Effects of short-term recovery periods on fluid-induced signaling in osteoblastic cells

It is well known that cyclic mechanical loading can produce an anabolic response in bone. In vivo studies have shown that the insertion of short-term recovery periods (10-15 s) into mechanical loading profiles led to an increased osteogenic response compared to continuous cyclic loading of bone. Although this is suggestive of temporal processing at the bone cell level, there is little evidence to support such a hypothesis. Therefore, the current study investigated the cellular mechanism of bone's response to rest inserted vs. continuous mechanical loading. Cell responses to rest inserted mechanical loading were quantified by applying oscillatory fluid flow (OFF) to osteoblastic cells and quantifying real-time intracellular calcium [Ca2+]i, prostaglandin E2 (PGE2) release, and osteopontin (OPN) mRNA levels. Cells were exposed to OFF (1 Hz) at shear stresses of 1 and 2 Pa with rest periods of 5, 10, and 15s inserted every 10 loading cycles. The insertion of 10 and 15s rest periods into the flow profile resulted in multiple [Ca2+]i responses by individual cells, increased [Ca2+]i response magnitudes, and increased overall percent of cells responding compared to continuously loaded control groups. We determined the source of the multiple calcium responses to be from intracellular stores. In addition, rest inserted OFF led to similar levels of PGE2 release and increased levels of relative OPN mRNA compared to cells exposed to continuous OFF. Our study suggests that the cellular mechanism of bone adaptation to rest inserted mechanical loading may involve modulation of intracellular levels of calcium (frequency, magnitude, percent of cells responding).     

Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity

Langerhans cells (LC) form a unique subset of dendritic cells (DC) in the epidermis but so far their in vivo functions in skin immunity and tolerance could not be determined, in particular in relation to dermal DC (dDC). Here, we exploit a novel diphtheria toxin (DT) receptor (DTR)/DT-based system to achieve inducible ablation of LC without affecting the skin environment. Within 24 h after intra-peritoneal injection of DT into Langerin-DTR mice LC are completely depleted from the epidermis and only begin to return 4 wk later. LC deletion occurs by apoptosis in the absence of inflammation and, in particular, the dDC compartment is not affected. In LC-depleted mice contact hypersensitivity (CHS) responses are significantly decreased, although ear swelling still occurs indicating that dDC can mediate CHS when necessary. Our results establish Langerin-DTR mice as a unique tool to study LC function in the steady state and to explore their relative importance compared with dDC in orchestrating skin immunity and tolerance.     

Inhibition of IKK-2 by 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarboxamides

A series of 21 novel 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarboxamides were synthesized and evaluated for the inhibition of IKK-2. In spite of their often modest activity on the enzyme, six selected analogs showed significant inhibition of the production of inflammatory cytokine IL-8 in IL-1beta stimulated rheumatoid arthritis-derived synovial fibroblasts, demonstrating their potential usefulness as NF-kappaB regulators.     

Larval Development and Vitellin-like Protein Expression in Palaemon elegans Larvae Following Xeno-oestrogen Exposure

Certain anthropogenic chemicals, most notably xeno-oestrogens,are known to have the potential to disrupt vertebrate endocrinesystems. For example, induction of the female-specific protein,vitellogenin, in male fish is a well-known effect of exposureto xeno-oestrogens and serves as a biomarker of such exposure.There have been few comparable studies of putative biomarkersof endocrine disruption in invertebrates. An exception is theupregulation of vitellin-like larval storage protein (LSP) expressionin the barnacle cypris larva following exposure to oestrogenicchemicals. The current study aimed to establish whether larvaeof the glass prawn, Palaemon elegans, are likewise susceptibleto xeno-oestrogen exposure. Using a polyclonal antiserum toP. elegans apolipovitellin, an 86 kDa polypeptide was detectedby western blotting in the larval and early postlarval stagesof this species. An indirect ELISA applied to the soluble proteinfraction of larval homogenates determined that the titre ofthis putative LSP ranged, depending on larval stage, from 0.48–0.67ng µg^–1. Exposure of P. elegans larvae to the xeno-oestrogen4-n-nonylphenol (4-NP), at 0.2–20 µg L^–1,resulted in a significant, concentration-independent increasein putative LSP levels of 5–18%. Conversely, exposureto the natural oestrogen, 17ß-oestradiol (E₂), at0.2 and 20 µg L^–1, led to a significant concentration-independentdecline (up to 11%) in LSP levels. Whether the effect of 4-NPresults from endocrine disruption is not known, however, anoestrogen receptor-mediated effect is unlikely. Other than aslight increase in larval mortality when exposed to 4-NP at2 µg L^–1, neither 4-NP nor E₂ significantly affecteddevelopment, growth or survival of P. elegans larvae.     

Testosterone and estradiol concentrations in serum, velvet skin, and growing antler bone of male white-tailed deer

The growth and mineralization of antlers correlate with the seasonal variation of serum androgens. Whereas seasonal levels of testosterone (T) in plasma are well established, steroid concentrations have not yet been determined in the tissues of growing antlers. Therefore, RIA was used to determine T and 17beta estradiol (E2) in serum, and three areas (tip, middle, and base) of the antler bone and the antler skin, called velvet. Blood and antler tissues of white-tailed deer (Odocoileus virginianus) were collected from May to August. The difference between levels of T and E2 among the sites was calculated using the square root transformation followed by a mixed model analysis with individual deer and an interaction of individual and year (individual(*)year) as a random factor. Concentrations of T in serum (799+/-82 pg/ml) were higher than T values in the velvet (589+/-58 pg/ml, P<0.01) and in the antler bone (538+/-58 pg/ml, P<0.001). Estradiol concentrations differed among antler tissues and serum (P<0.001) and between years (P<0.01). Estradiol concentrations in serum (25+/-25 pg/ml) were consistently lower than those in antler bone (208+/-11 pg/ml, P<0.001) and velvet (150+/-12 pg/ml, P<0.001). The E2:T ratio in serum was 1:10-60. The same ratio for the antler bone was only 1:2-3 and for the velvet 1:3.5. It is concluded that higher T and lower E2 concentrations found in plasma, as compared to antler bone or antler velvet, may indicate a partial metabolism of systemic androgens into estrogens xin the tissues of growing antlers.     

Stability of the major allergen Brazil nut 2S albumin (Ber e 1) to physiologically relevant in vitro gastrointestinal digestion

The major 2S albumin allergen from Brazil nuts, Ber e 1, was subjected to gastrointestinal digestion using a physiologically relevant in vitro model system either before or after heating (100 degrees C for 20 min). Whilst the albumin was cleaved into peptides, these were held together in a much larger structure even when digested by using a simulated phase 1 (gastric) followed by a phase 2 (duodenal) digestion system. Neither prior heating of Ber e 1 nor the presence of the physiological surfactant phosphatidylcholine affected the pattern of proteolysis. After 2 h of gastric digestion, approximately 25% of the allergen remained intact, approximately 50% corresponded to a large fragment of M(r) 6400, and the remainder comprised smaller peptides. During duodenal digestion, residual intact 2S albumin disappeared quickly, but a modified form of the 'large fragment' remained, even after 2 h of digestion, with a mass of approximately 5000 Da. The 'large fragment' comprised several smaller peptides that were identified, by using different MS techniques, as deriving from the large subunit. In particular, sequences corresponding to the hypervariable region (Q37-M47) and to another peptide (P42-P69), spanning the main immunoglobulin E epitope region of 2S albumin allergens, were found to be largely intact following phase 1 (gastric) digestion. They also contained previously identified putative T-cell epitopes. These findings indicate that the characteristic conserved skeleton of cysteine residues of 2S albumin family and, particularly, the intrachain disulphide bond pattern of the large subunit, play a critical role in holding the core protein structure together even after extensive proteolysis, and the resulting structures still contain potentially active B- and T-cell epitopes.     

TEMPERATURE-SENSITIVE MUTANTS OF ASCOBOLUS IMMERSUS

As a result of a search for genetic markers to be used in interallelic recombination studies, 23 temperature-sensitive mutants of Ascobolus immersus were isolated following UV-irradiation. Three of these failed to grow at 37 C but grew normally or, in one case with a colonial phenotype, at 25 C. Twenty mutants are like the wild type at 25 C but express a colonial phenotype at 37 C. Crosses of the mutants to wild type indicate that all are due to single-gene mutations. Crosses among certain of the mutants indicate that several different loci are affected.     

Flower preferences and pollen transport networks for cavity‐nesting solitary bees: Implications for the design of agri‐environment schemes

Floral foraging resources are valuable for pollinator conservation on farmland, and their provision is encouraged by agri‐environment schemes in many countries. Across Europe, wildflower seed mixtures are widely sown on farmland to encourage pollinators, but the extent to which key pollinator groups such as solitary bees exploit and benefit from these resources is unclear. We used high‐throughput sequencing of 164 pollen samples extracted from the brood cells of six common cavity‐nesting solitary bee species (Osmia bicornis, Osmia caerulescens, Megachile versicolor, Megachile ligniseca, Megachile centuncularis and Hylaeus confusus) which are widely distributed across the UK and Europe. We documented their pollen use across 19 farms in southern England, UK, revealing their forage plants and examining the structure of their pollen transport networks. Of the 32 plant species included currently in sown wildflower mixes, 15 were recorded as present within close foraging range of the bees on the study farms, but only Ranunculus acris L. was identified within the pollen samples. Rosa canina L. was the most commonly found of the 23 plant species identified in the pollen samples, suggesting that, in addition to providing a nesting resource for Megachile leafcutter bees, it may be an important forage plant for these species. Higher levels of connectance and nestedness were characteristic of pollen transport networks on farms with abundant floral resources, which may increase resilience to species loss. Our data suggest that plant species promoted currently by agri‐environment schemes are not optimal for solitary bee foraging. If a diverse community of pollinators is to be supported on UK and European farmland, additional species such as R. canina should be encouraged to meet the foraging requirements of solitary bees.     

The adaptive significance of monogamy in the golden-reumped elephant-shrew

Golden-rumped elephant-shrews Rhynchocyon chrysopygus are primarily monogamous, males and females defending joint territories for long periods and probably for life. However, males may occasionally solicit extra-pair copulations from neighbouring females and annex their territories if the resident male disappears for any reason. Males provide no direct paternal care, and the adaptive significance of monogamy in this species is unclear. Although it is possible that males provide some indirect benefits to the female and her offspring, these are unlikely to be substantial. Males and females spend little time together, so males are unlikely to provide additional protection from predators, and there is no evidence that the presence of a territorial male reduces the costs of territorial defence for the female. Females were able to breed successfully when mated bigamously, demonstrating that the undivided assistance of the male is not essential for successful rearing of offspring. The fact that male elephant-shrews occasionally attempt to defend more than one female suggests that polygyny would be beneficial for males. However, defending two territories is costly, resulting in increased activity and weight loss, and higher rates of intrusion by neighbouring males. Female elephant-shrews do not occupy particularly large home ranges for their body size. The results suggest that the high costs of defending a territory large enough to encompass the ranges of more than one female, combined with the greater probability of being cuckolded, make defending more than one female a poor option for male elephant-shrews.     

Use of Ac as an insertional mutagen in Arabidopsis

A pilot-scale transposon mutagenesis experiment using a modified autonomous Activator (Ac) element, AcΔNael, was carried out in Arabidopsis thaliana. Four different transformants carrying Ac elements in different and defined genomic locations were used to generate 1000 plants carrying approximately 500 independent germinal transposition events. These plants were then selfed and the 1000 families screened in tissue culture and soil for phenotypic mutants. Fifty different families segregated mutations in their progeny. Preliminary Southern blot analysis of 29 families which segregated mutant progeny, showed that 28 had a transposed Ac. Six of the families were further tested for linkage between the transposed Ac and the mutant phenotype, and instability of the putatively tagged locus. Two of the mutants were shown to be tagged as they were tightly linked to a transposed Ac, and somatic and germinal reversion was associated with loss of Ac. One other mutant locus was shown to be closely linked to a transposed Ac, and therefore was likely to be tagged. The remaining three mutations were not tagged as they were not linked to a transposed Ac. In two of the tagged mutants Ac had transposed to closely linked sites, while in a third mutant the co-segregating Ac had transposed to a site which was not tightly linked to the donor T-DNA. Multiple insertions into the DIF1 locus were found, due to the preferential transposition of Ac to a linked site.