Transcription factor Tfec contributes to the IL-4-inducible expression of a small group of genes in mouse macrophages including the granulocyte colony-stimulating factor receptor

Expression of the mouse transcription factor EC (Tfec) is restricted to the myeloid compartment, suggesting a function for Tfec in the development or function of these cells. However, mice lacking Tfec develop normally, indicating a redundant role for Tfec in myeloid cell development. We now report that Tfec is specifically induced in bone marrow-derived macrophages upon stimulation with the Th2 cytokines, IL-4 and IL-13, or LPS. LPS induced a rapid and transient up-regulation of Tfec mRNA expression and promoter activity, which was dependent on a functional NF-kappa B site. IL-4, however, induced a rapid, but long-lasting, increase in Tfec mRNA, which, in contrast to LPS stimulation, also resulted in detectable levels of Tfec protein. IL-4-induced transcription of Tfec was absent in macrophages lacking Stat6, and its promoter depended on two functional Stat6-binding sites. A global comparison of IL-4-induced genes in both wild-type and Tfec mutant macrophages revealed a surprisingly mild phenotype with only a few genes affected by Tfec deficiency. These included the G-CSFR (Csf3r) gene that was strongly up-regulated by IL-4 in wild-type macrophages and, to a lesser extent, in Tfec mutant macrophages. Our study also provides a general definition of the transcriptome in alternatively activated mouse macrophages and identifies a large number of novel genes characterizing this cell type.

Adipose tissue engineering based on the controlled release of fibroblast growth factor-2 in a collagen matrix

Adipose tissue forms when basement membrane extract ( Matrigel (TM)) and fibroblast growth factor-2 (FGF-2) are added to our mouse tissue engineering chamber model. A mouse tumor extract, Matrigel is unsuitable for human clinical application, and finding an alternative to Matrigel is essential. In this study we generated adipose tissue in the chamber model without using Matrigel by controlled release of FGF-2 in a type I collagen matrix. FGF-2 was impregnated into biodegradable gelatin microspheres for its slow release. The chambers were filled with these microspheres suspended in 60 mu L collagen gel. Injection of collagen containing free FGF-2 or collagen containing gelatin microspheres with buffer alone served as controls. When chambers were harvested 6 weeks after implantation, the volume and weight of the tissue obtained were higher in the group that received collagen and FGF-2 impregnated microspheres than in controls. Histologic analysis of tissue constructs showed the formation of de novo adipose tissue accompanied by angiogenesis. In contrast, control groups did not show extensive adipose tissue formation. In conclusion, this study has shown that de novo formation of adipose tissue can be achieved through controlled release of FGF-2 in collagen type I in the absence of Matrigel.

p-cresol as a reversible acylium ion scavenger in solid-phase peptide synthesis

In this work we have defined the nature of the p-cresol and p-thiocresol adducts generated from acylium ions during HF cleavage, following contemporary Boc/benzyl solid-phase peptide synthesis. Contrary to the results in previous reports, we found that both p-cresol and p-thiocresol predominantly form. aryl esters under typical cleavage conditions. Initially we investigated a number of small peptides containing either a single glutamate residue or a C-terminal long-chain amino acid which allowed us to unambiguously characterize the scavenged side products. Whereas, the p-cresol esters are stable at 0 degrees C they rearrange irreversibly at higher temperatures (5-20 degrees C) to form aryl ketones. By contrast, p-thiocresol esters do not undergo a Fries rearrangement but readily undergo further additions of p-thiocresol to form ketenebisthioacetals and trithio ortho esters, even at low temperatures. Importantly, we found by LC/MS and FT-ICR MS analysis that peptides containing p-cresol esters at glutamyl side chains are susceptible to amidation and fragmentation reactions at these sites during standard mild base workup procedures. The significance of these side reactions was further demonstrated in the synthesis of neutrophil immobilization factor, a 26-residue peptide, containing four glutamic acid residues. The side reactions were largely avoided by mild hydrogen peroxide-catalyzed hydrolysis which converted the p-cresol adducts to the free carboxylic acids in near quantitative yield. The choice of p-cresol as a reversible acylium ion scavenger when coupled with the simple workup conditions described is broadly applicable to Boc/benzyl peptide synthesis and will significantly enhance the quality of peptides produced.

Arterial heparan sulfate proteoglycans inhibit vascular smooth muscle cell proliferation and phenotype change in vitro and neointimal formation in vivo

Purpose: The aim of this study was to determine whether heparan sulfate proteoglycans (HSPGs) from the normal arterial wall inhibit neointimal formation after injury in vivo and smooth muscle cell (SMC) phenotype change and proliferation in vitro. Methods: Arterial HSPGs were extracted from rabbit aortae and separated by anion-exchange chromatography. The effect of HSPGs, applied in a periadventitial gel, on neointimal formation was assessed 14 days after balloon catheter injury of rabbit carotid arteries. Their effect on SMC phenotype and proliferation was measured by point-counting morphometry of the cytoplasmic volume fraction of myofilaments (Vvmyo) and H-3-thymidine incorporation in SMCs in culture. Results: Arterial HSPGs (680 mu g) reduced neointimal formation by 35% at 14 days after injury (P =.029), whereas 2000 mu g of the low-molecular-weight heparin Enoxaparin was ineffective. HSPGs at 34 mu g/mL maintained subconfluent primary cultured SMCs with the same high Vvmyo (52.1% +/- 13.8%) after 5 days in culture as did cells freshly isolated from the arterial wall (52.1% +/- 15.1%). In contrast, 100 mu g/mL Enoxaparin was ineffective in preventing phenotypic change over this time period (Vvmyo 38.9% +/- 14.6%, controls 35.9% +/- 12.8%). HSPGs also inhibited 3H-thymidine incorporation into primary cultured SMCs with an ID50 value of 0.4 mu g/mL compared with a value of 14 mu g/ml; for Enoxaparin (P

Effect of estrogen on vascular smooth muscle cells is dependent upon cellular phenotype

To investigate the growth-regulating action of estrogen on vascular smooth muscle cells (SMC), effects of beta-17-estradiol (beta-E-2) on phenotypic modulation and proliferation of rabbit aortic SMC were observed in vitro. At 10(-8) M, beta-E-2 significantly slowed the decrease in volume fraction of myofilaments (V(v)myo) of freshly dispersed SMCs in primary culture, indicating an inhibitory effect of beta-E-2 On spontaneous phenotypic modulation of SMC from a contractile to a synthetic phenotype. Freshly dispersed SMCs treated with beta-E-2 also had a relatively longer quiescent phase than control cells before intense proliferation occurred. This was in contrast to SMCs in passage 2-3 (synthetic state), where beta-E-2-treated cells replicated significantly faster than untreated cells. beta-E-2 also markedly enhanced the serum-induced DNA synthesis of synthetic SMCs in a concentration-dependent manner within physiological range (10(-10) to 10-8 M). These findings indicate that the growth-regulating effect of estrogen on vascular SMC is dependent on the cell's phenotypic stare. It delays the cell cycle re-entry of the contractile SMCs by retarding their phenotypic modulation however, once cells have modulated to the synthetic phenotype, it promotes their replication. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

Enhanced downregulation of the p75 nerve growth factor receptor by cholesteryl and bis-cholesteryl antisense oligonucleotides

The effects of conjugating cholesterol to either or both ends of a phosphorothioate (PS) oligonucleotide were analyzed in terms of cellular uptake and antisense efficacy. The oligo sequence was directed against the p75 nerve growth factor receptor (p75), and was tested in differentiated PC12 cells, which express high levels of this protein. The addition of a single cholesteryl group to the 5'-end significantly increased cellular uptake and improved p75 mRNA downregulation compared with the unmodified PS oligo, However, only a minor degree of downregulation of p75 protein was obtained with 5' cholesteryl oligos, Three different linkers was used to attach the 5' cholesteryl group but were found not to have any impact on efficacy. Addition of a single cholesteryl group to the 3'-end led to greater p75 mRNA downregulation (31%) and p75 protein downregulation (28%) than occurred with the 5' cholesteryl oligos. The biggest improvement in antisense efficacy, both at the mRNA and protein levels, was obtained from the conjugation of cholesterol to both ends of the oligo. One of the bis-cholesteryl oligos was nearly as effective as cycloheximide at decreasing synthesis of p75, The bis-cholesteryl oligos also displayed significant efficacy at 1 mu M, whereas the other oligos required 5 mu M to be effective. The enhanced efficacy of bis-cholesteryl oligos is likely to be due to a combination of enhanced cellular uptake and resistance to both 5' and 3' exonucleases.

Fibroblast growth factor receptor expression in outer hair cells of rat cochlea

FIBROBLAST growth factors (FGFs) are critical for normal development of the organ of Corti, and may also protect hair cells from ototoxic damage. Four different fibroblast growth factors are known, three of which have different splice variants in the extracellular immunoglobin-like (Ig) III FGF-binding domain, giving different patterns of sensitivity to the different FGFs. Analysis of a cDNA library of rat outer hair cells by the polymerase chain reaction, using isoform specific primers, showed expression only of FGF receptor 3, splice variant IIIc. This allows us to predict the pattern of sensitivity to applied FGFs, may be useful in targeting outer hair cells selectively during an FGF-based strategy for cochlear therapy. (C) 1998 Lippincott Williams & Wilkins.

Role of the 6-20 disulfide bridge in the structure and activity of epidermal growth factor

Two synthetic analogues of murine epidermal. growth factor, [Abu6, 20] mEGF4-48 (where Abu denotes amino-butyric acid) and [G1, M3, K21, H40] mEGF1-48, have been investigated by NMR spectroscopy. [Abu6, 20] mEGF4-48 was designed to determine the contribution of the 6-20 disulfide bridge to the structure and function of mEGF The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. Significant structural differences were observed near the N-terminus, however, with the direction of the polypeptide chain between residues four and nine being altered such that these residues were now located on the opposite face of the main beta-sheet from their position in native mEGF Thermal denaturation experiments also showed that the structure of [Abu6, 20] mEGF4-48 was less stable than that of mEGF. Removal of this disulfide bridge resulted in a significant loss of both mitogenic activity in Balb/c 3T3 cells and receptor binding on A431 cells compared with native mEGF and mEGF4-48, implying that the structural changes in [Abu6, 20] mEGF4-48, although limited to the N-terminus, were sufficient to interfere with receptor binding. The loss of binding affinity probably arose mainly from steric interactions of the dislocated N-terminal region with part of the receptor binding surface of EGF [G1, M3, K21, H40] mEGF1-48 was also synthesized in order to compare the synthetic polypeptide with the corresponding product of recombinant expression. Its mitogenic activity in Balb/c 3T3 cells was similar to that of native mEGF and analysis of its H-1 chemical shifts suggested that its structure was also very similar to native.

The solution structure of the N-terminal zinc finger of GATA-1 reveals a specific binding face for the transcriptional co-factor FOG

Zinc fingers (ZnFs) are generally regarded as DNA-binding motifs. However, a number of recent reports have implicated particular ZnFs in the mediation of protein-protein interactions. The N-terminal ZnF of GATA-1 (NF) is one such finger, having been shown to interact with a number of other proteins, including the recently discovered transcriptional co-factor FOG. Here we solve the three-dimensional structure of the NF in solution using multidimensional H-1/N-15 NMR spectroscopy, and we use H-1/N-15 spin relation measurements to investigate its backbone dynamics. The structure consists of two distorted beta-hairpins and a single alpha-helix, and is similar to that of the C-terminal ZnF of chicken GATA-1. Comparisons of the NF structure with those of other C-4-type zinc binding motifs, including hormone receptor and LIM domains, also reveal substantial structural homology. Finally, we use the structure to map the spatial locations of NF residues shown by mutagenesis to be essential for FOG binding, and demonstrate that these residues all lie on a single face of the NE Notably, this face is well removed from the putative DNA-binding face of the NE an observation which is suggestive of simultaneous roles for the NF; that is, stabilisation of GATA-1 DNA complexes and recruitment of FOG to GATA-1-controlled promoter regions.

A cell type-specific constitutive point mutant of the common beta-subunit of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors requires the GM-CSF receptor alpha-subunit for activation

The high affinity receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a cytokine-specific alpha-subunit (hGMR alpha) and a common signal-transducing beta-subunit (hpc) that is shared with the interleukin-3 and -5 receptors, We have previously identified a constitutively active extracellular point mutant of hpc, I374N, that can confer factor independence on murine FDC-P1 cells but not BAF-B03 or CTLL-2 cells (Jenkins, B. J., D'Andrea, R. J., and Gonda, T. J. (1995) EMBO J. 14, 4276-4287), This restricted activity suggested the involvement of cell type-specific signaling molecules in the activation of this mutant. We report here that one such molecule is the mouse GMR alpha (mGMR alpha) subunit, since introduction of mGMR alpha, but not hGMR alpha, into BAF-B03 or CTLL-2 cells expressing the I374N mutant conferred factor independence, Experiments utilizing mouse/human chimeric GMR alpha subunits indicated that the species specificity lies in the extracellular domain of GMRa. Importantly, the requirement for mGMR alpha correlated with the ability of I374N (but not wild-type hpc) to constitutively associate with mGMRa. Expression of I374N in human factor-dependent UT7 cells also led to factor-independent proliferation, with concomitant up-regulation of hGMR alpha surface expression. Taken together, these findings suggest a critical role for association with GMR alpha in the constitutive activity of I374N.

Sustained gene expression in transplanted skin fibroblasts in rats

Retrovirus-mediated gene transfer into adult skin fibroblasts has provided measurable amounts of therapeutic proteins in animal models. However, the major problem emerging from these experiments was a limited time of vector encoded gene expression once transduced cells were engrafted We hypothesized that sustained transduced gene expression in quiescent fibroblasts in vivo might be obtained by using a fibronectin (Fn) promoter. Fibronectin plays a key role in cell adhesion, migration and wound healing and is up-regulated in quiescent fibroblasts. Retroviral vectors containing human adenosine deaminase (ADA) cDNA linked to rat fibronectin promoter (LNFnA) or viral LTR promoter (LASN) were compared for their ability to express ADA from transduced primary rat skin fibroblasts in vivo. Skin grafts formed from fibroblasts transduced with LNFnA showed strong human ADA enzyme activity from 1 week to 3 months. In contrast, skin grafts containing LASN-transduced fibroblasts tested positive for human ADA for weeks 1 and 2, were faintly positive at week 3 and showed no human ADA expression at 1, 2 and 3 months. Thus, a fibronectin promoter provided sustained transduced gene expression at high levels for at least 3 months in transplanted rat skin fibroblasts, perhaps permitting the targeting of this tissue for human gene therapy.

Low-molecular-weight peptidic and cyclic antagonists of the receptor for the complement factor C5a

Activation of the human complement system of plasma proteins during immunological host defense can result in overproduction of potent proinflammatory peptides such as the anaphylatoxin C5a. Excessive levels of C5a are associated with numerous immunoinflammatory diseases, but there is as yet no clinically available antagonist to regulate the effects of C5a. We now describe a series of small molecules derived from the C-terminus of C5a, some of which are the most potent low-molecular-weight C5a receptor antagonists reported to date for the human polymorphonuclear leukocyte (PMN) C5a receptor. H-1 NMR spectroscopy was used to determine solution structures for two cyclic antagonists and to indicate that antagonism is related to a turn conformation, which can be stabilized in cyclic molecules that are preorganized for receptor binding. While several cyclic derivatives were of similar antagonistic potency, the most potent antagonist was a hexapeptide-derived macrocycle AcF[OPdChaWR] with an IC50 = 20 nM against a maximal concentration of C5a (100 nM) on intact human PMNs. Such potent C5a antagonists may be useful probes to investigate the role of C5a in host defenses and to develop therapeutic agents for the treatment of many currently intractable inflammatory conditions.

Distribution of interleukin-2,-4,-10, tumour necrosis factor-alpha and transforming growth factor-beta mRNAs in oral lichen planus

In the present study. MRNA for the cytokines interleukin-2 (IL-2), IL-4, IL-10 tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor beta-1 (TGF-beta-1) were investigated in oral lichen planus (OLP) lesions using in situ hybridization with S-35-labelled oligonucleotide probes on frozen tissue sections. In addition, the expression of interferon-gamma (IFN-gamma), IL-10 and IL-4 mRNAs was analysed in cultured lesional T lymphocytes from oral lichen planus by polymerase chain reaction. Cells expressing mRNA for IL-2, IL-4, IL-10, TNF-alpha and TGF-beta(1) were found in all the biopsies studied. Approximately 1-2% of the total number of infiltrating cells in the lesions were positive for each of the different cytokine mRNAs. Most biopsies contained basement membrane-oriented, mRNA-positive cells. In the cultured T-cell lines, message for IFN-gamma was detected in all the patients, IL-10 in all but one, and IL-4 in just one of the seven patients investigated. The results suggest that mRNA for both pro- and anti-inflammatory cytokines, i.e., mixed T-helper 1 (T(H)1) and T(H)2 cytokine profiles, are generated simultaneously by a limited number of cells in chronic lesions of OLP. (C) 1999 Elsevier Science Ltd. All rights reserved.

Changing patterns of coronary heart disease in the hunter region of New South Wales, Australia

A population-based observational study of men acid women aged 35-69 years in the Hunter Region of New South Wales, Australia, was conducted to assess the impact. of risk-factor modification and increased drug therapy on the trends in major coronary events and case fatality. From 1985 to 1993, there were 3006 coronary deaths and 6450 nonfatal major coronary events. Rates of death and nonfatal myocardial infarction declined, but there was an increase in hospital admissions for prolonged chess pain. Reductions in cigarette smoking, diastolic blood pressure, total cholesterol, and increased use of aspirin can fully explain the 3.3% (95% confidence interval [CI] 2.4, 4.2) average annual reduction in rates of major coronary events for men and the 4.1% (95% CI 2.7, 5.5) reduction for women. In contrast, increased use of aspirin, beta-blockers, fibrinolytic therapy, and angiotensin-converting enzyme inhibitors explain less than hall of the 8.9% (95% CI 5.9, 11.8) and 6.9% (95% CI 2.7, 10.9) average annual reduction in case fatality in hospital for men and women, respectively. These trends suggest a decline in severity of coronary heart disease consistent with reductions in risk-factor levels and improved acute medical treatment. J CLIN EPIDEMIOL 52;8:761-771, 1999. (C) 1999 Elsevier Science Inc.