Angiogenesis is Differentially Modulated by Anthocyanin and Phenolic Acid Extracts from Wild Blueberry (V. angustifolium) Through PI3K Pathway.

Wild blueberries rank very high in anthocyanins (ACNs) and phenolic acids (PA) among other berries. Previous work from our group has documented their beneficial effects. In this study, human umbilical vein endothelial cells were used, and ACNs, PAs, and their combination (ACNs:PAs) at concentrations of 0.002, 8, 15, 60, and 120 µg/mL were tested for endothelial tube formation. Treatment with ACNs decreased, while treatment with PAs and ACNs:PAs increased overall endothelial cell tube formation compared to control. Endothelial cells exposed to ACNs downregulated gene expression of AKT1 and endothelial nitric oxide synthase (eNOS), while PAs upregulated AKT1 and vascular endothelial growth factor (VEGF) gene expression. Combination of ACNs:PAs decreased gene expression of AKT1 and eNOS, while protein levels of AKT1 increased. In summary, based on the type of wild blueberry extract, angiogenesis is differentially modulated and is concentration dependent. Further experiments will delineate the mechanism(s) of the differential action of the aforementioned extracts on angiogenesis.

Preclinical evaluation of [18F]FB-A20FMDV2 as a selective marker for measuring αVß6 integrin occupancy using positron emission tomography in rodent lung.

PURPOSE: Integrin αvß6 belongs to the RGD subset of the integrin family, and its expression levels are a prognostic and theranostic factor in some types of cancer and pulmonary fibrosis. This paper describes the GMP radiolabelling of the synthetic 20 amino acid peptide A20FMDV2 (NAVPNLRGDLQVLAQKVART), derived from the foot-and-mouth disease virus, and characterises the use of [18F]FB-A20FMDV2 as a high affinity, specific and selective PET radioligand for the quantitation and visualisation of αvß6 in rodent lung to support human translational studies. METHODS: The synthesis of [18F]FB-A20FMDV2 was performed using a fully automated and GMP-compliant process. Sprague-Dawley rats were used to perform homologous (unlabelled FB-A20FMDV2) and heterologous (anti-αvß6 antibody 8G6) blocking studies. In order to generate a dosimetry estimate, tissue residence times were generated, and associated tissue exposure and effective dose were calculated using the Organ Level Internal Dose Assessment/Exponential Modelling (OLINDA/EXM) software. RESULTS: [18F]FB-A20FMDV2 synthesis was accomplished in 180 min providing ~800 MBq of [18F]FB-A20FMDV2 with a molar activity of up to 150 GBq/µmol and high radiochemical purity (> 97%). Following i.v. administration to rats, [18F]FB-A20FMDV2 was rapidly metabolised with intact radiotracer representing 5% of the total radioactivity present in rat plasma at 30 min. For the homologous and heterologous block in rats, lung-to-heart SUV ratios at 30-60 min post-administration of [18F]FB-A20FMDV2 were reduced by 38.9 ± 6.9% and 56 ± 19.2% for homologous and heterologous block, respectively. Rodent biodistribution and dosimetry calculations using OLINDA/EXM provided a whole body effective dose in humans 33.5 µSv/MBq. CONCLUSION: [18F]FB-A20FMDV2 represents a specific and selective PET ligand to measure drug-associated αvß6 integrin occupancy in lung. The effective dose, extrapolated from rodent data, is in line with typical values for compounds labelled with fluorine-18 and combined with the novel fully automated and GMP-compliant synthesis and allows for clinical use in translational studies.

18F-Trifluoromethanesulfinate Enables Direct C-H 18F-Trifluoromethylation of Native Aromatic Residues in Peptides.

18F labeling strategies for unmodified peptides with [18F]fluoride require 18F-labeled prosthetics for bioconjugation more often with cysteine thiols or lysine amines. Here we explore selective radical chemistry to target aromatic residues applying C-H 18F-trifluoromethylation. We report a one-step route to [18F]CF3SO2NH4 from [18F]fluoride and its application to direct [18F]CF3 incorporation at tryptophan or tyrosine residues using unmodified peptides as complex as recombinant human insulin. The fully automated radiosynthesis of octreotide[Trp(2-CF218F)] enables in vivo positron emission tomography imaging.

Phenolic and anthocyanin fractions from wild blueberries (V. angustifolium) differentially modulate endothelial cell migration partially through RHOA and RAC1.

The present study investigates the effect of anthocyanin (ACN), phenolic acid (PA) fractions, and their combination (ACNs:PAs) from wild blueberry powder (Vaccinum angustifolium) on the speed of endothelial cell migration, gene expression, and protein levels of RAC1 and RHOA associated with acute exposure to different concentrations of ACNs and PAs. Time-lapse videos were analyzed and endothelial cell speed was calculated. Treatment with ACNs at 60 µg/mL inhibited endothelial cell migration rate ( P ≤ 0.05) while treatment with PAs at 0.002 µg/mL ( P ≤ 0.0001), 60 µg/mL ( P ≤ 0.0001), and 120 µg/mL ( P ≤ 0.01) significantly increased endothelial cell migration rate compared with control. Moreover, exposure of HUVECs to ACNs:PAs at 8:8 µg/mL ( P ≤ 0.05) and 60:60 µg/mL increased ( P ≤ 0.001) endothelial cell migration. Gene expression of RAC1 and RHOA significantly increased 2 hours after exposure with all treatments. No effect of the above fractions was observed on the protein levels of RAC1 and RHOA. Findings suggest that endothelial cell migration is differentially modulated based on the type of blueberry extract (ACN or PA fraction) and is concentration-dependent. Future studies should determine the mechanism of the differential action of the above fractions on endothelial cell migration.

In vivo imaging of microglial activation by positron emission tomography with [(11)C]PBR28 in the 5XFAD model of Alzheimer's disease.

Microglial activation has been linked with deficits in neuronal function and synaptic plasticity in Alzheimer's disease (AD). The mitochondrial translocator protein (TSPO) is known to be upregulated in reactive microglia. Accurate visualization and quantification of microglial density by PET imaging using the TSPO tracer [(11)C]-R-PK11195 has been challenging due to the limitations of the ligand. In this study, it was aimed to evaluate the new TSPO tracer [(11)C]PBR28 as a marker for microglial activation in the 5XFAD transgenic mouse model of AD. Dynamic PET scans were acquired following intravenous administration of [(11)C]PBR28 in 6-month-old 5XFAD mice and in wild-type controls. Autoradiography with [(3)H]PBR28 was carried out in the same brains to further confirm the distribution of the radioligand. In addition, immunohistochemistry was performed on adjacent brain sections of the same mice to evaluate the co-localization of TSPO with microglia. PET imaging revealed that brain uptake of [(11)C]PBR28 in 5XFAD mice was increased compared with control mice. Moreover, binding of [(3)H]PBR28, measured by autoradiography, was enriched in cortical and hippocampal brain regions, coinciding with the positive staining of the microglial marker Iba-1 and amyloid deposits in the same areas. Furthermore, double-staining using antibodies against TSPO demonstrated co-localization of TSPO with microglia and not with astrocytes in 5XFAD mice and human post-mortem AD brains. The data provided support of the suitability of [(11)C]PBR28 as a tool for in vivo monitoring of microglial activation and assessment of treatment response in future studies using animal models of AD.

Comparison of [corrected] actin- and glass-supported phospholipid bilayer diffusion coefficients.

The formation of biomimetic lipid membranes has the potential to provide insights into cellular lipid membrane dynamics. The construction of such membranes necessitates not only the utilization of appropriate lipids, but also physiologically relevant substrate/support materials. The substrate materials employed have been shown to have demonstrable effects on the behavior of the overlying lipid membrane, and thus must be studied before use as a model cushion support. To our knowledge, we report the formation and investigation of a novel actin protein-supported lipid membrane. Specifically, inner leaflet lateral mobility of globular actin-supported DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayers, deposited via the Langmuir-Blodgett/Langmuir Schaefer methodology, was investigated by z-scan fluorescence correlation spectroscopy across a temperature range of 20-44°C. The actin substrate was found to decrease the diffusion coefficient when compared to an identical membrane supported on glass. The depression of the diffusion coefficient occurred across all measured temperatures. These results indicated that the actin substrate exerted a direct effect on the fluidity of the lipid membrane and highlighted the fact that the choice of substrate/support is critical in studies of model lipid membranes.

A localized tolerance in the substrate specificity of the fluorinase enzyme enables "last-step" 18F†fluorination of a RGD peptide under ambient aqueous conditions.

A strategy for last-step (18)F fluorination of bioconjugated peptides is reported that exploits an "Achilles heel" in the substrate specificity of the fluorinase enzyme. An acetylene functionality at the C-2 position of the adenosine substrate projects from the active site into the solvent. The fluorinase catalyzes a transhalogenation of 5'-chlorodeoxy-2-ethynyladenosine (ClDEA) to 5'-fluorodeoxy-2-ethynyladenosine (FDEA). Extending a polyethylene glycol linker from the terminus of the acetylene allows the presentation of bioconjugation cargo to the enzyme for (18)F labelling. The method uses an aqueous solution (H2(18)O) of [(18)F]fluoride generated by the cyclotron and has the capacity to isotopically label peptides of choice for positron emission tomography (PET).

Characterising the plasma-target occupancy relationship of the neurokinin antagonist GSK1144814 with PET.

GSK1144814 is a potent, insurmountable antagonist at human NK1 and NK3 receptors. Understanding the relationship between plasma pharmacokinetics and receptor occupancy in the human brain, was crucial for dose selection in future clinical studies. GSK1144814 occupancy data were acquired in parallel with the first-time-in-human safety and tolerability study. [¹¹C]GR-205171 a selective NK1 receptor PET ligand was used to estimate NK1 occupancy at several time-points following single dose administration of GSK1144814. The time-plasma concentration-occupancy relationship post-single dose administration was assessed, and used to predict the plasma concentration-occupancy relationship following repeat dose administration. Repeat dose predictions were tested in a subsequent cohort of subjects examined following approximately 7 and 14 days dosing with GSK1144814. GSK1144814 was shown to demonstrate a dose-dependent occupancy of the NK1 receptor with an estimated in vivo EC50~0.9 ng/mL in the human brain. A direct relationship was seen between the GSK1144814 plasma concentration and its occupancy of the brain NK1 receptor, indicating that in future clinical trials the occupancy of brain receptors can be accurately inferred from the measured plasma concentration. Our data provided support for the further progression of this compound and have optimised the likely therapeutic dose range.

Technologies: preclinical imaging for drug development.

Preclinical imaging with magnetic resonance imaging (MRI), computerised tomography (CT), ultrasound (US), positron emission tomography (PET) or single-photon emission computed tomography (SPECT) enable non-invasive measures of tissue structure, function or metabolism in vivo. The technologies can add value to preclinical studies by enabling dynamic pharmacological observations on the same animal and because of possibilities for relatively direct clinical translation. Potential benefits from the application of preclinical imaging should be considered routinely in drug development.

Cofilin-1 inactivation leads to proteinuria--studies in zebrafish, mice and humans.

BACKGROUND: Podocytes are highly specialized epithelial cells on the visceral side of the glomerulus. Their interdigitating primary and secondary foot processes contain an actin based contractile apparatus that can adjust to changes in the glomerular perfusion pressure. Thus, the dynamic regulation of actin bundles in the foot processes is critical for maintenance of a well functioning glomerular filtration barrier. Since the actin binding protein, cofilin-1, plays a significant role in the regulation of actin dynamics, we examined its role in podocytes to determine the impact of cofilin-1 dysfunction on glomerular filtration. METHODS AND FINDINGS: We evaluated zebrafish pronephros function by dextran clearance and structure by TEM in cofilin-1 morphant and mutant zebrafish and we found that cofilin-1 deficiency led to foot process effacement and proteinuria. In vitro studies in murine and human podocytes revealed that PMA stimulation induced activation of cofilin-1, whereas treatment with TGF-ß resulted in cofilin-1 inactivation. Silencing of cofilin-1 led to an accumulation of F-actin fibers and significantly decreased podocyte migration ability. When we analyzed normal and diseased murine and human glomerular tissues to determine cofilin-1 localization and activity in podocytes, we found that in normal kidney tissues unphosphorylated, active cofilin-1 was distributed throughout the cell. However, in glomerular diseases that affect podocytes, cofilin-1 was inactivated by phosphorylation and observed in the nucleus. CONCLUSIONS: Based on these in vitro and in vivo studies we concluded cofilin-1 is an essential regulator for actin filament recycling that is required for the dynamic nature of podocyte foot processes. Therefore, we describe a novel pathomechanism of proteinuria development.

Evaluation of 11C-GSK189254 as a novel radioligand for the H3 receptor in humans using PET.

UNLABELLED: The histamine H(3) receptor is implicated in the pathophysiology of several central nervous system disorders. N-methyl-6-(3-cyclobutyl-2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-yloxy)-nicotamide (GSK189254) is a highly potent, selective, and brain-penetrant H(3) receptor antagonist. Previous studies in the pig using PET have shown that (11)C-GSK189254 uptake in H(3)-rich regions of the brain can be blocked by the selective H(3) antagonist ciproxifan. The purpose of the present study was to evaluate (11)C-GSK189254 as a PET radioligand for human studies and to determine the dose-receptor occupancy relationship of GSK189254 in the human brain. METHODS: Dynamic PET scans were obtained in healthy subjects over 90 min after intravenous administration of approximately 370 MBq of (11)C-GSK189254. Blood samples were taken throughout the scans to derive the arterial plasma parent input function. Each subject was scanned twice, either with tracer alone (test-retest) or before and after a single oral dose of GSK189254 (10-100 microg). Data were analyzed by compartmental analysis, and regional receptor-occupancy estimates were obtained by graphical analysis of changes in the total volumes of distribution (V(T)) of the radioligand. RESULTS: (11)C-GSK189254 readily entered the brain; its regional brain distribution reflected the known distribution of H(3) receptors, with high binding in the caudate and putamen, intermediate binding in cortical regions, and low binding in the cerebellum. GSK189254 displayed a high receptor affinity, and a marked reduction in V(T) was apparent at all the doses tested. The oral dose equaling 50% occupancy of the available receptor sites (ED(50)) was estimated as 4.33 microg. Additional data on plasma pharmacokinetics after oral dosing and the plasma free fraction gave a corresponding estimate of the free concentration of GSK189254 required to occupy 50% of the available receptor sites (EC(50)) (0.011 nM). The test-retest data showed reductions in regional V(T) on the second scan in all subjects. A nonlinear compartmental analysis of this effect demonstrated that this reduction was consistent with carryover of a tracer mass dose effect with an estimated in vivo apparent dissociation constant of 0.010 nM, close to the independent estimate of the plasma EC(50). CONCLUSION: (11)C-GSK189254 can be used to quantify H(3) receptor availability in humans in vivo using PET but requires high specific activity; the possibility of tracer mass dose effects should be carefully analyzed.

Cytokine-induced F-actin reorganization in endothelial cells involves RhoA activation.

Acute ischemic kidney injury results in marked increases in local and systemic cytokine levels. IL-1alpha, IL-6, and TNF-alpha orchestrate various inflammatory reactions influencing endothelial permeability by altering cell-to-cell and cell-to-extracellular matrix attachments. To explore the role of actin and the regulatory proteins RhoA and cofilin in this process, microvascular endothelial cells (MS1) were exposed to individual cytokines or a cytokine cocktail. Within minutes, a marked, time-dependent redistribution of the actin cytoskeleton occurred with the formation of long, dense F-actin basal stress fibers. The concentration of F-actin, normalized to nuclear staining, significantly increased compared with untreated cells (up 20%, P < or = 0.05). Western blot analysis of MS1 lysates incubated with the cytokine cocktail for 4 h showed an increase in phosphorylated/inactive cofilin (up 25 +/- 15%, P < or = 0.05) and RhoA activation (up to 227 +/- 26% increase, P < or = 0.05) compared with untreated cells. Decreasing RhoA levels using small interfering RNA blocked the effect of cytokines on stress fiber organization. Treatment with Y-27632, an inhibitor of the RhoA effector p160-ROCK, decreased levels of phosphorylated cofilin and reduced stress fiber fluorescence by 22%. In cells treated with Y-27632 followed by treatment with the cytokine cocktail, stress fiber levels were similar to control cells and cofilin phosphorylation was 55% of control levels. Taken together, these studies demonstrate cytokine stimulation of RhoA, which in turn leads to cofilin phosphorylation and formation of numerous basal actin stress fibers. These results suggest cytokines signal through the Rho-ROCK pathway, but also through another pathway to affect actin dynamics.

Fluorescent labeling of renal cells in vivo.

In vivo fluorescence imaging, using confocal or multiphoton microscopes, provides a powerful method to analyze kidney function in experimental animals. In this review, the preparation used for physiological studies in rats is described. A variety of fluorescent probes are available to study glomerular permeability, renal blood flow, peritubular capillary permeability, cell ion concentrations, tubule transport properties, and the functional status of renal cells. We have recently used micropuncture techniques and an adenovirus vector to accomplish gene transfer into kidney tubule and endothelial cells; this new methodology will allow the dynamic study of fluorescently-labeled proteins. Two examples of the use of two-photon fluorescence microscopy to study renal pathophysiology, namely polycystic kidney disease and renal ischemia, are presented. Software is available to quantify data collected from in vivo imaging experiments and to construct 3-dimensional images of renal structures. Two-photon or confocal microscopy offers many opportunities for a better understanding of kidney function in health and disease.

Cofilin mediates ATP depletion-induced endothelial cell actin alterations.

Ischemia and sepsis lead to endothelial cell damage, resulting in compromised microvascular flow in many organs. Much remains to be determined regarding the intracellular structural events that lead to endothelial cell dysfunction. To investigate potential actin cytoskeletal-related mechanisms, ATP depletion was induced in mouse pancreatic microvascular endothelial cells (MS1). Fluorescent imaging and biochemical studies demonstrated a rapid and progressive increase in F-actin along with a decrease in G-actin at 60 min. Confocal microscopic analysis showed ATP depletion resulted in destruction of actin stress fibers and accumulation of F-actin aggregates. We hypothesized these actin alterations were secondary to dephosphorylation/activation of actin-depolymerizing factor (ADF)/cofilin proteins. Cofilin, the predominant isoform expressed in MS1 cells, was rapidly dephosphorylated/activated during ATP depletion. To directly investigate the role of cofilin activation on the actin cytoskeleton during ischemia, MS1 cells were infected with adenoviruses containing the cDNAs for wild-type Xenopus laevis ADF/cofilin green fluorescent protein [XAC(wt)-GFP], GFP, and the constitutively active and inactive isoforms XAC(S3A)-GFP and XAC(S3E)-GFP. The rate and extent of cortical actin destruction and actin aggregate formation were increased in ATP-depleted XAC(wt)-GFP- and XAC(S3A)-GFP-expressing cells, whereas increased actin stress fibers were observed in XAC(S3E)-GFP-expressing cells. To investigate the upstream signaling pathway of ADF/cofilin, LIM kinase 1-GFP (LIMK1-GFP) was expressed in MS1 cells. Cells expressing LIMK1-GFP protein had higher levels of phosphorylated ADF/cofilin, increased stress fibers, and delayed F-actin cytoskeleton destruction during ATP depletion. These results strongly support the importance of cofilin regulation in ischemia-induced endothelial cell actin cytoskeleton alterations leading to cell damage and microvascular dysfunction.

Micropuncture gene delivery and intravital two-photon visualization of protein expression in rat kidney.

Understanding molecular mechanisms of pathophysiology and disease processes requires the development of new methods for studying proteins in animal tissues and organs. Here, we describe a method for adenoviral-mediated gene transfer into tubule or endothelial cells of the rat kidney. The left kidney of an anesthetized rat was exposed and the lumens of superficial proximal tubules or vascular welling points were microinfused, usually for 20 min. The microinfusion solution contained adenovirus with a cDNA construct of either 1) Xenopus laevis actin depolymerizing factor/cofilin [XAC; wt-green fluorescent protein (GFP)], 2) actin-GFP, or 3) GFP. Sudan black-stained castor oil, injected into nearby tubules, allowed us to localize the microinfused structures for subsequent visualization. Two days later, the rat was anesthetized and the kidneys were fixed for tissue imaging or the left kidney was observed in vivo using two-photon microscopy. Expression of GFP and GFP-chimeric proteins was clearly seen in epithelial cells of the injected proximal tubules and the expressed proteins were localized similarly to their endogenously expressed counterparts. Only a minority of the cells in the virally exposed regions, however, expressed these proteins. Endothelial cells also expressed XAC-GFP after injection of the virus cDNA construct into vascular welling points. An advantage of the proximal tubule and vascular micropuncture approaches is that only minute amounts of virus are required to achieve protein expression in vivo. This micropuncture approach to gene transfer of the virus cDNA construct and intravital two-photon microscopy should be applicable to study of the behavior of any fluorescently tagged protein in the kidney and shows promise in studying renal physiology and pathophysiology.

Renal ischemia induces tropomyosin dissociation-destabilizing microvilli microfilaments.

Ischemic-induced cell injury results in rapid duration-dependent actin-depolymerizing factor (ADF)/cofilin-mediated disruption of the apical microvilli microfilament cores. Because intestinal microvillar microfilaments are bound and stabilized in the terminal web by the actin-binding protein tropomyosin, we questioned whether a protective effect of tropomyosin localization to the terminal web of the proximal tubule microfilament cores is disrupted during ischemic injury. With tropomyosin-specific antibodies, we examined rat cortical sections under physiological conditions and following ischemic injury by confocal microscopy. In addition, Western blot analysis of cortical extracts and urine was undertaken. Our studies demonstrated the presence of tropomyosin isoforms in the proximal tubule microvillar terminal web under physiological conditions and their dissociation in response to 25 min of ischemic injury. This correlated with the excretion of tropomyosin-containing plasma membrane vesicles in urine from ischemic rats. In addition, we noted increased tropomyosin Triton X-100 solubility following ischemia in cortical extracts. These studies suggest tropomyosin binds to and stabilizes the microvillar microfilament core in the terminal web under physiological conditions. With the onset of ischemic injury, we propose that tropomyosin dissociates from the microfilament core providing access to microfilaments in the terminal web for F-actin binding, severing and depolymerizing actions of ADF/cofilin proteins.

ADF/cofilin mediates actin cytoskeletal alterations in LLC-PK cells during ATP depletion.

Ischemic injury induces actin cytoskeleton disruption and aggregation, but mechanisms affecting these changes remain unclear. To determine the role of actin-depolymerizing factor (ADF)/ cofilin participation in ischemic-induced actin cytoskeletal breakdown, we utilized porcine kidney cultured cells, LLC-PK(A4.8), and adenovirus containing wild-type (wt), constitutively active, and inactive Xenopus ADF/cofilin linked to green fluorescence protein [XAC(wt)-GFP] in an ATP depletion model. High adenoviral infectivity (70%) in LLC-PK(A4.8) cells resulted in linearly increasing XAC(wt)-GFP and phosphorylated (p)XAC(wt)-GFP (inactive) expression. ATP depletion rapidly induced dephosphorylation, and, therefore, activation, of endogenous pcofilin as well as pXAC(wt)-GFP in conjunction with the formation of fluorescent XAC(wt)-GFP/actin aggregates and rods. No significant actin cytoskeletal alterations occurred with short-term ATP depletion of LLC-PK(A4.8) cells expressing GFP or the constitutively inactive mutant XAC(S3E)-GFP, but cells expressing the constitutively active mutant demonstrated nearly instantaneous actin disruption with aggregate and rod formation. Confocal image three-dimensional volume reconstructions of normal and ATP-depleted LLC-PK(A4.8) cells demonstrated that 25 min of ATP depletion induced a rapid increase in XAC(wt)-GFP apical and basal signal in addition to XAC-GFP/actin aggregate formation. These data demonstrate XAC(wt)-GFP participates in ischemia-induced actin cytoskeletal alterations and determines the rate and extent of these ATP depletion-induced cellular alterations.

Organization of the promoter region of the human NF-IL6 gene.

In monocyte/macrophages, the human NF-IL6 gene was activated by LPS or PMA. However, a robust response required stimulation of cells with both LPS and PMA. To examine the molecular basis of this response, we isolated human genomic DNA and determined the nucleotide sequence of a segment (6.4 kb) that included the transcription initiation site of the gene. The unique sequences in the 6.4-kb DNA include several potential transcription factor-binding elements that may explain the molecular basis of the activation of the human NF-IL6 gene by signaling molecules that control the immune and inflammatory responses. Deletion analysis localized an LPS+PMA responsive region downstream position -287, with respect to the transcription initiation site of the NF-IL6 gene. The responsive region includes a potential site for interactions with CREB and a region (-287 to -247) that interacts with SP1 and SP3. In functional assays, the potential CREB site responded to cellular stimulation. The region that interacted with SP1 and SP3 augmented the overall level of activity produced in response to LPS+PMA.