A dual negative regulation model of Toll-like receptor 4 signaling for endotoxin preconditioning in human endotoxemia

We discuss a model illustrating how the outcome of repeated endotoxin administration experiments can emerge as a natural consequence of the tightly regulated signaling pathways and also highlight the importance of a dual negative feedback regulation including PI3K/Akt and IRAK-M (IRAK3). We identify the relative time scales of the onset and the magnitude of the stimulus as key determinants of outcome in repeated administration experiments. The results of our simulations involve potentiated response, tolerance, and protective tolerance. Moreover, the knockout of negative regulators shows that IRAK-M is a necessary and sufficient factor for generation of endotoxin tolerance (ET). The effects of the knockout of IRAK-M gene or administration of PI3K inhibitor do yield predictions that have been verified experimentally. Finally, the pretreatment with PI3K inhibitor reveals the interaction between these two negative regulations.     

Aflatoxicosis in rabbits: Effectiveness of Egyptian raw bentonite in prevention or diminution the detrimental effects of naturally aflatoxin contaminated diets

Thirty five females and 15 males of New Zealand White mature rabbits about 6 months of age, were assigned to 1–5 dietary treatments (7 does+3 bucks for each): uncontaminated control diet, naturally aflatoxin contaminated diet without or with 1,2 and 3% bentonite. Rabbit fed with the aflatoxin-diet had a decreased (P<0.01 or 0.05) physical semen characteristics of bucks and a reproductive performance traits of does. The values of conception rate (%), gestation length (days), litter size (n) and litter weights (g) at birth and viability (%) of litters of doe rabbits, fed with the aflatoxin-diet, recorded, respectively: 64.5; 31.0; 4.4; 275.0 and 57.1 versus 85.6; 30.3; 7.9; 508.0; and 100 for those fed with the uncontaminated diet. Addition of bentonite to the aflatoxin contaminated diet improved in general the physical semen characteristics of buck and reproductive performance traits of doe rabbits. The results of the study demonstrate that adding 1% of Egyptian raw bentonite to the naturally aflatoxin contaminated rabbit diets can provide an effective, cheap and safe practical technique for preventing the aflatoxicosis in mature rabbits.     

Comparison of a specific two-site immunoradiometric assay with radioimmunoassay for rat/human CRF-41

We report the development of an immunoradiometric assay (IRMA) for the specific measurement of corticotrophin releasing factor (CRF-41) which uses two antibodies directed to opposite ends of the CRF-41 molecule. In this assay, 125I-labelled affinity purified rabbit anti-(CRF 36-41) immunoglobulin (IgG) and a guinea-pig anti-(CRF 1-20) serum are simultaneously added to 200 microliter volumes of standard or unknown. After 16 h incubation at room temperature, free and CRF-bound guinea-pig antibodies are precipitated using affinity purified sheep anti-(guinea-pig Fc region) IgG coupled to solid phase Dynospheres. Radioactive rabbit anti-(CRF 36-41) is only precipitated in tubes containing CRF-41, since the peptide acts as a link between the 125I-labelled rabbit IgG and the unlabelled guinea-pig CRF-specific antibodies. Precipitated counts are directly proportional to the concentration of CRF-41 in the sample. This CRF IRMA is compared with two radioimmunoassays (RIA) using the N- and C-terminal CRF antisera employed in the IRMA and found to be more sensitive, specific and rapid to perform. The CRF-41 content of rat and human hypothalamic extracts is the same whether measured by IRMA or conventional RIA. Sephadex G50 chromatography of rat hypothalamic extracts reveals two peaks, detected equally by IRMA and RIA, with a main peak in the elution position of synthetic CRF-41, and a smaller void peak. This is the case whether the hypothalamic extracts are prepared from adrenalectomised or sham-operated rats, non-stressed or subjected to ether stress. Re-chromatography of pooled void peaks under dissociating conditions gives the elution profile of synthetic CRF-41, indicating that the large molecular weight 'CRF-41' peak is not a CRF-41 precursor, but is due to CRF-41 associating non-covalently with large molecular weight proteins.     

Rapid mapping and identification of mutations in Caenorhabditis elegans by restriction site-associated DNA mapping and genomic interval pull-down sequencing

Forward genetic screens provide a powerful approach for inferring gene function on the basis of the phenotypes associated with mutated genes. However, determining the causal mutation by traditional mapping and candidate gene sequencing is often the rate-limiting step, especially when analyzing many mutants. We report two genomic approaches for more rapidly determining the identity of the affected genes in Caenorhabditis elegans mutants. First, we report our use of restriction site-associated DNA (RAD) polymorphism markers for rapidly mapping mutations after chemical mutagenesis and mutant isolation. Second, we describe our use of genomic interval pull-down sequencing (GIPS) to selectively capture and sequence megabase-sized portions of a mutant genome. Together, these two methods provide a rapid and cost-effective approach for positional cloning of C. elegans mutant loci, and are also applicable to other genetic model systems.     

Cytoplasmic filaments and cellular wound healing in amoeba proteus

The flexibility and self-healing properties of animal cell surface membranes are well known. These properties have been best exploited in various micrurgical studies on living cells (2, 3), especially in amoebae (7, 20). During nuclear transplantation in amoebae, the hole in the membrane through which a nucleus passes can have a diameter of 20-30 μm, and yet such holes are quickly sealed, although some cytoplasm usually escapes during the transfer. While enucleating amoebae in previous studies, we found that if a very small portion of a nucleus was pushed through the membrane and exposed to the external medium, the amoeba expelled such a nucleus on its own accord. When this happened, a new membrane appeared to form around the embedded portion of the nucleus and no visible loss of cytoplasm occurred during nuclear extrusion. In the present study, we examined amoebae that were at different stages of expelling partially exposed nuclei, to follow the sequence of events during the apparent new membrane formation. Unexpectedly, we found that a new membrane is not formed around the nucleus from inside but a hole is sealed primarily by a constriction of the existing membrane, and that cytoplasmic filaments are responsible for the prevention of the loss of cytoplasm.     

Movements of beluga whales (Delphinapterus leucas) in Bristol Bay,Alaska

We describe the annual distribution of beluga whales (Delphinapterus leucas) in Bristol Bay, Alaska, using data from 31 satellite‐linked transmitters during 2002–2011. Bristol Bay has one of the largest and best studied Pacific salmon (Oncorhynchus spp.) fisheries in the world, allowing us to link the seasonal distribution of belugas to that of salmon. During salmon migrations, beluga movements were restricted to river entrances. Belugas generally did not relocate to different river entrances or change bays during peak salmon periods. However, the location of belugas was not related to the number of salmon passing counting towers, suggesting that belugas were either selecting locations that were good for catching salmon or there were simply more salmon than belugas needed to supply their nutritional needs. The distribution of belugas expanded after salmon runs ended, and was greatest in winter when belugas ranged beyond the inner bays, traveling as far west as Cape Constantine. Belugas continued to frequent the inner bays in winter whenever sea ice conditions allowed, e.g., when winds moved sea ice offshore; however, they were never located south of the southern ice edge in open water or outside of Bristol Bay.     

Curcumin induces human cathelicidin antimicrobial peptide gene expression through a vitamin D receptor-independent pathway

The vitamin D receptor (VDR) mediates the pleiotropic biologic effects of 1α,25 dihydroxy-vitamin D₃. Recent in vitro studies suggested that curcumin and polyunsaturated fatty acids (PUFAs) also bind to VDR with low affinity. As potential ligands for the VDR, we hypothesized that curcumin and PUFAs would induce expression of known VDR target genes in cells. In this study, we tested whether these compounds regulated two important VDR target genes — human cathelicidin antimicrobial peptide (CAMP) and 1,25-dihydroxyvitamin D₃ 24-hydroxylase (CYP24A1) — in human monocytic cell line U937, colon cancer cell line HT-29 and keratinocyte cell line HaCaT. We demonstrated that PUFAs failed to induce CAMP or CYP24A1 mRNA expression in all three cell lines, but curcumin up-regulated CAMP mRNA and protein levels in U937 cells. Curcumin treatment induced CAMP promoter activity from a luciferase reporter construct lacking the VDR binding site and did not increase binding of the VDR to the CAMP promoter as determined by chromatin immunoprecipitation assays. These findings indicate that induction of CAMP by curcumin occurs through a vitamin D receptor-independent manner. We conclude that PUFAs and curcumin do not function as ligands for the VDR.