Alane Gray

Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling

Vertebrates and invertebrates initiate a series of defence mechanisms following infection by Gram-negative bacteria by sensing the presence of lipopolysaccharide (LPS), a major component of the cell wall of the invading pathogen. In humans, monocytes and macrophages respond to LPS by inducing the expression of cytokines, cell-adhesion proteins, and enzymes involved in the production of small proinflammatory mediators. Under pathophysiological conditions, LPS exposure can lead to an often fatal syndrome known as septic shock. Sensitive responses of myeloid cells to LPS require a plasma protein called LPS-binding protein and the glycosylphosphatidylinositol-anchored membrane protein CD14. However, the mechanism by which the LPS signal is transduced across the plasma membrane remains unknown. Here we show that Toll-like receptor 2 (TLR2) is a signalling receptor that is activated by LPS in a response that depends on LPS-binding protein and is enhanced by CD14. A region in the intracellular domain of TLR2 with homology to a portion of the interleukin (IL)-1 receptor that is implicated in the activation of the IL-1–receptor-associated kinase is required for this response. Our results indicate that TLR2 is a direct mediator of signalling by LPS.

Nucleotide sequence of epidermal growth factor cDNA predicts a 128,000-molecular weight protein precursor

Epidermal growth factor (EGF) has a profound effect on the differentiation of specific cells in vivo, and has been shown to be a potent mitogenic factor for a variety of cultured cells, of both ectodermal and mesodermal origin (see ref. 1 for review). This 53-amino acid polypeptide of known sequence2 contains six cysteine residues3, which are thought to form three intrachain disulphide bonds4. Urogastrone, a polypeptide bearing anti-gastric secretory activity isolated from human urine5, which is presumably synthesized in submandibular and Brunners glands6,7, shares extensive sequence homology (70%) with EGF and may represent the human EGF equivalent. Here we present the sequence of a mouse EGF cDNA clone, which suggests that EGF is synthesized as a large protein precursor of 1,168 amino acids. Our data indicate that the discrepancy between EGF levels in male and female mouse submaxillary glands (MSGs) is due to different EGF mRNA levels in these tissues, and suggest that precursor EGF processing may differ from that described previously for other polypeptide hormones.

Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon of E. coli.

Abstract We have constructed recombinant plasmids containing the entire Escherichia coli rrnB ribosomal RNA operon and segments thereof. Cloning of the 7.5-kb BamHI fragment, from λrifd18 which contains this operon, in plasmid vectors pBR 313 or pBR 322 is described. The 3.2-kb Eco RI Bam HI fragment containing the 3′ two-thirds of the 23 S rRNA gene, the 5S rRNA gene, and the terminator region has been cloned separately in pBR 313. As the nucleotide sequences of pBR 322 and the 7.5-kb fragment carrying the rrnB operon have been established, the entire 11.9-kb sequence of pKK 3535 is now known. This makes possible precise rearrangements and site-specific alterations of the ribosomal RNA operon; thus, pKK 3535 becomes a powerful tool for studies such as initiation and termination of transcription, processing of rRNA precursors, and investigations of the structure, function, and assembly of the ribosome itself. A detailed physical map of pKK 3535 is presented.

The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation

Vascular development is a complex but orderly process that is tightly regulated. A number of secreted factors produced by surrounding cells regulate endothelial cell (EC) differentiation, proliferation, migration and coalescence into cord-like structures. Vascular cords then undergo tubulogenesis to form vessels with a central lumen. But little is known about how tubulogenesis is regulated in vivo. Here we report the identification and characterization of a new EC-derived secreted factor, EGF-like domain 7 (Egfl7). Egfl7 is expressed at high levels in the vasculature associated with tissue proliferation, and is downregulated in most of the mature vessels in normal adult tissues. Loss of Egfl7 function in zebrafish embryos specifically blocks vascular tubulogenesis. We uncover a dynamic process during which gradual separation and proper spatial arrangement of the angioblasts allow subsequent assembly of vascular tubes. This process fails to take place in Egfl7 knockdown embryos, leading to the failure of vascular tube formation. Our study defines a regulator that controls a specific and important step in vasculogenesis.

Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymphoma: Target and Linker-Drug Selection

Antibody-drug conjugates (ADC), potent cytotoxic drugs covalently linked to antibodies via chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Here, we systematically examine the potential targets and linker-drug combinations that could provide an optimal ADC for the treatment for non-Hodgkins lymphoma. We identified seven antigens (CD19, CD20, CD21, CD22, CD72, CD79b, and CD180) for potential treatment of non-Hodgkins lymphoma with ADCs. ADCs with cleavable linkers mediated in vivo efficacy via all these targets; ADCs with uncleavable linkers were only effective when targeted to CD22 and CD79b. In target-independent safety studies in rats, the uncleavable linker ADCs showed reduced toxicity, presumably due to the reduced release of free drug or other toxic metabolites into the circulation. Thus, our data suggest that ADCs with cleavable linkers work on a broad range of targets, and for specific targets, ADCs with uncleavable linkers provide a promising opportunity to improve the therapeutic window for ADCs in humans.

Differential signalling potential of insulin- and IGF-1-receptor cytoplasmic domains

The human receptors for insulin‐like growth factor 1 (IGF‐1) and insulin, and two chimeric receptors consisting of ligand‐binding, extracellular insulin receptor and intracellular IGF‐1 receptor structures, have been expressed in NIH‐3T3 fibroblasts. All four receptor types were synthesized, processed and transported to the cell surface to form high‐affinity binding sites. All normal and chimeric receptors had an active tyrosine kinase which was regulated by homologous or heterologous ligands respectively. In addition, cell surface receptors were internalized efficiently and subjected to accelerated degradation in the presence of ligand. While all four types of receptor stimulated glucose transport with similar efficiency, they displayed significant differences in their mitogenic signalling potentials. Receptors with an IGF‐1 receptor cytoplasmic domain were 10 times more active in stimulating DNA synthesis than the insulin receptor. In NIH‐3T3 cells overexpressing wild‐type and chimeric receptors, maximal growth responses obtained with IGF‐1 or insulin alone were equivalent to those obtained with 10% fetal calf serum. We conclude that in the cell system employed the receptors for IGF‐1 and insulin mediate short‐term responses similarly, but display distinct characteristics in their long‐term mitogenic signalling potentials.

EGFL7 regulates the collective migration of endothelial cells by restricting their spatial distribution

During sprouting angiogenesis, groups of endothelial cells (ECs) migrate together in units called sprouts. In this study, we demonstrate that the vascular-specific secreted factor EGFL7 regulates the proper spatial organization of ECs within each sprout and influences their collective movement. In the homozygous Egfl7-knockout mice, vascular development is delayed in many organs despite normal EC proliferation, and 50% of the knockout embryos die in utero. ECs in the mutant vasculatures form abnormal aggregates and the vascular basement membrane marker collagen IV is mislocalized, suggesting that ECs fail to recognize the proper spatial position of their neighbors. Although the migratory ability of individual ECs in isolation is not affected by the loss of EGFL7, the aberrant spatial organization of ECs in the mutant tissues decreases their collective movement. Using in vitro and in vivo analyses, we showed that EGFL7 is a component of the interstitial extracellular matrix deposited on the basal sides of sprouts, a location suitable for conveying positional information to neighboring ECs. Taken together, we propose that EGFL7 defines the optimal path of EC movement by assuring the correct positioning of each EC in a nascent sprout.

Isolation of the human insulin-like growth factor I gene using a single synthetic DNA probe

A single synthetic oligonucleotide was employed as hybridization probe to detect and enable isolation of the human insulin‐like growth factor I (IGF‐I) gene from a human genomic DNA library. The synthetic oligonucleotide probe coded for the B‐chain of IGF‐I and was designed for expression in Escherichia coli. Despite numerous interspersed mismatches, the synthetic probe hybridized specifically with seven recombinant lambda phage containing almost the entire B‐chain region of the human IGF‐I gene. The usefulness of this approach was further demonstrated by the detection of lambda phage containing human preproinsulin, using A and B chain synthetic oligonucleotides, 90 and 63 nucleotides in length, as hybridization probes. The nucleotide sequence of the human IGF‐I exon suggests that IGF‐I is synthesized as a larger precursor molecule.

Hepatocytes Express Nerve Growth Factor during Liver Injury: Evidence for Paracrine Regulation of Hepatic Stellate Cell Apoptosis

A key feature of recovery from liver fibrosis is hepatic stellate cell (HSC) apoptosis, which serves the dual function of removing the major source of neomatrix and tissue inhibitors of metalloproteinases thereby facilitating matrix degradation. The mechanisms regulating HSC apoptosis remain undefined but may include the interaction of nerve growth factor (NGF) with its receptor, p75, on HSC. In this study, by TaqMan polymerase chain reaction in situ hybridization and immunohistochemistry, we demonstrate that NGF is expressed by hepatocytes during fibrotic injury. Peak hepatocyte expression of NGF (48 hours after CCl(4) injection) coincides with maximal rate of apoptosis of HSC by terminal dUTP nick-end labeling staining. Addition of recombinant NGF to HSC in tissue culture causes a dose-dependent increase in apoptosis. NGF regulates nuclear factor (NF)-kappaB activity, reducing p50/p65 binding detected by electromobility shift assay and reduced NF-kappaB CAT reporter activities from both basal unstimulated levels and after NF-kappaB induction by tumor necrosis factor. In each case, a relative reduction in NF-kappaB binding was associated with a significant increase in caspase 3 activity. These data provide evidence that NGF is expressed during fibrotic liver injury and may regulate number of activated HSCs via induction of apoptosis.

Nucleotide sequence of a portion of human chromosome 9 containing a leukocyte interferon gene cluster.

The nucleotide sequence of a 9937 base-pair portion of human chromosome 9, which contains two complete leukocyte interferon genes (LeIF-L and J), the complete intergenic region, and part of a third related possible pseudogene (LeIF-M), has been determined. The coding regions of the L and J genes are separated by 4363 nucleotides. The coding regions for the putative L and J interferons are 96% homologous and are each surrounded by about 3500 nucleotides of flanking sequences, which are also highly homologous. The L and J genes and their respective flanking sequences comprise a 4000 nucleotide leukocyte interferon gene repeat unit; the L gene repeat unit contains two major insertions not present in the J gene repeat unit. The J gene repeat unit is flanked by sequence features reminiscent of those found surrounding transposable elements. Both the L and J gene repeat units are embedded within sequences that are highly repeated in the human genome. Structural features identified within this portion of chromosome 9 may have been important for the generation of this interferon gene cluster.