Receptors on the brush border membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis delta-endotoxins

To investigate the biochemical basis of the differences in the insecticidal spectrum of Bacillus thuringiensis insecticidal crystal proteins (ICPs), we performed membrane binding and toxicity assays with three different ICPs and three lepidopteran species. The three ICPs have different toxicity patterns in the three selected target species. Binding studies with these 125I-labeled ICPs revealed high-affinity saturable binding to brush border membrane vesicles of the sensitive species. ICPs with no toxicity against a given species did not bind saturably to vesicles of that species. Together with previous data that showed a correlation between toxicity and ICP binding, our data support the statement that differences in midgut ICP receptors are a major determinant of differences in the insecticidal spectrum of the entire lepidopteran-specific ICP family. Receptor site heterogeneity in the insect midgut occurs frequently and results in sensitivity to more than one type of ICP.     

Perspectives in the new Russulales

The Russulales is one of 12 major lineages recently elucidated by molecular sequence data in the homobasidiomycetes. The order is morphologically most diverse, containing a remarkable variety of sporophore forms including resupinate, discoid, effused-reflexed, clavarioid, pileate, or gasteroid and hymenophore configurations from smooth, poroid, hydnoid, lamellate, to labyrinthoid. Functionally these fungi are primarily saprotrophs but others are ectomycorrhizal, root parasites and insect symbionts. A phylogenetic analysis of the nuclear 5.8S, ITS2 and large-subunit rDNA genes comprises the best information to date on relationships of taxa within the Russulales. Two large sister groups encompassing 11-13 major clades have been recovered within the Russulales. Based on molecular and morphological data 12 families and approximately 80 genera have been identified, although placement of many taxa has not yet been determined. The two clades containing ectomycorrhizal taxa, corresponding to the Russulaceae and the Albatrellaceae, represent the greatest diversity of sporophore morphologies. The primarily pileate lamellate family Russulaceae is nested with resupinate species and also contains pileate sequestrate, gasteroid annulate and pleurotoid forms. Albatrellaceae similarly contains resupinate poroid, pileate poroid and pileate labyrinthoid sporophores. Presence of gloeoplerous hyphae containing fluid that typically stains black in sulfoaldehyde compounds is a synapomorphy for the Russulales. Amyloid reactions in spore or hyphal walls that occur frequently throughout the Russulales often are perceived as an obvious synapomorphy but are inconsistent. Approaches including sequencing functional genes, analysis of gene expression and biochemical analysis across the entire order are needed.     

HLA-DR/DQ Transgenic, class II deficient mice as a novel model to select for HSP T cell epitopes with immunotherapeutic or preventative vaccine potential

Protective immunity against mycobacteria is dependent on antigen/MHC class II specific, CD4⁺ Th1 cells. HLA-DR3-restricted Th1 cells respond to a subset of mycobacterial antigens, including the immunodominant hsp65, and recognize a single epitope in hsp65, notably p1-20. Altered peptide ligands (APL) of p1-20 can inhibit p1-20/hsp65-induced proliferation of DR3-restricted T cells in an allele specific mannerin vitro. In order to develop a preclinical model in which p1-20 APL can be testedin vivo in the context of HLA, we have used murine class II deficient, HLA transgenic (Ab⁰) mice, in which all CD4⁺ T cells are restricted by the tg HLA molecule. BCG-immunized DR3.Ab⁰ and DQ8.Ab⁰ mice both responded well to hsp65. Furthermore, DR3.Ab⁰ mice recognized precisely the same p1-20 epitope as DR3-restricted human T cells, whereas DQ8.Ab⁰ mice responded to a different set of hsp65 peptides. This shows that (i) the same immunodominant protein and peptide epitope are recognized by T cells from DR3.Ab⁰ mice and DR3⁺ humans and (ii) indicates the major role of HLA-polymorphism in controlling the human T cell response to mycobacterial antigens. Thus, HLA-transgenic, Ab⁰ mice provide a novel, preclinical model system to analyze APL and vaccines in the context of HLA polymorphism.     

A straightforward approach to isolate DNA sequences with potential linkage to the retinoblastoma locus

Summary From a human-Chinese hamster somatic cell hybrid a clone was derived containing chromosome 13 in duplicate as its only human material. This clone was used to construct a human chromosome 13-specific recombinant DNA-library. Overlapping Sau3AI DNA sequences (11.9–17.2 kb) from the cell hybrid were inserted into the lambda phage vector EMBL4. From eleven recombinants having a human insert thirteen putative unique DNA sequences were isolated and cloned into the plasmid vector pBR329. A human-mouse hybrid containing a human chromosome 13 with a deletion of 13q14 and lacking its undeleted homologue was constructed to be used in a selection procedure for DNA sequences belonging to band q14. Three probes originating from two different phages were assigned to 13q14 because they did not hybridise to DNA from this cell hybrid. One of these 13q14 probes detects a low frequency (2/44) Msp I restriction fragment length polymorphism. The probes are now being used for screening a cosmid library to find adjacent polymorphic sequences with a RFLP information content suitable for application in the diagnosis of hereditary retinoblastoma.Preliminary reports of these experiments were presented at the 8th International Workshop on Human Gene Mapping, Helsinki, August 4–10, 1985, and the 13th International Congress of Biochemistry, Amsterdam, August 25–30, 1985 (Scheffer et al. 1985a,b)     

Microarray analysis of 1alpha,25-dihydroxyvitamin D3-treated MC3T3-E1 cells

The active form of Vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], demonstrates potent antiproliferative actions on normal as well as on malignant cell types by blocking the transition from the G1- to the S-phase of the cell cycle. Key target genes for 1,25-(OH)(2)D(3) in this non-classic effect remain largely unknown. Therefore, this study aims to identify genes that, through changes in expression after 1,25-(OH)(2)D(3) treatment, contribute to the observed antiproliferative effect. cDNA microarrays containing 4600 genes were used to investigate changes in gene expression in MC3T3-E1 mouse osteoblasts at 6 and at 12h after treatment with 1,25-(OH)(2)D(3) (10(-8)M), preceding (6h) or coinciding with (12h) the G1/S block in these cells. Approximately one fifth of the genes that were significantly down-regulated after a 12h incubation period with 1,25-(OH)(2)D(3) were genes involved in the DNA replication process, a basic process for cell growth that starts at the end of G1-phase and continues in S-phase. Down-regulation of these genes by 1,25-(OH)(2)D(3) was confirmed by quantitative RT-PCR in MC3T3-E1. In conclusion, cDNA microarrays revealed that treatment of MC3T3-E1 cells with 1,25-(OH)(2)D(3) resulted in the down-regulation of DNA replication genes in parallel with the observed G1/S-arrest.     

Common variants in the type 2 diabetes KCNQ1 gene are associated with impairments in insulin secretion during hyperglycaemic glucose clamp

Background

Genome-wide association studies in Japanese populations recently identified common variants in the KCNQ1 gene to be associated with type 2 diabetes. We examined the association of these variants within KCNQ1 with type 2 diabetes in a Dutch population, investigated their effects on insulin secretion and metabolic traits and on the risk of developing complications in type 2 diabetes patients.

Methodology

The KCNQ1 variants rs151290, rs2237892, and rs2237895 were genotyped in a total of 4620 type 2 diabetes patients and 5285 healthy controls from the Netherlands. Data on macrovascular complications, nephropathy and retinopathy were available in a subset of diabetic patients. Association between genotype and insulin secretion/action was assessed in the additional sample of 335 individuals who underwent a hyperglycaemic clamp.

Principal Findings

We found that all the genotyped KCNQ1 variants were significantly associated with type 2 diabetes in our Dutch population, and the association of rs151290 was the strongest (OR 1.20, 95% CI 1.07–1.35, p = 0.002). The risk C-allele of rs151290 was nominally associated with reduced first-phase glucose-stimulated insulin secretion, while the non-risk T-allele of rs2237892 was significantly correlated with increased second-phase glucose-stimulated insulin secretion (p = 0.025 and 0.0016, respectively). In addition, the risk C-allele of rs2237892 was associated with higher LDL and total cholesterol levels (p = 0.015 and 0.003, respectively). We found no evidence for an association of KCNQ1 with diabetic complications.

Conclusions

Common variants in the KCNQ1 gene are associated with type 2 diabetes in a Dutch population, which can be explained at least in part by an effect on insulin secretion. Furthermore, our data suggest that KCNQ1 is also associated with lipid metabolism.     

Altered expression of key players in vitamin D metabolism and signaling in malignant and benign thyroid tumors

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the active form of vitamin D, mediates antitumor effects in various cancers. The expression of key players in vitamin D signaling in thyroid tumors was investigated. Vitamin D receptor (VDR) and CYP27B1 and CYP24A1 (respectively activating and catabolizing vitamin D) expression was studied (RT-PCR, immunohistochemistry) in normal thyroid, follicular adenoma (FA), differentiated thyroid cancer (DTC) consisting of the papillary (PTC) and follicular (FTC) subtype, and anaplastic thyroid cancer (ATC). VDR, CYP27B1, and CYP24A1 expression was increased in FA and DTC compared with normal thyroid. However, in PTC with lymph node metastasis, VDR and CYP24A1 were decreased compared with non-metastasized PTC. In ATC, VDR expression was often lost, whereas CYP27B1/CYP24A1 expression was comparable to DTC. Moreover, ATC with high Ki67 expression (>30%) or distant metastases at diagnosis was characterized by more negative VDR/CYP24A1/CYP27B1 staining. In conclusion, increased expression of key players involved in local 1,25(OH)(2)D(3) signaling was demonstrated in benign and differentiated malignant thyroid tumors, but a decrease was observed for local nodal and especially distant metastasis, suggesting a local antitumor response of 1,25(OH)(2)D(3) in early cancer stages. These findings advocate further studies with 1,25(OH)(2)D(3) and analogs in persistent and recurrent iodine-refractory DTC.     

Changes in peripheral immune cell numbers and functions in octogenarian walkers – an acute exercise study

Background

Age-related changes of the immune system, termed immunosenescence, may underlie the increased risk of infections and morbidity in the elderly. Little is known about the effects of acute exercise on peripheral immune parameters in octogenarians. Therefore, we investigated acute exercise-induced changes in phenotype and function of the immune system in octogenarians participating in the 2013 edition of the Nijmegen Four Days Marches. Blood sampling was performed at baseline and immediately after 4 days of the walking exercise (30 km/day). A comprehensive set of adaptive and innate immune traits were enumerated and analyzed by flow-cytometry. Peripheral blood mononuclear cells, isolated before and after walking were stimulated with LPS and supernatants were analysed for IL-1β, IL-6, IL-8 and TNF-α concentrations by ELISA. CMV serostatus was determined by ELISA.

Results

The walking exercise induced a clear leucocytosis with numerical increases of granulocytes, monocytes and lymphocytes. These exercise-induced changes were most profound in CMV seropositive subjects. Within lymphocytes, numerical increases of particularly CD4+ T cells were noted. Further T cell differentiation analysis revealed profound increases of naïve CD4+ T cells, including naïve Treg. Significant increases were also noted for CD4+ memory T cell subsets. In contrast, only slight increases in naïve and memory CD8+ T cell subsets were detected. Exercise did not affect markers of immune exhaustion in memory T cell subsets. NK cells demonstrated a numerical decline and a change in cellular composition with a selective decrease of the mature CD56^dim NK cells. The latter was seen in CMV seronegative subjects only. Also, a higher IL-6 and IL-8 production capacity of LPS-stimulated PBMC was seen after walking.

Conclusion

In this exceptional cohort of octogenarian walkers, acute exercise induced changes in immune cell numbers and functions. A clear response of CD4+ T cells, rather than CD8+ T cells or NK cells was noted. Remarkably, the response to exercise within the CD4+ T cell compartment was dominated by naïve CD4+ subsets.

     

Changes in Permeability of Brush Border Membrane Vesicles from Spodoptera littoralis Midgut Induced by Insecticidal Crystal Proteins from Bacillus thuringiensis

Bacillus thuringiensis insecticidal crystal proteins (ICPs) are thought to induce pore formation in midgut cell membranes of susceptible insects. Cry1Ca, which is significantly active in Spodoptera littoralis, made brush border membrane vesicles permeable to KCl (osmotic swelling was monitored by the light scattering technique); the marginally active ICPs Cry1Aa, Cry1Ab, and Cry1Ac did not.