Characterization of yeast artificial chromosomes containing interleukin genes on human chromosome 5.

To understand better the organization and linkage of the interleukin genes, IL4 and IL5, we prepared long-range restriction maps of five yeast artificial chromosomes (YACs) containing IL5. We determined that IL4 and IL5 are within 100-170 kb, and that the regions surrounding these genes contain several GC-rich areas. Fluorescence in situ chromosomal analysis demonstrated that three of the five YAC clones contain non-contiguous genomic sequences originating from multiple human chromosomes.     

Refinement of bovine chromosome 2 linkage map near the mh locus reveals rearrangements between the bovine and human genomes

The locus responsible for the appearance of muscular hypertrophy (mh) in double muscled cattle breeds has recently been shown to encode a secreted growth factor designated myostatin (MSTN). This conclusion was based in part on the placement of MSTN in the interval to which mh had been mapped on bovine chromosome 2 (BTA2). During the mapping phase of the study, numerous yeast artificial chromosome (YAC) clones were isolated that contained genetic markers closely linked to mh. Other YACs and cosmids were identified that contained genes selected from human chromosome 2q (HSA2q), with the goal of defining the position of breakpoints in conserved synteny between the bovine and human comparative maps, thereby permitting accurate selection of positional candidate genes. An efficient subcloning procedure was developed to obtain microsatellites (ms) from YAC clones, to increase the number of informative meioses in herds segregating for mh. The same procedure was used to place the human orthologues of engrailed-1 (EN1), interleukin 1 beta (IL1B), and paired-box-containing 8 (PAX8) genes on the cattle map to further define the positions of breakpoints in conserved synteny and gene order. Twenty-three of 28 ms identified from YAC subclone libraries were informative in the mapping families. Seven mapped to the centromeric end of BTA2, which contains the mh locus, improving marker density and informativeness. The two MSTN and four EN1 gene-associated ms markers developed from YACs, map to positions 1·5 and 61·6 cm in the BTA2 linkage group, respectively. In addition, ms markers developed from cosmids containing either IL1B or PAX8, map to positions 56·6 and 56·9 cm in the BTA11 linkage group, respectively. These linkage data confirm the location and orientation of orthologous segments of HSA2q that were previously indistinguishable on the bovine map, and demonstrates the presence of microrearrangements of gene order (segments <10 cm ) and conserved synteny between the human and bovine genomes.     

Mutation analysis of β-thalassemia genes in a German family reveals a rare transversion in the first intron

Summary The gene for human interleukin 7 (IL7) maps to chromosome 8 by Southern analysis of a somatic cell hybrid panel and to 8q12-q13 by in situ hybridization.     

Genetic mapping near the myd locus on mouse Chromosome 8

Myodystrophy (myd), an autosomal recessive mutation of the mouse characterized by progressive weakness and dystrophic muscle histology, maps to the central portion of Chromosome (Chr) 8 (Lane et al. J. Hered 67, 135, 1976). This portion of Chr 8 contains the genes for a mitochondrial uncoupling protein (Ucp) and kallikrein (Kal3), which map to distal 4q in the human, providing evidence for a segment of homology. Characteristics of the myd phenotype coupled with this homology suggest that myd may be a mouse homolog of facioscapulohumeral muscular dystrophy (FSHD), which maps to human 4q35. We have confirmed and expanded the region of mouse 8-human 4 homology by generating a map of Chr 8 in an interspecific backcross of C57BL/6J and a partially inbred strain derived from M. spretus. The map is comprised of the genes for Ucp, coagulation factor XI (Cf11), and chloride channel 5 (Clc5), all of which have homologs on distal human 4q, 15 microsatellite loci, and the membrane cofactor protein pseudogene (Mcp-ps). To place myd in the genetic map, 75 affected progeny from an intersubspecific backcross of animals heterozygous for myd with Mus musculus castaneus were genotyped with Chr 8 microsatellite loci. The mutation maps between D8Mit30 and D8Mit75, an interval that is flanked by genes with human homologs at distal 4q. These results are consistent with the possibility that myd is the mouse homolog of FSHD.     

Synthesis and Expression of Some Genes for Human Cytokines

Abstract

Intronless genes for human mature interleukin 1a (IL1a) and its receptor antagonist (IL1ra) have been synthesized and efficiently expressed in a specially devised bacterial plasmid vector as part of a versatile two-cistron prokaryotic expression system.     

Polymorphism of the Interleukin- and Interleukin Receptor Genes: Population Distribution and Association with Atopic Asthma

Population distribution and pathogenetic significance for bronchial asthma (BA) of the eight polymorphic variants of six interleukin- (IL) and interleukin receptor genes, C-589T, G/C 3"-UTRIL4, C-703T IL5 T113M IL9 Q551R, 150V IL4RA, G-80A IL5RA, and G1972A IL5RB, was examined. In the population samples of Russians, Tajiks, Buryats, and Tuvinians racial and ethnic specificity of these polymorphisms was established. These specific features were manifested as population-specific genetic portraits in respect of polymorphic allele frequencies. Analysis of the BA patients and their relatives from Tomsk by use of transmission/disequilibrium test (TDT) revealed the presence of a statistically significant association between the C-703 IL5 allele and the disease (P= 0.005). This is the first evidence of an association between the IL5 gene polymorphism and BA.     

Mapping of the interleukin 5 receptor gene to human Chromosome 3 p25–p26 and to mouse Chromosome 6 close to the Raf-1 locus with polymorphic tandem repeat sequences

Simple-sequence tandem repeat sequences in the 3 UTR of interleukin 5 (IL5)-receptor gene of human and mouse are polymorphic in their length among humans and different strains of mice. In 20 different human Epstein-Barr virus (EBV)-transformed cell lines, six alleles of IL5R could be distinguished. In the mouse, three different alleles are found. With the human-specific IL5R tandem repeat marker in human-rodent somatic cell hybrids, the IL5R gene was mapped to human Chromosome (Chr) 3 p25–p26. With the mouse-specific IL5R tandem repeat sequence in recombinant inbred strains of mice, the Il5r gene was mapped to the distal part of mouse Chr 6 close to the Raf-1 locus.     

Loci of the immune system are implicated in diabetes frequency and age at onset of diabetes in BB rats

The spontaneously diabetic BB rat is a well-established animal model in diabetes research developing an insulin-dependent type-1 diabetes mellitus closely resembling human diabetes. By several crossing studies using BB/OK rats it has been demonstrated that beside the MHC class-II genes of the RT1^u haplotype, Iddm1, and the lymphopenia, Iddm2, at least two additional non-MHC genes located on chromosomes 6 (Iddm4) and 18 (Iddm3) are involved in diabetes development. In addition, there are at least three genes located on chromosomes 6 (Dm1), 8 (Dm2) and 10 (Dm3) influencing the age at onset of diabetes. Comparing the homologous regions between rat and human, it is shown that most diabetogenic genes lie on human chromosomes near genes involved in immune processes providing human geneticist with new candidate regions for the analysis of diabetogenic non-MHC genes in human type-1 diabetes.     

Lipopolysaccharide enhances asymmetrical production of cytokines and nitric oxide by left and right cerebral cortical microglial cells in BALB/C mice

Lipopolysaccharide (LPS)‐induced inflammatory factors production by the cerebral cortical glial cells in two sides of the murine brain are different. To determine if microglial cells, a subset of glial cells, are involved in asymmetric production, interleukin‐6 (IL‐6), interleukin‐1β (IL‐1β) and nitric oxide (NO) responses to LPS by microglial cells in the right and left cerebral cortices were examined. Primary microglial cells were isolated from BALB/C neonatal mice, treated with LPS (10 µg ml^?1) for 24 h and examined for IL‐6, IL‐1β and NO production. At untreated state, the levels of IL‐6, IL‐1β and NO showed no statistical difference between left and right. However, after LPS treatment, the levels of IL‐6, IL‐1β and NO for the right microglial cells was statistically significant higher than the left (P < 0·05). Our results denote that enhanced production of IL‐6, IL‐1β and NO after LPS treatment in microglia is directly proportional to their basal‐state levels, and right cortical microglia produce higher levels of IL‐6, IL‐1β and NO than left cortical microglia. Copyright © 2011 John Wiley & Sons, Ltd.     

Mapping the midkine family of developmentally regulated signaling molecules

Midkine (Mdk) and heparin-binding neurotrophic factor (Hbnf)/pleiotrophin (Ptn) comprise the Midkine family of developmentally regulated signaling molecules. We have determined the chromosomal localization of these genes in the mouse by use of singlestrand conformation polymorphisms (SSCPs), which facilitated the typing of Mdk and Hbnf alleles in recombinant inbred (RI) strains and interspecific backcrosses. Mapping was performed relative to other cloned genes, as well as simple sequence length polymorphisms (SSLPs) in the interspecific backcrosses. Mdk maps to mouse Chromosome (Chr) 2, linked to the Hoxd gene cluster. Hbnf maps to proximal mouse Chr 6, linked to the Cftr and Cpa genes. Comparative mapping of human MDK and HBNF employing species-specific polymerase chain reaction (PCR) primers and human monochromosomal somatic cell hybrids assigns MDK to human Chr 11 and HBNF to human Chr 7q32-qter.     

A novel tumor-promoting mechanism of IL6 and the therapeutic efficacy of tocilizumab: Hypoxia-induced IL6 is a potent autophagy initiator in glioblastoma via the p-STAT3-MIR155-3p-CREBRF pathway

Hypoxia induces protective autophagy in glioblastoma cells and new therapeutic avenues that target this process may improve the outcome for glioblastoma patients. Recent studies have suggested that the autophagic process is upregulated in glioblastomas in response to extensive hypoxia. Hypoxia also induces the upregulation of a specific set of proteins and microRNAs (miRNAs) in a variety of cell types. IL6 (interleukin 6), an inflammatory autocrine and paracrine cytokine that is overexpressed in glioblastoma, has been reported to be a biomarker for poor prognosis because of its tumor-promoting effects. Here, we describe a novel tumor-promoting mechanism of IL6, whereby hypoxia-induced IL6 acts as a potent initiator of autophagy in glioblastoma via the phosphorylated (p)-STAT3-MIR155-3p pathway. IL6 and p-STAT3 levels correlated with the abundance of autophagic cells and HIF1A levels in human glioma tissues and with the grade of human glioma, whereas inhibition of exogenous or endogenous IL6 repressed autophagy in glioblastoma cells in vitro. Knockdown of endogenous MIR155-3p inhibited IL6-induced autophagy, and enforced expression of MIR155-3p restored the anti-autophagic activity of IL6 inhibitors. We show that the hypoxia-IL6-p-STAT3-MIR155-3p-CREBRF-CREB3-ATG5 pathway plays a central role in malignant glioma progression, with blockade of the IL6 receptor by tocilizumab demonstrating a certain level of therapeutic efficacy in a xenograft model in vivo, especially in combination with temozolomide. Moreover, tocilizumab inhibits autophagy by promoting tumor apoptosis. Collectively, our findings provide new insight into the molecular mechanisms underlying hypoxia-induced glioma cell autophagy and point toward a possible efficacious adjuvant therapy for glioblastoma patients.     

Time-dependent mRNA expression of selected pro-inflammatory factors in the endometrium of primiparous cows postpartum

Background  

Inflammatory processes and infections of the uterine wall must be accepted as a physiological event in dairy cows after calving. This might result in clinical or subclinical endometritis which is assumed to impair reproductive performance in the current lactation. Several cytokines and acute phase proteins have been discussed as local and systemic mediators of these inflammatory processes. The aim of the present study was to investigate the endometrial mRNA expression of the chemokine CXC ligand 5 (CXCL5), interleukin 1β (IL1B), IL6, IL8, tumour necrosis factor alpha (TNF), prostaglandin-endoperoxide synthase 2 (PTGS2) and haptoglobin (HP) in the postpartum period.     

Immunomodulatory effects of Bacillus subtilis (natto) B4 spores on murine macrophages

To investigate the immunomodulatory effects of Bacillus subtilis (B. subtilis) (natto) B4 spores on murine macrophage, RAW 264.7 cells were cultured alone or with B subtilis (natto) B4 spores at 37°C for 12 hrs, then both cells and culture supernatants were collected for analyses. Exposure of RAW 264.7 cells to B. subtilis (natto) B4 spores had no significant effects on macrophage viability and amounts of extracellular lactate dehydrogenase (LDH). However, it remarkably increased the activities of acid phosphatase (ACP), lactate dehydrogenase (LDH) and inducible nitric oxide synthase (iNOS) in cells and the amounts of nitric oxide (NO) and cytokines (tumor necrosis factor‐alpha, interferon‐gamma, interleukin [IL]‐1 beta, IL‐6, IL‐12, IL‐10 and macrophage inflammatory protein‐2) in culture supernatants. These results demonstrate that B. subtilis (natto) B4 spores are harmless to murine macrophages and can stimulate their activation through up‐regulation of ACP and LDH activities and enhance their immune function by increasing iNOS activity and stimulating NO and cytokine production. The above findings suggest that B. subtilis (natto) B4 spores have immunomodulatory effects on macrophages.     

High Local Concentrations and Effects on Differentiation Implicate Interleukin‐6 as a Paracrine Regulator

Objective: To examine the possibility that interleukin‐6 (IL‐6) can act as a paracrine regulator in adipose tissue by examining effects on adipogenic genes and measuring interstitial IL‐6 concentrations in situ. Research Methods and Procedures: Circulating and interstitial IL‐6 concentrations in abdominal and femoral adipose tissue were measured using the calibrated microdialysis technique in 20 healthy male subjects. The effects of adipose cell enlargement on gene expression and IL‐6 secretion were examined, as well as the effect of IL‐6 in vitro on gene expression of adiponectin and other markers of adipocyte differentiation. Results: The IL‐6 concentration in the interstitial fluid was ～100‐fold higher than that in plasma, suggesting that IL‐6 may be a paracrine regulator of adipose tissue. This was further supported by the finding that adding IL‐6 in vitro at similar concentrations down‐regulated the expression of adiponectin, aP2, and PPARγ‐2 in cultured human adipose tissue. In addition, gene expression and release of IL‐6, both in vivo and in vitro, correlated with adipose cell size. Discussion: These data suggest that IL‐6 may be a paracrine regulator of adipose tissue. Furthermore, increased adipose tissue production of IL‐6 after hypertrophic enlargement of the adipose cells may detrimentally affect systemic insulin action by inducing adipose tissue dysfunction with impaired differentiation of the pre‐adipocytes and/or adipocytes and lower adiponectin.     

Preliminary evaluation of selected inflammatory cytokine gene expression in lymphocytes isolated from whole human blood infected with trans-anethole-treated Staphylococcus aureus Newman strain

In our previous study based on a whole-blood model of sepsis infected with trans-anethole (TA)-treated Staphylococcus aureus, we have found that innate immune response was more effective in comparison to non-treated cells. Due to the previous observation, in the current preliminary study, a primary adaptive immune response was analysed. This study was conducted to evaluate the expression of selected cytokine (IL1B, IL2, IL6, IL10, TNF, TGFB1, IFNG) and Toll-like receptor (TLR2) genes in lymphocytes isolated from whole human blood infected with S. aureus Newman strain treated with TA. The lymphocytes were isolated by density gradient centrifugation from blood samples infected with S. aureus, as well as from non-infected samples. Gene expression was measured using quantitative real-time PCR. The lymphocytes isolated from the blood infected with TA-treated staphylococcal cells demonstrated significantly greater IL10, IL1B, IL6, TNF and TLR2 expression. Hence, it is possible that the previously observed changes in the surface structure of TA-treated S. aureus Newman strain may significantly increase the relative expression of IL10, IL1B, IL6, TNF and TLR2 genes in lymphocytes; however, further studies are needed.     

Genomic structure, mapping, and expression analysis of the mammalian Lunatic, Manic, and Radical fringe genes

The three members of the mammalian fringe gene family, Manic fringe (Mfng), Radical fringe (Rfng), and Lunatic fringe (Lfng), were identified on the basis of their similarity to Drosophila fringe (fng) and their participation in the evolutionarily conserved Notch receptor signaling pathway. Fringe genes encode pioneer secretory proteins with weak similarity to glycosyltransferases. Both expression patterns and functional studies support an important role for Fringe genes in patterning during embryonic development and an association with cellular transformation. We have now further characterized the expression and determined the chromosomal localization and genomic structure of the mouse Mfng, Rfng, and Lfng genes; the genomic structure and conceptual open reading frame of the human RFNG gene; and the refined chromosomal localization of the three human fringe genes. The mouse Fringe genes are expressed in the embryo and in adult tissues. The mouse and human Fringe family members map to three different chromosomes in regions of conserved synteny: Mfng maps to mouse Chr 15, and MFNG maps to human Chr 22q13.1 in the region of two cancer-associated loci; Lfng maps to mouse Chr 5, and LFNG maps to human Chr 7p22; Rfng maps to mouse Chr 11, and RFNG maps to human Chr 17q25 in the minimal region for a familial psoriasis susceptibility locus. Characterization of the genomic loci of the Fringe gene family members reveals a conserved genomic organization of 8 exons. Comparative analysis of mammalian Fringe genomic organization suggests that the first exon is evolutionarily labile and that the Fringe genes have a genomic structure distinct from those of previously characterized glycosyltransferases. Received: 19 February 1999 / Accepted: 22 February 1999     

The linkage map of sheep Chromosome 6 compared with orthologous regions in other species

The genetic linkage map of sheep Chromosome (Chr) 6 has been extended to include 35 loci with the addition of 11 RFLP and 12 microsatellite loci. The sex-averaged linkage map now spans 154 cM from phosphodiesterase cyclic GMP beta polypeptide (PDE6B) to OarCP125, an anonymous sheep microsatellite. The male and female map lengths, at 180 cM and 132 cM respectively, did not differ significantly. The physical assignment of PDE6B to Chr 6q33-qter orientates the linkage map on sheep Chr 6 with PDE6B near the telomere and OarCP125 towards the centromere. The order and genetic distances between loci are similar for the sheep Chr 6 and cattle Chr 6 maps, except for the position of the casein genes. The sheep Chr 6 linkage map is also comparable to portions of human Chr 4, mouse Chrs 5 and 3, and pig Chr 8. The synteny between sheep Chr 6 and human Chr 4 has been extended from PDE6B (4p16.3) to epidermal growth factor (EGF, 4q25-q27). However, a region from platelet-derived growth factor receptor α polypeptide (PDGFRA) to bone morphogenetic protein 3 (BMP3), which spans 19 cM on sheep Chr 6, appears to be inverted with respect to the human and mouse loci. Other differences in the gene order between sheep, pig, and mouse suggest more complex rearrangements. Received: 16 August 1995 / Accepted: 12 December 1995