GCP-2/CXCL6 synergizes with other endothelial cell-derived chemokines in neutrophil mobilization and is associated with angiogenesis in gastrointestinal tumors

The precise role of chemokines in neovascularization during inflammation or tumor growth is not yet fully understood. We show here that the chemokines granulocyte chemotactic protein-2 (GCP-2/CXCL6), interleukin-8 (IL-8/CXCL8), and monocyte chemotactic protein-1 (MCP-1/CCL2) are co-induced in microvascular endothelial cells after stimulation with pro-inflammatory stimuli. In contrast with its weak proliferative effect on endothelial cells, GCP-2 synergized with MCP-1 in neutrophil chemotaxis. This synergy may represent a mechanism for tumor development and metastasis by providing efficient leukocyte infiltration in the absence of exogenous immune modulators. To mimic endothelial cell-derived GCP-2 in vivo, GCP-2 was intravenously injected and shown to provoke a dose-dependent systemic response, composed of an immediate granulopenia, followed by a profound granulocytosis. By immunohistochemistry, GCP-2 was further shown to be expressed by endothelial cells from human patients with gastrointestinal (GI) malignancies. GCP-2 staining correlated with leukocyte infiltration into the tumor and with the expression of the matrix metalloproteinase-9 (MMP-9/gelatinase B). Together with previous findings, these data suggest that the production of GCP-2 by endothelial cells within the tumor can contribute to tumor development through neovascularization due to endothelial cell chemotaxis and to tumor cell invasion and metastasis by attracting and activating neutrophils loaded with proteases that promote matrix degradation.     

Salvianolic acid B attenuates MMP-2 and MMP-9 expression in vivo in apolipoprotein-E-deficient mouse aorta and in vitro in LPS-treated human aortic smooth muscle cells

Salvianolic acid B (Sal B), a water-soluble antioxidant derived from a Chinese medicinal herb, is believed to have multiple therapeutic and preventive against human vascular diseases, including atherosclerosis and restenosis. To elucidate the underlying cellular mechanisms, we produced hypercholesterolemia by feeding apo-E-deficient mice a 0.15% cholesterol diet and inflammation in human aortic smooth muscle cells (HASMCs) with the endotoxin lipopolysaccharide (LPS), focusing on the metallopreteinases MMP-2 and MMP-9, the relevant signal transduction pathways and the effects of Sal B. Immunohistochemical analyses indicated apo-E-deficient mice fed a 0.15% cholesterol diet for 12 weeks exhibited thickened intima and elevated levels of MMP-2 and MMP-9 in aortic sections, both of which were attenuated by 0.3% Sal B dietary supplement. Western blotting and zymography analyses indicated that unstimulated HASMCs exhibited basal levels of protein and activity levels for MMP-2 and barely detectable levels for MMP-9, both of which were markedly upregulated by LPS, which also induced cell migration. Sal B significantly attenuated upregulations of both MMPs as well as the LPS-induced cell migration through the inactivation of MMP-2 and MMP-9 protein synthesis as well as the downregulation of the extracellular-signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH(2)-terminal kinase (JNK). These results demonstrate that Sal B has anti-migration properties on smooth muscle cells and may explain its anti-atherosclerotic properties. This novel mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis.     

L-nucleotides and 8-methylguanine of d(C1m8G2C3G4C5LG6LC7G8C9G10)2 act cooperatively to promote a left-handed helix under physiological salt conditions

The structure and thermal stability of a hetero chiral decaoligodeoxyribonucleotide duplex d(C1m8 G2C3G4C5LG6LC7G8C9G10)d(C11m8G12C13G14C15LG16LC17G18C19G20) (O1) with two contiguous pairs of enantiomeric 2'-deoxy-L-ribonucleotides (C5LG6L/C15LG16L) at its centre and an 8-methylguanine at position 2/12 was analysed by circular dichroism, NMR and molecular modelling. O1 resolves in a left-handed helical structure already at low salt concentration (0.1 M NaCl). The central L2-sugar portion assumes a B* left-handed conformation (mirror-image of right-handed B-DNA) while its flanking D4-sugar portions adopt the known Z left-handed conformation. The resulting Z4-B2*-Z4 structure (left-handed helix) is the reverse of that of B4-Z2*-B4 (right-handed helix) displayed by the nearly related decaoligodeoxyribonucleotide d(mC1G2mC3G4C5L G6LmC7G8mC9G10)2, at the same low salt concentration (0.1 M NaCl). In the same experimental conditions, d(C1m8G2C3G4C5G6C7G8C9G10)2 (O2), the stereoregular version of O1, resolves into a right-handed B-DNA helix. Thus, both the 8-methylguanine and the enantiomeric step CLpGL at the centre of the molecule are needed to induce left-handed helicity. Remarkably, in the various heterochiral decaoligodeoxyribonucleotides so far analysed by us, when the central CLpGL adopts the B* (respectively Z*) conformation, then the adjacent steps automatically resolves in the Z (respectively B) conformation. This allows a good optimisation of the base-base stackings and base-sugar van der Waals interactions at the ZB*/B*Z (respectively BZ*/Z*B) junctions so that the Z4-B2*-Z4 (respectively B4-Z2*-B4) helix displays a Tm (approximately 65 degrees C) that is only 5 degrees C lower than the one of its homochiral counterpart. Here we anticipate that a large variety of DNA helices can be generated at low salt concentration by manipulating internal factors such as sugar configuration, duplex length, nucleotide composition and base methylation. These helices can constitute powerful tools for structural and biological investigations, especially as they can be used in physiological conditions.     

Distribution of antigenic determinants recognized by three monoclonal antibodies (Q2/70, Q5/6 and Q5/13) on human Ia-like alloantigens and on their subunits

The distribution of antigenic determinants recognized by the anti-Ia-like antigen monoclonal antibodies (MoAb) Q2/70, Q5/6 and Q5/13 on molecules coded for by theDR locus and by non-DR loci was investigated using a binding assay with¹²⁵I-labeled Ia-like antigens isolated from four B lymphoid cell lines. The determinants reacting with the MoAb Q2/70 and Q5/13 are expressed on all DR alloantigens tested and on BR4X7 specificities, while those reacting with the MoAb Q5/6 are not detectable on DRw7 and BR4X7 molecules. None of the monoclonal antibodies reacted with DC1 molecules. The MoAb Q5/6 and Q5/13 reacted with the isolatedβ subunit of the Ia-like antigenic complex, while the MoAb Q2/70 did not react with the isolated chains.     

Distribution of antigenic determinants recognized by three monoclonal antibodies (Q2/70, Q5/6 and Q5/13) on human Ia-like alloantigens and on their subunits

The distribution of antigenic determinants recognized by the anti-Ia-like antigen monoclonal antibodies (MoAb) Q2/70, Q5/6 and Q5/13 on molecules coded for by the DR locus and by non-DR loci was investigated using a binding assay with 125I-labeled Ia-like antigens isolated from four B lymphoid cell lines. The determinants reacting with the MoAb Q2/70 and Q5/13 are expressed on all DR alloantigens tested and on BR4X7 specificities, while those reacting with the MoAb Q5/6 are not detectable on DRw7 and BR4X7 molecules. None of the monoclonal antibodies reacted with DC1 molecules. The MoAb Q5/6 and Q5/13 reacted with the isolated beta subunit of the Ia-like antigenic complex, while the MoAb Q2/70 did not react with the isolated chains.     

cDNA cloning of human muskelin and localisation of the muskelin (MKLN1) gene to human chromosome 7q32 and mouse chromosome 6 B1/B2 by physical mapping and FISH

Muskelin is a novel intracellular protein, identified in mouse cells, that acts as a mediator of cell spreading and cytoskeletal responses to the extracellular matrix component thrombospondin-1. We report that human muskelin is 98% identical with mouse muskelin at the amino acid level. The human muskelin gene is located at 7q32. The mouse muskelin gene maps syntenically at chromosome 6 B1/B2.     

Thermochemotherapy effect of nanosized As2O3/Fe3O4 complex on experimental mouse tumors and its influence on the expression of CD44v6, VEGF-C and MMP-9

Background  

Both thermotherapy and arsenic have been shown to be active against a broad spectrum of cancers. To reduce the limitations of conventional thermotherapy, improve therapeutic anticancer activity, reduce the toxicity of arsenic on normal tissue, and increase tissue-specific delivery, we prepared a nanosized As₂O₃/Fe₃O₄ complex (Fe₃O₄ magnetic nanoparticles encapsulated in As₂O₃). We assessed the thermodynamic characteristics of this complex and validated the hyperthermia effect, when combined with magnetic fluid hyperthermia (MFH), on xenograft HeLa cells (human cervical cancer cell line) in nude mice. We also measured the effect on the expression of CD44v6, VEGF-C, and MMP-9 which were related to cancer and/or metastasis.     

BMP-9 downregulates StAR expression and progesterone production by activating both SMAD1/5/8 and SMAD2/3 signaling pathways in human granulosa-lutein cells obtained from gonadotropins induced ovarian cycles

Bone morphogenetic proteins (BMPs) are expressed in different cell types of the human ovarian follicle and play important roles in the regulation of ovarian function. BMP-9, also known as growth differentiation factor-2 (GDF-2), belongs to the transforming growth factor-beta (TGF-β) superfamily. BMP-9 is mainly synthesized in the liver and secreted into the blood which allows it to regulate various physiological and pathological functions. To date, the expression of BMP-9 in the human ovary and its function in human granulosa cells remains unknown. In the present study, we detect the protein expression of BMP-9 in the human follicular fluid. Using the primary culture of human granulosa-lutein (hGL) cells obtained from patients undergoing in vitro fertilization as a cell model, we show that treatment with BMP-9 downregulates steroidogenic acute regulatory protein (StAR) expression and suppresses progesterone (P4) production. The expression levels of the P450 side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) are not affected by BMP-9 treatment. Mechanistically, treatment of hGL cells with BMP-9 activates both SMAD1/5/8 and SMAD2/3 signaling pathways. Blocking the activations of SMAD1/5/8 and SMAD2/3 by pharmacological inhibitors or knockdown of SMAD4 attenuates the inhibitory effects of BMP-9 on StAR expression and P4 production. This study reveals a novel function of BMP-9 in the regulation of ovarian steroidogenesis.     

The gene encoding TBC1D1 with homology to the tre-2/USP6 oncogene, BUB2, and cdc16 maps to mouse chromosome 5 and human chromosome 4

TBC1D1 is the founding member of a family of related proteins with homology to tre-2/UPS6, BUB2, and cdc16 and containing the tbc box motif of 180-220 amino acids. This protein family is thought to have a role in differentiation and in regulating cell growth. We set out to map the TBC1D1 gene in mouse and human. Segregation analysis of a TBC1D1 RFLP in two independent mouse RI (recombinant inbred) lines reveals that mouse Tbc1d1 is closely linked to Pgm1 on chromosome 5. The human TBC1D1 gene was assigned to human chromosome 4p15.1-->4q21 using Southern blot analyses of genomic DNAs from rodent-human somatic cell lines. A human-specific genomic fragment was observed in the somatic cell lines containing human chromosome 4 or the 4p15.1-->4q21 region of the chromosome. TBC1D1 maps to the region containing the ortholog of mouse Pgm1 adding another locus to this long region of conserved synteny between mouse and man.     

Fluorescence in situ hybridization establishes homology between human and silvered leaf monkey chromosomes,reveals reciprocal translocations between chromosomes homologous to human Y/5, 1/9, and 6/16, and delineates an X1X2Y1Y2/X1X1X2X2 sex-chromosome system

We employed in situ hybridization of chromosome-specific DNA probes (“chromosome painting”) of all human chromosomes to establish homologies between the human and the silvered lead monkey karyotypes (Presbytis cristata 2n=44). The 24 human paints gave 30 signals on the haploid female chromosome set and 34 signals on the haploid male chromosome set. This difference is due to a reciprocal translocation between the Y and an autosome homologous to human chromosome 5. This Y/autosome reciprocal translocation which is unique among catarrhine primates has produced a X₁X₂Y₁Y₂/X₁X₁X₂X₂ sex-chromosome system. Although most human syntenic groups have been maintained in the silvered leaf monkey chromosomes homologous to human chromosomes 14 and 15, 21 and 22 have experienced Robertsonian fusions. Further, the multiple FISH signals provided by libraries to human chromosomes 1/9, 6/16 indicate that these chromosomes have been split by reciprocal translocations. G-banding analysis shows three different forms of chromosome 1 (X₂) which differ by a complex series of inversions in the 10 individuals karyotyped. Comparisons with the hybridization patterns in hylobatids (gibbons and siamang) demonstrate that resemblances in chromosomal morphology and banding previously taken to indicate a special phylogenetic relationship between gibbons and colobines are due to convergence. A. J. Phys. Anthropol. 102:315–327, 1997. © 1997 Wiley-Liss, Inc.