC), anion exchange fast protein liquid chromatography (FPLC), fluorescence spectroscopy and dynamic light scattering. Analysis of the data showed strong cross correlations between many of the parameters of oxidation. Oxidation was more rapid for lower concentrations than for higher concentrations of LDL, despite the same ratio of copper to LDL being employed. Chemical kinetics analysis of the GC data suggested that 7beta-hydroxycholesterol formation occurred as a first order (or pseudo first order) consecutive reaction to the oxidation of linoleate. The first order rate constants for decomposition of linoleate and production of 7beta-hydroxycholesterol correlated closely with the theoretically-calculated times between collision of LDL particles. LDL particle diameter, measured by dynamic light scattering, increased by ca. 50% over 24 h oxidation, suggesting unfolding of apo B-100. Prolonged oxidation of LDL at low concentration suggested that the radical chain reaction was able to propagate, albeit slowly, on cholesterol after all the polyunsaturated fatty acid was consumed. For higher concentrations of LDL, prolonged oxidation resulted in partial aggregation. These findings are applicable to preparing oxidised LDL with different degrees of oxidation, under controlled conditions, for studying its biological properties.

Toxicity of polyunsaturated fatty acid esters for human monocyte-macrophages: the anomalous behaviour of cholesteryl linolenate.

We have investigated the toxicity to human monocytemacrophages, and susceptibility to oxidation, of different individual dietary fatty acids in cholesterol esters and triglycerides, added to the cell cultures as coacervates with bovine serum albumin. Toxicity was assessed using release of radioactivity from cells preloaded with tritiated adenine. Lipid oxidation was measured by gas chromatography (GC). The triglycerides showed a direct relationship between toxicity and increasing unsaturation, which in turn correlated with increasing susceptibility to oxidation. Triolein (18:1; omega-9) and trilinolein (18:2; omega-6) were non-toxic. Trilinolenin (18:3; omega-3) was toxic only after prolonged incubation. Triarachidonin (20:4; omega-6), trieicosapentaenoin (20:5; omega-3) and tridocosahexaenoin (22:6; omega-3) were profoundly and rapidly toxic. There was a similar relationship between toxicity and increasing unsaturation for most of the cholesterol esters, but cholesteryl linolenate was apparently anomalous, being non-toxic in spite of possessing three double bonds and being extensively oxidised. Probucol and DL-alpha-tocopherol conferred protection against the toxicity of cholesteryl arachidonate and triarachidonin. The oxidation in these experiments was largely independent of the presence of cells. GC indicated that formation of 7-oxysterols might contribute to the toxicity of cholesteryl linoleate. The toxicity of triglycerides suggests that polyunsaturated fatty acid peroxidation products are also toxic. Possible mechanisms of cytotoxicity and relevance to atherosclerosis are discussed.

Depletion of alpha-tocopherol in human atherosclerotic lesions.

Estimations of alpha-tocopherol content were made on a series of human necropsy samples of normal arterial wall and of atherosclerotic lesions. The results were compared with stage of lesion, shown by histology, and with the amounts of cholesterol and hydroxycholesterols in the same lesions. The ratio of alpha-tocopherol to cholesterol levels varied widely in normal arterial wall but was consistently low in lesions, especially in lesions rich in macrophage foam cells. The results suggested that significant accumulation of hydroxycholesterols, found almost exclusively in lesions, only occurred when alpha-tocopherol levels were low in relation to the cholesterol content. This suggests that oxidative activity in the lesion may lead to significant oxidation of constituents of low-density lipoprotein only after alpha-tocopherol has been depleted.

Oxidation of low-density lipoprotein by human monocyte-macrophages results in toxicity to the oxidising culture.

Human monocyte-macrophage cultures were exposed to native low density lipoprotein (LDL) for up to 24 h in Ham's F10 medium and the extent of cell-mediated LDL oxidation was determined by measurement of electrophoretic mobility on agarose gels and measurement of lipids and oxidised lipids (including 7 beta-hydroxycholesterol) by GC. After an initial lag phase, which varied from 2-8 h, there was a steady increase in oxidation over 24 h. No-cell control incubations showed minimal increases in oxidation over 24 h. Significant toxicity, measured as release of radioactivity from macrophages pre-loaded with tritiated adenine, was observed in the cells when they oxidised LDL and the extent of radioactivity release correlated closely with the extent of LDL oxidation. Inhibition of oxidation using alpha-tocopherol or probucol reduced toxicity within the oxidising culture. This self-inflicted toxicity may help to explain the origin and enlargement of the lipid core of advanced atherosclerotic lesions.