Obesity shifts house dust mite-induced airway cellular infiltration from eosinophils to macrophages: effects of glucocorticoid treatment.

Although classically characterized by chronic airway inflammation with eosinophil infiltration, asthma is a complex and multifactorial condition with numerous clinical phenotypes. Epidemiological studies strongly support the link between obesity and asthma and suggest that obesity precedes and promotes asthma development, increases asthma severity, and reduces steroid responsivity. Using a house dust mite (HDM) model of airway hyperresponsiveness in C57BL/6 mice, we examined the effects of diet-induced obesity on allergic airway inflammation and its treatment with dexamethasone. When compared to lean mice treated with HDM, obese-HDM mice had reduced plasma adiponectin, an anti-inflammatory adipokine, lower eosinophil and higher macrophage infiltration into the lungs and bronchoalveolar lavage (BAL) fluid, increased expression of total, M1, and M2 macrophage markers in the lungs, and enhanced Th2 and non-Th2 cytokine expression in the lungs. While Th2-associated responses in obese-HDM mice were suppressed by systemic dexamethasone, several Th2-independent responses, including total and M1 macrophage markers in the lungs, and lung CXC-motif ligand 1 (CXCL1) levels, were not improved following dexamethasone treatment. Thus, HDM combined with obesity promotes mixed localized inflammatory responses (e.g., M1, M2, Th1, and Th2) and shifts the cellular infiltration from eosinophils to macrophages, which are less sensitive to dexamethasone regulation. Because obese asthmatics exhibit more severe symptoms, lack a predominance of Th2 biomarkers, and are predicted to experience more steroid resistance when compared to lean asthmatics, this model could be used to study blunted steroid responses in obese-HDM mice and to define the macrophages found in the lungs.

Photodynamic therapy for circumscribed choroidal haemangioma: a case report.

Choroidal haemangioma is a benign tumour with visual acuity diminution due to subretinal fluid accumulation. There are many modalities of treatment of this visually disabling syndrome, some of them being argon laser photocoagulation, cryotherapy, external beam irradiation, proton beam radiotherapy, episcleral plaque radiotherapy and transpupillary thermotherapy. Another new modality of treatment with remarkable success rate is photodynamic therapy. In this modality a photosensitiser is injected intravenously followed by irradiation of a specific wave length for a specified time period. The photosensitiser concentrates within the vascular channels and after irradiation these channels are irreversibly obliterated. A 62 years old female patient of choroidal haemangioma, who presented in eye outpatient department was treated with the standard protocol used for photodynamic therapy. On follow-up of this patient it was found that there was improvement in the visual acuity from 6/12 in the left eye (affected eye) to 6/9. Not only was there an improvement in the visual acuity but there was anatomical improvement too as was evident by regressed cystoid macular oedema and circumscribed choroidal haemangioma. After six months of follow-up there was no leakage of dye with digital fluorescein angiography and indocyanine green.

Physiological activities of carbon monoxide-releasing molecules: Ca ira.

In this issue of British Journal of Pharmacology, Megías and colleagues demonstrate how preincubation of human colonic Caco-2 cells with CORM-2, a carbon monoxide releasing molecule (CO-RM), reduces the expression of inducible nitric oxide synthase, interleukin (IL)-6 and IL-8 caused by proinflammatory cytokines. A role for IL-6 in the regulation of metalloproteinase (MMP)-7 expression by CORM-2 is described. However, it is the demonstration that CORM-2 inhibits MMP-7 or matrilysin expression, which is most intriguing as this small MMP has been implicated in carcinogenesis. Thus, CO-RMs appear to now possess chemoprotective properties and, in this particular case, may influence inflammation-induced colon carcinogenesis via modulation of nuclear factors participating in the transcription of genes implicated in the development of intestinal inflammation and cancer. This report opens yet another door for research involving these exciting molecules and it is now clear that further discoveries of the beneficial properties of CO-RMs will go on.

Calpain inhibitor-1 reduces renal ischemia/reperfusion injury in the rat.

BACKGROUND: Activation of the cysteine protease calpain has been implicated in renal ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of calpain inhibitor-1 (Cal I-1) in an in vivo model of renal I/R injury. METHODS: Male Wistar rats were administered Cal I-1 (10 mg/kg, IP) 30 minutes before undergoing bilateral renal ischemia (45 minutes) followed by reperfusion (6 hours). Plasma concentrations of urea, creatinine, Na(+), gamma-glutamyl transferase (gamma GT), aspartate aminotransferase (AST) and urinary Na(+), glutathione S-transferase (GST), and N-acetyl-beta-D-glucosaminidase (NAG) were measured for the assessment of renal dysfunction and I/R injury. Creatinine clearance (C(Cr)) and fractional excretion of Na(+) (FE(Na)) were used as indicators of glomerular and tubular function, respectively. Kidney myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured for assessment of neutrophil infiltration and lipid peroxidation, respectively. Renal sections were used for histologic grading of renal injury and for immunohistochemical localization of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). RESULTS: Cal I-1 significantly reduced I/R-mediated increases in urea, creatinine, gamma GT, AST, NAG, and FE(Na) and significantly improved C(Cr). Cal I-1 also significantly reduced kidney MPO activity and MDA levels. Cal I-1 also reduced histologic evidence of I/R-mediated renal damage and caused a substantial reduction in the expression of iNOS and COX-2, both of which involve activation of nuclear factor-kappa B (NF-kappa B). CONCLUSIONS: : These results suggest that Cal I-1 reduces the renal dysfunction and injury associated with I/R of the kidney. We suggest that the mechanism could involve the inhibition of I/R-mediated activation of NF-kappa B.

Calpain inhibitor I reduces the activation of nuclear factor-kappaB and organ injury/dysfunction in hemorrhagic shock.

There is limited evidence that inhibition of the activity of the cytosolic cysteine protease calpain reduces ischemia/reperfusion injury. The multiple organ injury associated with hemorrhagic shock is due at least in part to ischemia (during hemorrhage) and reperfusion (during resuscitation) of target organs. Here we investigate the effects of calpain inhibitor I on the organ injury (kidney, liver, pancreas, lung, intestine) and dysfunction (kidney) associated with hemorrhagic shock in the anesthetized rat. Hemorrhage and resuscitation with shed blood resulted in an increase in calpain activity (heart), activation of NF-kappaB (kidney), expression of iNOS and COX-2 (kidney), and the development of multiple organ injury and dysfunction, all of which were attenuated by calpain inhibitor I (10 mg/kg i.p.), administered 30 min prior to hemorrhage. Chymostatin, a serine protease inhibitor that does not prevent the activation of NF-kappaB, had no effect on the organ injury/failure caused by hemorrhagic shock. Pretreatment (for 1 h) of murine macrophages or rat aortic smooth muscle cells (activated with endotoxin) with calpain inhibitor I attenuated the binding of activated NF-kappaB to DNA and the degradation of IkappaBalpha, IkappaBbeta, and IkappaBvarepsilon. Selective inhibition of iNOS activity with L-NIL reduced the circulatory failure and liver injury, while selective inhibition of COX-2 activity with SC58635 reduced the renal dysfunction and liver injury caused by hemorrhagic shock. Thus, we provide evidence that the mechanisms by which calpain inhibitor I reduces the circulatory failure as well as the organ injury and dysfunction in hemorrhagic shock include 1) inhibition of calpain activity, 2) inhibition of the activation of NF-kappaB and thus prevention of the expression of NFkappaB-dependent genes, 3) prevention of the expression of iNOS, and 4) prevention of the expression of COX-2. Inhibition of calpain activity may represent a novel therapeutic approach for the therapy of hemorrhagic shock.

Formation and characterization of the trimeric form of the fusion protein of Semliki Forest Virus.

Enveloped animal viruses infect cells via fusion of the viral membrane with a host cell membrane. Fusion is mediated by a viral envelope glycoprotein, which for a number of enveloped animal viruses rearranges itself during fusion to form a trimeric alpha-helical coiled-coil structure. This conformational change from the metastable, nonfusogenic form of the spike protein to the highly stable form involved in fusion can be induced by physiological activators of virus fusion and also by a variety of destabilizing conditions. The E1 spike protein subunit of Semliki Forest virus (SFV) triggers membrane fusion upon exposure to mildly acidic pH and forms a homotrimer that appears necessary for fusion. We have here demonstrated that formation of the E1 homotrimer was efficiently triggered under low-pH conditions but not by perturbants such as heat or urea, despite their induction of generalized conformational changes in the E1 and E2 subunits and partial exposure of an acid-specific E1 epitope. We used a sensitive fluorescence assay to show that neither heat nor urea treatment triggered SFV-liposome fusion at neutral pH, although either treatment inactivated subsequent low-pH-triggered fusion activity. Once formed, the low-pH-induced E1 homotrimer was very stable and was only dissociated under harsh conditions such as heating in sodium dodecyl sulfate. Taken together, these data, as well as protein structure predictions, suggest a model in which the less stable native E1 subunit specifically responds to low pH to form the more stable E1 homotrimer via conformational changes different from those of the coiled-coil type of fusion proteins.

Lipoteichoic acid induces delayed protection in the rat heart: A comparison with endotoxin.

Classic ischemic preconditioning transiently (30 to 120 minutes) protects the myocardium against subsequent lethal ischemia/reperfusion injury. After dissipation of this acute protection, a second window of protection (SWOP) appears 12 to 24 hours later; this SWOP lasts up to 3 days. Several triggers induce a SWOP, including brief repetitive cycles of coronary artery occlusion, rapid ventricular pacing, stimulation of adenosine A(1) receptors, and administration of wall fragments of Gram-negative bacteria, such as lipopolysaccharide (LPS). The aim of this study was to investigate whether lipoteichoic acid (LTA), a cell wall fragment of Gram-positive bacteria, can induce a SWOP in a rat model of left anterior descending coronary artery (LAD) occlusion (25 minutes) and reperfusion (2 hours). Thus, 166 male Wistar rats were pretreated (2 to 24 hours) with saline, LTA (1 mg/kg IP), or LPS (1 mg/kg IP) and subjected to LAD occlusion/reperfusion. Pretreatment with LTA or LPS for 16 hours led to a substantial, approximately 65%, reduction in infarct size and a reduction in the release of cardiac troponin T into the plasma. The dose of LTA used had no toxic effect (on any of the parameters studied), whereas the same dose of LPS caused a time-dependent activation of the coagulation system and liver injury. By use of RNase protection assays, it was determined that LPS caused a time-dependent induction of tumor necrosis factor-alpha, interleukin-1beta, and manganese superoxide dismutase mRNA content in the heart, whereas LTA failed to induce manganese superoxide dismutase. LPS also caused an upregulation of the expression of intercellular adhesion molecule-1 and P-selectin, whereas LTA downregulated these molecules and attenuated the accumulation of polymorphonuclear granulocytes caused by myocardial ischemia/reperfusion. This study demonstrates for the first time that pretreatment with LTA at 8 to 24 hours before myocardial ischemia significantly reduces (1) infarct size, (2) cardiac troponin T, and (3) the histological signs of tissue injury in rats subjected to LAD occlusion and reperfusion. The mechanism(s) underlying the observed cardioprotective effects of LTA warrants further investigation but is likely to be related to its ability to inhibit the interactions between the coronary vascular endothelium and polymorphonuclear granulocytes. Therefore, LTA represents a novel and promising agent capable of enhancing myocardial tolerance to ischemia/reperfusion injury.

Biochemical consequences of a mutation that controls the cholesterol dependence of Semliki Forest virus fusion.

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-triggered membrane fusion reaction that requires cholesterol and sphingolipid in the target membrane. Cholesterol-depleted insect cells are highly resistant to alphavirus infection and were used to select srf-3, an SFV mutant that is approximately 100-fold less cholesterol dependent for infection due to a single amino acid change in the E1 spike subunit, proline 226 to serine. Sensitive lipid-mixing assays here demonstrated that the in vitro fusion of srf-3 and wild-type (wt) virus with cholesterol-containing liposomes had comparable kinetics, activation energies, and sphingolipid dependence. In contrast, srf-3 fusion with sterol-free liposomes was significantly more efficient than that of wt virus. Thus, the srf-3 mutation does not affect its general fusion properties with purified lipid bilayers but causes a marked and specific reduction in cholesterol dependence. Upon exposure to low pH, the E1 spike subunit undergoes distinct conformational changes, resulting in the exposure of an acid conformation-specific epitope and formation of an E1 homotrimer. These conformational changes were strongly cholesterol and sphingolipid dependent for wt SFV and strikingly less cholesterol dependent for srf-3. Our results thus demonstrate the functional importance of fusogenic E1 conformational changes in the control of SFV cholesterol dependence.

An epitope of the Semliki Forest virus fusion protein exposed during virus-membrane fusion.

Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by acidic pH in the endocytic pathway. Fusion is mediated by the spike protein E1 subunit, an integral membrane protein that contains the viral fusion peptide and forms a stable homotrimer during fusion. We have characterized four monoclonal antibodies (MAbs) specific for the acid conformation of E1. These MAbs did not inhibit fusion, suggesting that they bind to an E1 region different from the fusion peptide. Competition analyses demonstrated that all four MAbs bound to spatially related sites on acid-treated virions or isolated spike proteins. To map the binding site, we selected for virus mutants resistant to one of the MAbs, E1a-1. One virus isolate, SFV 4-2, showed reduced binding of three acid-specific MAbs including E1a-1, while its binding of one acid-specific MAb as well as non-acid-specific MAbs to E1 and E2 was unchanged. The SFV 4-2 mutant was fully infectious, formed the E1 homotrimer, and had the wild-type pH dependence of infection. Sequence analysis demonstrated that the relevant mutation in SFV 4-2 was a change of E1 glycine 157 to arginine (G157R). Decreased binding of MAb E1a-1 was observed under a wide range of assay conditions, strongly suggesting that the E1 G157R mutation directly affects the MAb binding site. These data thus localize an E1 region that is normally hidden in the neutral pH structure and becomes exposed as part of the reorganization of the spike protein to its fusion-active conformation.

Inhibitors of poly (ADP-ribose) synthetase protect rat proximal tubular cells against oxidant stress.

BACKGROUND: The generation of reactive oxygen species (ROS) has been implicated in the pathogenesis of renal ischemia-reperfusion injury. ROS produce DNA strand breaks that lead to the activation of the DNA-repair enzyme poly (ADP-ribose) synthetase (PARS). Excessive PARS activation results in the depletion of its substrate, nicotinamide adenine dinucleotide (NAD) and subsequently of adenosine 5'-triphosphate (ATP), leading to cellular dysfunction and eventual cell death. The aim of this study was to investigate the effect of various PARS inhibitors on the cellular injury and death of rat renal proximal tubular (PT) cells exposed to hydrogen peroxide (H2O2). METHODS: Rat PT cell cultures were incubated with H2O2 (1 mM) either in the presence or absence of the PARS inhibitors 3-aminobenzamide (3-AB, 3 mM), 1,5-dihydroxyisoquinoline (0.3 mM) or nicotinamide (Nic, 3 mM), or increasing concentrations of desferrioxamine (0.03 to 3 mM) or catalase (0.03 to 3 U/ml). Cellular injury and death were determined using the MTT and lactate dehydrogenase (LDH) assays, respectively. H2O2-mediated PARS activation in rat PT cells and the effects of PARS inhibitors on PARS activity were determined by measurement of the incorporation of [3H]NAD into nuclear proteins. RESULTS: Incubation of rat PT cells with H2O2 significantly inhibited mitochondrial respiration and increased LDH release, respectively. Both desferrioxamine and catalase reduced H2O2-mediated cellular injury and death. All three PARS inhibitors significantly attenuated the H2O2-mediated decrease in mitochondrial respiration and the increase in LDH release. Incubation with H2O2 produced a significant increase in PARS activity that was significantly reduced by all PARS inhibitors. 3-Aminobenzoic acid (3 mM) and nicotinic acid (3 mM), structural analogs of 3-AB and Nic, respectively, which did not inhibit PARS activity, did not reduce the H2O2-mediated injury and necrosis in cultures of rat PT cells. CONCLUSION: We propose that PARS activation contributes to ROS-mediated injury of rat PT cells and, therefore, to the cellular injury and cell death associated with conditions of oxidant stress in the kidney.

Selective antagonism of the AT1 receptor inhibits angiotensin II stimulated DNA and protein synthesis in primary cultures of human proximal tubular cells.

The hypertrophy of renal proximal tubular cells occurs as an adaptive response to a variety of stimuli and may be involved with the progression of renal disease. Angiotensin II acting alone or in combination with other growth factors has been implicated in this process. The aims of this study were to identify the role of both angiotensin II and the angiotensin receptor subtypes in DNA synthesis and protein synthesis in human renal proximal tubular cells. Primary cultures of human renal proximal tubular cells were incubated with angiotensin II (10(-10) M, 10(-8) M, 10(-6) M) for 24 to 120 hours either alone or in combination with losartan, PD123319 or 8-bromo-cAMP. Incubation of human proximal tubular cells with angiotensin II (10(-10) M, 10(-8) M) induced a significant early increase in [3H]thymidine uptake by 19% and 56% (P < 0.01), respectively, and a later increase in total protein content by 30% (P < 0.01). The effect of angiotensin II upon DNA and protein synthesis was inhibited by 8-bromo-cAMP and losartan but not by PD 123319, indicating that the responses are mediated via the AT1 receptor and dependent upon the inhibition of adenylate cyclase.

A general genetic approach in Escherichia coli for determining the mechanism(s) of action of tumoricidal agents: application to DMP 840, a tumoricidal agent.

We describe here a simple and easily manipulatable Escherichia coli-based genetic system that permits us to identify bacterial gene products that modulate the sensitivity of bacteria to tumoricidal agents, such as DMP 840, a bisnaphthalimide drug. To the extent that the action of these agents is conserved, these studies may expand our understanding agents is conserved, these studies may expand our understanding of how the agents work in mammalian cells. The approach briefly is to use a library of E. coli genes that are overexpressed in a high copy number vector to select bacterial clones that are resistant to the cytotoxic effects of drugs. AtolC bacterial mutant is used to maximize permeability of cells to hydrophobic organic molecules. By using DMP 840 to model the system, we have identified two genes, designated mdaA and mdaB, that impart resistance to DMP 840 when they are expressed at elevated levels. mdaB maps to E. coli map coordinate 66, is located between the parE and parC genes, and encodes a protein of 22 kDa. mdaA maps to E. coli map coordinate 18, is located adjacent to the glutaredoxin (grx) gene, and encodes a protein of 24 kDa. Specific and regulatable overproduction of both of these proteins correlates with DMP 840 resistance. Overproduction of the MdaB protein also imparts resistance to two mammalian topoisomerase inhibitors, Adriamycin and etoposide. In contrast, overproduction of the MdaA protein produces resistance only to Adriamycin. Based on its drug-resistance properties and its location between genes that encode the two subunits of the bacterial topoisomerase IV, we suggest that mdaB acts by modulating topoisomerase IV activity. The location of the mdaA gene adjacent to grx suggests it acts by a drug detoxification mechanism.

Characterization of Syrian hamster gastric mucosal H+,K+-ATPase.

Employing a simple one-step sucrose gradient fractionation method, gastric mucosal membrane of Syrian hamster was prepared and demonstrated to be specifically enriched in H+,K+-ATPase activity. The preparation is practically devoid of other ATP hydrolyzing activity and contains high K+-stimulated ATPase activity of at least 4-5 fold compared to basal ATPase activity. The H+,K+-ATPase showed hydroxylamine-sensitive phosphorylation and K+-dependent dephosphorylation of the phosphoenzyme, characteristic inhibition by vanadate, omeprazole and SCH 28080, and nigericin-reversible K+-dependent H+-transport--properties characteristic of gastric proton pump. One notable difference with H+,K+-ATPase of other species has been the observation of valinomycin-independent H+ transport in such membrane vesicles. It is proposed that such H+,K+-ATPase-rich hamster gastric mucosal membrane preparation might provide a unique model to study physiological aspects of H+,K+-ATPase function in relation to HCl secretion.

Atrial natriuretic factor modulates nitric oxide production: an ANF-C receptor-mediated effect.

OBJECTIVES: To investigate the possible immunomodulatory and regulatory functions of atrial natriuretic factor (ANF) and the natriuretic peptide C (NPR-C) receptor in the control of cytokine-stimulated nitric oxide production in primary cultures of human proximal tubular cells. METHODS: Freshly prepared human proximal tubular cells were seeded on plastic plates and allowed to reach confluence. The confluent cells were then incubated with ANF or cyclic(4-23)ANF (c(4-23)ANF) alone, or preincubated with ANF or c(4-23)ANF before incubation with the nitric oxide-stimulating combination of cytokines interleukin-1 beta (10 u/ml), tumour necrosis factor-alpha (10 ng/ml) and interferon-gamma (100 u/ml). RESULTS: In the present series of experiments we have found that incubation of primary cultures of human proximal tubular cells with ANF or c(4-23)ANF stimulates nitric oxide production dose-dependently. Paradoxically, ANF acting via the NPR-C receptor also inhibits cytokine activation of the enzyme-inducible nitric oxide synthase via a cyclic GMP-independent mechanism. Both of these effects were reproduced by the NPR-C receptor-specific ligand c(4-23)ANF. CONCLUSIONS: These findings represent novel actions of ANF mediated via the NPR-C receptor. The results also provide a simple model system in which to study the subcellular mechanisms of NPR-C receptor activation.

Using cell-fractionation and photochemical crosslinking methods to determine the cellular binding site(s) of the antitumor drug DMP 840.

In order to understand its mechanism of action we have begun an effort to better define the cellular target of action of the experimental antitumor agent DMP 840 (NSC D640430; (R,R)-2,2'-(1,2-ethanediylbis(imino-(1-methyl-2,1-ethanediyl)))-bi s(5- nitro-1H-benz(de)isoquinoline-1,3-(2H)-dione) dimethanesulfonate). Using a combination of gentle cell fractionation procedures and a previously unidentified photochemical crosslinking reaction, we have shown that after the drug is added to cultured Clone A cells, more than 80% of the drug that is found associated with cells partitions to the chromatin-containing structural framework of the cell and that the primary target after crosslinking with 360 nm light is DNA. While DMP 840 photoreacts quite efficiently with purified RNA in vitro, no photoattachment of the drug to RNA was observed in cells. In vitro photochemical studies also reveal that while GC-rich DNA is a preferred target for drug interaction, AT-rich DNA is more active in the photochemical crosslinking reaction. These results suggest that DMP 840 probably kills cells by interfering with DNA-metabolic processes, and that the drug and its derivatives are likely to be useful photoactive molecular probes for investigating higher order chromatin structures in cells.

Purification of an active TATA-binding protein-containing factor using a monoclonal antibody that recognizes the human TATA-binding protein.

The human TATA-binding protein was expressed in Escherichia coli as a fusion with an N-terminal hexahistidine sequence, partially purified, and used to raise monoclonal antibodies. More than 50 hybridoma clones producing antibodies that reacted in immunoblot assays with HeLa cell TATA-binding protein and its bacterially synthesized derivative were identified. All antibodies examined recognized epitopes within the N-terminal 159 amino acids of the human TATA-binding protein. Further characterization of one monoclonal antibody, MTBP-6, established that it immunoprecipitates both native HeLa cell TATA-binding protein and TATA-binding protein extracted from cells in the presence of 0.5% SDS. Antibody MTBP-6 immunoprecipitates of native, human cell TATA-binding protein contained the TATA-binding protein and additional polypeptides. Immunoprecipitation of both the TATA-binding protein and several additional polypeptides was specifically blocked by bacterially synthesized, hexahistidine-tagged TATA-binding protein, suggesting that MTBP-6 can efficiently recognize the TATA-binding protein in TFIID and other complexes. Consistent with this conclusion, immunoaffinity chromatography on antibody MTBP-6 permitted purification, in active form, of a TATA-binding protein-containing factor required for transcription by RNA polymerase III. These properties suggest that MTBP-6 will be a useful reagent for the purification and characterization of the multiple TBP-containing complexes present in human cells.

Specific transcription from the adenovirus E2E promoter by RNA polymerase III requires a subpopulation of TFIID.

The early E2 (E2E) promoter of adenovirus type 2 possesses a TATA-like element and binding sites for the factors E2F and ATF. This promoter is transcribed by RNA polymerase II in high salt nuclear extracts, but by RNA polymerase III in standard nuclear extracts, as judged by sensitivity to low and high, respectively, concentrations of alpha-amanitin. Transcription by the two RNA polymerases initiated at the same site and depended, in both cases, on the TATA-like sequence and upstream elements. However, RNA polymerase III transcripts, unlike those synthesized by RNA polymerase II, terminated at two runs of Ts downstream of the initiation site. Although they are not essential, sequences downstream of the initiation site increased the efficiency of E2E transcription by RNA polymerase III. Such RNA polymerase III dependent transcription required a subpopulation of the general transcription factor, TFIID: TFIID that binds weakly to phosphocellulose (0.3 M eluate) complemented a TFIID-depleted extract to restore RNAp III transcription, whereas TFIID tightly associated with phosphocellulose (1 M eluate) was unable to do so.

DNA-binding properties of an adenovirus 289R E1A protein.

An adenovirus 2 289 amino acid (289R) E1A protein purified from Escherichia coli has been shown to interact with DNA by two independent methods. UV-crosslinking of complexes containing unmodified, uniformly 32P-labelled DNA and purified E1A protein induced efficient labelling of the protein with covalently attached oligonucleotides, indicating that the E1A protein itself contacts DNA. Discrete nucleoprotein species were also observed when E1A protein--DNA complexes were analysed by gel electrophoresis. Although the 289R E1A protein exhibited no significant binding to single-stranded DNA or to RNA, no evidence for its sequence-specific binding to double-stranded DNA was obtained with either assay. Identification of the sites of covalent attachment of 32P-labelled oligonucleotides by partial proteolysis of the crosslinked E1A protein indicated that the interaction of this protein with DNA is mediated via domain(s) in the C-terminal half of the protein. Such previously unrecognized DNA-binding activity is likely to contribute to the regulatory activities of this important adenoviral protein.

Adenovirus type 2 endopeptidase: an unusual phosphoprotein enzyme matured by autocatalysis.

A 19-kDa protein, present in low copy number in purified adenovirus type 2, has been characterized. Several criteria were used to establish that this protein is neither a degradation product of the known structural proteins of the virion nor a minor, unusually modified, form of protein VII. This 19-kDa protein, unlike other virion proteins, possesses alkali-resistant phosphoamino acids. Analysis by partial proteolysis indicated that it is related to a 23-kDa phosphoprotein present in H2ts-1 virions assembled in infected cells maintained at 39 degrees C. Affinity labeling with [3H]diisopropyl fluorophosphate showed that the 19-kDa protein contains the active site for a serine protease. We, therefore, conclude that the 19-kDa protein is the active form of the adenovirus-encoded endopeptidase, defined by the H2ts-1 mutation, and is synthesized as a 23-kDa precursor that appears to mature by autocatalysis.

Partition of E1A proteins between soluble and structural fractions of adenovirus-infected and -transformed cells.

The partition of E1A proteins between soluble and structural framework fractions of human cells infected or transformed by subgroup C adenoviruses was investigated by using gentle cell fractionation conditions. A polyclonal antibody raised against a trpE-E1A fusion protein (K.R. Spindler, D.S.E. Rosser, and A. J. Berk, J. Virol. 132-141, 1984) synthesized in Escherichia coli was used to measure the steady-state levels of E1A proteins recovered in the various fractions by immunoblotting. The relative concentration of E1A proteins recovered in the soluble fraction of adenovirus type 2-infected cells was at least fivefold greater than the relative concentration in the corresponding fraction of transformed 293 cells. The observed distribution of E1A proteins was not altered by the sulfhydryl-blocking reagent N-ethylmaleimide. E1A proteins were recovered in nuclear matrix, chromatin, and cytoskeleton fractions after further fractionation of the structural framework fraction. However, the E1A protein species that could be identified by one-dimensional gel electrophoresis were not uniformly distributed among the subcellular fractions examined. The results obtained when fractionation was performed in the presence of the oxidation catalysts Cu2+ or (ortho-phenanthroline)2 Cu2+ indicate that E1A proteins can be efficiently cross-linked, via disulfide bonds, to the structural framework of both adenovirus-infected and adenovirus-transformed cells.