Merozoites of the human malaria parasite, Plasmodium falciparum, when treated with cytochalasin B, will attach irreversibly to red cells with formation of a vestigial internal (parasitophorous) vacuole, but they are inhibited from moving into the cell. The existence of an actin-based motile mechanism is implied. Immunoblotting, peptide mapping and the DNase inhibition assay have been used to show that the merozoite contains actin. It makes up an estimated 0.3% of the total parasite protein and is partitioned in the ratio of about 1:2 between the cytosolic and particulate protein fractions. In the former it is unpolymerised and in the latter filamentous. Most of the anti-actin-reactive protein in the soluble fraction and about 20% of that in the pellet has an apparent molecular weight of 55,000 and reacts with an anti-ubiquitin antibody; it is thus evidently ubiquitinyl actin, or arthrin, which has so far been detected only in insect flight muscle.

Human papillomaviruses (HPVs) are present in virtually all cervical cancers. An important step in the development of malignant disease, including cervical cancer, involves a loss of sensitivity to transforming growth factor beta (TGF-beta). HPV type 16 (HPV16) early gene expression, including that of the E6 and E7 oncoprotein genes, is under the control of the upstream regulatory region (URR), and E6 and E7 expression in HPV16-immortalized human epithelial cells is inhibited at the transcriptional level by TGF-beta. While the URR contains a myriad of transcription factor binding sites, including seven binding sites for nuclear factor I (NFI), the specific sequences within the URR or the transcription factors responsible for TGF-beta modulation of the URR remain unknown. To identify potential transcription factors and binding sites involved in TGF-beta modulation of the URR, we performed DNase I footprint analysis on the HPV16 URR using nuclear extracts from TGF-beta-sensitive HPV16-immortalized human keratinocytes (HKc/HPV16) treated with and without TGF-beta. Differentially protected regions were found to be located around NFI binding sites. Electrophoretic mobility shift assays, using the NFI binding sites as probes, showed decreased binding upon TGF-beta treatment. This decrease in binding was not due to reduced NFI protein or NFI mRNA levels. Mutational analysis of individual and multiple NFI binding sites in the URR defined their role in TGF-beta sensitivity of the promoter. Overexpression of the NFI family members in HKc/HPV16 decreased the ability of TGF-beta to inhibit the URR. Since the oncoprotein Ski has been shown to interact with and increase the transcriptional activity of NFI and since cellular Ski levels are decreased by TGF-beta treatment, we explored the possibility that Ski may provide a link between TGF-beta signaling and NFI activity. Anti-NFI antibodies coimmunoprecipitated endogenous Ski in nuclear extracts from HKc/HPV16, confirming that NFI and Ski interact in these cells. Ski levels dramatically decreased upon TGF-beta treatment of HKc/HPV16, and overexpression of Ski eliminated the ability of TGF-beta to inhibit the URR. Based on these studies, we propose that TGF-beta inhibition of HPV16 early gene expression is mediated by a decrease in Ski levels, which in turn dramatically reduces NFI activity.

Human serum vitamin D binding protein (hDBP), a 58-kDa inter-alpha-globulin, is known to bind, monomeric actin (G-actin) in equimolar quantities. Using monoclonal and polyclonal anti-hDBP antibodies, hDBP, and radioiodinated actin, we developed a reliable saturation assay for actin bound to hDBP. By utilizing this assay, kinetic analysis, and ultracentrifugal sedimentation in sucrose gradients, these proteins' binding affinities (Kd = 10(-9) M) were demonstrated to be 10- to 100-fold greater than earlier estimates. At 4 degrees C, hDBP has an association rate constant of 2.2 x 10(4) M-1 s-1 and a rate of dissociation displaying a t1/2 of 22 h. This high affinity binding was largely unaffected by conditions favoring actin filament formation (1 mM MgCl2 and/or 50 mM KCl), by the range of pH from 6.8 to 8.6 or by temperatures from 4 to 37 degrees C. Compared with ATP-alpha-actin, a 2-fold decrease of binding affinity was observed for the nonmuscle isoactins (beta,gamma), ADP-G-alpha-actin, and N'-ethylmaleimide-modified G-alpha-actin. The 25-hydroxyvitamin D3 and 1 alpha,25-dihydroxyvitamin D3 holo-sterol forms of hDBP bound actin in a manner indistinguishable from the apo-sterol hDBP. The common polymorphisms of hDBP (DBP1 slow, DBP1 fast, and DBP2) were shown to have an equal avidity for G-actin binding. Human platelet profilin competed with hDBP for binding to G-actin, but was 1000-fold less potent (Ki = 1.9 microM). When platelet profilactin was incubated with hDBP, profilin was liberated and hDBP-actin complexes formed. DNase I, which forms a triprotein complex with hDBP-G actin, did not alter the affinity of binding of actin by hDBP. The very high affinity binding observed, which was largely unaffected by the state of G-actin, pH, and ionic conditions, appears to support a constitutive role for plasma DBP in the sequestration of actin monomers, as well as actin from actin-profilin complexes, that are liberated during cell injury.

Group A streptococcal (GAS) serology is used for the diagnosis of post-streptococcal diseases, such as acute rheumatic fever, and occasionally for the diagnosis of streptococcal pharyngitis. Experts recommend that the upper limits of normal for streptococcal serology be determined for individual populations because of differences in the epidemiology of GAS between populations. Therefore, we performed a study to determine the values of the upper limit of normal for anti-streptolysin O (ASO) and anti-DNase B (ADB) titers in Fiji. Participants with a history of GAS disease, including pharyngitis or impetigo, were excluded. A total of 424 serum samples from people of all ages (with a sample enriched for school-aged children) were tested for their ASO and ADB titers. Reference values, including titers that were 80% of the upper limit of normal, were obtained by regression analysis by use of a curve-fitting method instead of the traditional nonparametric approach. Normal values for both the ASO titer and the ADB titer rose sharply during early childhood and then declined gradually with age. The estimated titers that were 80% of the upper limit or normal at age 10 years were 276 IU/ml for ASO and 499 IU/ml for ADB. Data from our study are similar to those found in countries with temperate climates, suggesting that a uniform upper limit of normal for streptococcal serology may be able to be applied globally.

In the heart, mRNA accumulations for sarcomeric actins and myosin heavy chains (MHC) are subject to diverse regulatorial processes. To study cardiac contractile protein transcriptional regulations, an in vitro transcription system using nonenzymatically isolated rat cardiac nuclei was characterized. Transcription was shown to be rapid and continuous during the first 20 min of incubation and 5.4-fold less than that seen from comparably isolated hepatocyte nuclei. Neither RNase nor DNase activities were detectable. Direct transcriptional analyses of the alpha- and beta-MHC and cardiac and skeletal alpha-actin genes from cardiac nuclei were performed. In 23-24-day-old rats, significant levels of transcription were seen for alpha-MHC and for the sarcomeric alpha-actins. beta-MHC was just detectable, and no positive signals were ever seen for fibronectin. We then compared the perecentages of MHC and sarcomeric alpha-actin expressions determined from 1) the transcriptional assays and 2) total isolated RNA (alpha-MHC: 90.1 +/- 4.8% (transcription), 93.0 +/- 4.7% (accumulation); beta-MHC: 9.9 +/- 4.8%, 7.0 +/- 4.7%; cardiac alpha-actin: 84.0 +/- 2.5%, 84.9 +/- 2.5%; skeletal alpha-actin: 16.1 +/- 2.5%, 15.0 +/- 2.5%). The results support the conclusion that the primary mechanisms controlling the accumulations of these gene products are transcriptional. Additionally, we show that an anti-sense mRNA showing strong homology or identity with the 5' end of the beta-MHC gene is transcribed in cardiac nuclei but not in hepatocyte nuclei.

OBJECTIVE

To describe the clinical features of rheumatic fever and to assess the Jones criteria in a population and setting similar to that in many developing countries.

METHODS

The charts of 555 cases of confirmed acute rheumatic fever in 367 patients (97% Aboriginal) and more than 200 possible rheumatic fever cases from the tropical "Top End" of Australia's Northern Territory were reviewed retrospectively.

RESULTS

Most clinical features were similar to classic descriptions. However, monoarthritis occurred in 17% of confirmed non-chorea cases and 35% of unconfirmed cases, including up to 27 in whom the diagnosis was missed because monoarthritis is not a major manifestation. Only 71% and 25% of confirmed non-chorea cases would have had fever using cut off values of 38 degrees C and 39 degrees C, respectively. In 17% of confirmed non-chorea cases, anti-DNase B titres were raised but antistreptolysin O titres were normal. Although features of recurrences tended to correlate with initial episodes, there were numerous exceptions.

CONCLUSIONS

Monoarthritis and low grade fever are important manifestations of rheumatic fever in this population. Streptococcal serology results may support a possible role for pyoderma in rheumatic fever pathogenesis. When recurrences of rheumatic fever are common, the absence of carditis at the first episode does not reliably predict the absence of carditis with recurrences.

Pioglitazone preserves pancreatic beta-cell morphology and function in diabetic animal models. In this study, we investigated the molecular mechanisms by which pioglitazone protects beta-cells in diabetic db/db mice. In addition to the morphological analysis of the islets, gene expression profiles of the pancreatic islet were analyzed using laser capture microdissection and were compared with real-time RT-PCR of db/db and nondiabetic m/m mice treated with or without pioglitazone for 2 wk or 2 days. Pioglitazone treatment (2 wk) ameliorated dysmetabolism, increased islet insulin content, restored glucose-stimulated insulin secretion, and preserved beta-cell mass in db/db mice but had no significant effects in m/m mice. Pioglitazone upregulated genes that promote cell differentiation/proliferation in diabetic and nondiabetic mice. In db/db mice, pioglitazone downregulated the apoptosis-promoting caspase-activated DNase gene and upregulated anti-apoptosis-related genes. The above-mentioned effects of pioglitazone treatment were also observed after 2 days of treatment. By contrast, the oxidative stress-promoting NADPH oxidase gene was downregulated, and antioxidative stress-related genes were upregulated, in db/db mice treated with pioglitazone for 2 wk, rather than 2 days. Morphometric results for proliferative cell number antigen and 4-hydroxy-2-noneal modified protein were consistent with the results of gene expression analysis. The present results strongly suggest that pioglitazone preserves beta-cell mass in diabetic mice mostly by two ways; directly, by acceleration of cell differentiation/proliferation and suppression of apoptosis (acute effect); and indirectly, by deceleration of oxidative stress because of amelioration of the underlying metabolic disorder (chronic effect).

In B cells, the Igh gene locus contains several DNase I-hypersensitive (hs) sites with enhancer activity. These include the 3' Igh enhancers, which are located downstream of the Calpha gene(s) in both mouse and human. In vivo experiments have implicated murine 3' enhancers, hs3B and/or hs4, in class switching and somatic hypermutation. We previously reported that murine hs4 was regulated by NF-kappaB, octamer binding proteins, and Pax5 (B cell-specific activator protein). In this study we report that human hs4 is regulated differently. EMSAs and Western analysis of normal B cells before and after stimulation with anti-IgM plus anti-CD40 showed the same complex binding pattern formed by NF-kappaB, Oct-1, and Oct-2 (but not by Pax5). A similar EMSA pattern was detected in mature human B cell lines (BL-2, Ramos, and HS-Sultan) and in diffuse large B cell lymphoma cell lines, although yin yang 1 protein (YY1) binding was also observed. We have confirmed the in vivo association of these transcription factors with hs4 in B cells by chromatin immunoprecipitation assays. The diffuse large B cell lymphoma cell lines had a distinctive slow-migrating complex containing YY1 associated with Rel-B. We have confirmed by endogenous coimmunoprecipitation an association of YY1 with Rel-B, but not with other NF-kappaB family members. Transient transfection assays showed robust hs4 enhancer activity in the mature B cell lines, which was dependent on synergistic interactions between NF-kappaB and octamer binding proteins. In addition, human hs4 enhancer activity required Oct-2 and correlated with expression of Oct coactivator from B cells (OCA-B).

OBJECTIVE

To follow-up prospectively patients with arthritis after infection with beta-haemolytic streptococci of Lancefield group A (beta HSA), with emphasis on clinical characteristics and serological features. We additionally investigated whether these patients, though often fulfilling the Jones' criteria for acute rheumatic fever (ARF), had a disease with clinical characteristics different from ARF.

METHODS

We performed a systematic prospective observational study of consecutive patients at a Dutch Outpatient Clinic and Department of Rheumatology, with arthritis after throat infection with beta HSA presenting to rheumatologist or internist from September 1992 until September 1996. Main outcome measures were clinical and biochemical characteristics with special reference to carditis.

RESULTS

A total of 23 patients (21 Dutch, two Turkish; female/male ratio 15/8; mean (SD) age 42 (14) years) with predominantly non-migratory arthritis were included. A positive throat swab culture was obtained in 17%. All patients showed a significant rise of antistreptolysine-O (ASO; normal < 200 i.u. mL-1) and antideoxyribonuclease-B (anti-DNase-B; normal < 200 i.u. mL-1) titre. The mean (SEM) maximal ASO was 1305 (195) i.u. mL-1, and anti-DNase-B titre 980 (115) i.u. mL-1. Arthritis was present in mean (SEM) 5.4 (0.9) joints: 2.2 (0.7) small, 3.2 (0.4) larger joints. The arthritis was monarticular in 23% of the patients, oligoarticular in 35%, and polyarticular in 43%. Skin abnormalities were present in 12 patients: erythema nodosum in seven patients (30%), and erythema multiforme in five patients (22%). A transient cholestatic hepatitis was found in four patients (17%). In two patients a transient first-degree conduction block was found; however, neither echocardiography nor clinical course supported carditis. All patients were advised to receive monthly penicillin prophylaxis during a period of 2 years. Two patients refused to follow medical advice: in one a non-migratory arthritis recurred 15 months after the first episode of arthritis.

CONCLUSIONS

Commonly, arthritis secondary to beta HSA infection in the Netherlands, a prosperous West-European country with State Welfare, is not accompanied by carditis, contrary to literature on classical ARF. Other factors discriminating it from ARF are the age of onset, the non-migratory character of the arthritis, the high frequency of erythema nodosum and multiforme, as well as the presence of transient hepatitis. As arthritis is the hallmark of this syndrome, post-streptococcal reactive arthritis (PSRA) is the most proper name for this disease entity. Whether penicillin profylaxis is needed in PSRA, as it is in ARF, warrants further prospective investigation.

Tumor necrosis factor (TNF) down-regulates the production of bone matrix proteins by osteoblasts, thereby inhibiting bone formation. Osteocalcin, the major noncollagenous protein in bone, is inhibited by TNF at the transcriptional level. Mapping studies were undertaken to characterize the TNF-responsive element (TNFRE) in the osteocalcin promoter. Deletion analysis localized the TNFRE to the -522/-511 region, which contains a 9-bp palindromic motif (AGGCTGCCT). Promoter segments containing this sequence down-regulated a heterologous simian virus 40 promoter. Site-specific mutagenesis of the TNFRE eliminated TNF down-regulation. Mobility shift assays demonstrated that a constitutively expressed nuclear factor bound to the TNFRE; this factor was tentatively identified as the p50 homodimer of NF-kappa B. TNF stimulation induced a second TNFRE-binding protein which displaced the constitutive factor. The TNF-induced protein was not inhibitable by the NF-kappa B consensus sequence and was unreactive with anti-NF-kappa B antiserum. DNase footprinting demonstrated that both factors protected the -522/-501 portion of the promoter, consistent with the results of mapping studies and competitive mobility shift assays. It is hypothesized that the generalized catabolic activities of TNF in infectious and malignant diseases may be regulated via this novel element.

OBJECTIVE

Nuclear components targeted by autoantibodies are a characteristic feature of the autoimmune disease systemic lupus erythematosus (SLE). The nucleosome, a major autoantigen, is released in patients with SLE as a result of a disturbed apoptosis and/or an insufficient clearance of apoptotic debris. During apoptosis the nucleosome is modified, thereby creating more immunogenic epitopes. Subsequently, epitope spreading will lead to the formation of autoantibodies against unmodified chromatin components. However, characterization of B cell epitopes specific for apoptotic chromatin modifications is hampered by the fact that the existing monoclonal antibodies (mAbs) were originally selected on non-apoptotic chromatin. Here, we describe a novel approach for generating mAbs from lupus mice that are specific for apoptosis-induced chromatin modifications.

METHODS

Hybridomas were generated from pre-diseased and diseased lupus mice using standard fusion methods. Selection occurred on isolated apoptotic chromatin. Antibodies were further characterized by ELISA, western blot and immunofluorescence staining with apoptotic and non-apoptotic chromatin/cells. In addition, reactivity was determined with subnucleosomal complexes and with nucleosomes treated with trypsin or DNase I. Finally, reactivity was determined with cells treated with the histone deacetylase inhibitor TSA.

RESULTS

Most generated mAbs appeared to be nucleosome specific with a clear preference for apoptotic nucleosomes compared to normal nucleosomes. Although the exact elucidation of the epitopes of these mAbs specific for apoptosis-associated nucleosome modifications remains a major challenge, the epitopes contain both DNA and histones, whereby the histone tails play a role in establishing the epitopes. Most importantly, the conformational epitopes of these nucleosome-specific antibodies seem to contain acetylated residues.

CONCLUSIONS

Our approach, yielding a new panel of anti-apoptotic-chromatin antibodies, should facilitate the discovery of more apoptosis-induced chromatin modifications and their identification as key autoantigens in the pathogenesis of SLE.

The Gram-positive intracellular pathogen Listeria monocytogenes is endowed with 17 sets of genes encoding two-component systems. L. monocytogenes is closely related to the Gram-positive model bacterium Bacillus subtilis, in which we have shown previously that the DegS/DegU system plays a central role in controlling stationary phase adaptive responses, including degradative enzyme synthesis and competence. Although an orthologue of the DegU response regulator is present in L. monocytogenes, the gene encoding the cognate DegS kinase is conspicuously absent. We have inactivated the degU gene of L. monocytogenes and shown that DegU negatively regulates its own synthesis. Direct binding of L. monocytogenes DegU to its own promoter region was shown in vitro by gel mobility shift and DNase I footprinting experiments. DegU was also shown to bind upstream from the motB operon, which also encodes the GmaR anti-repressor of flagellar synthesis. In contrast to the situation in B. subtilis, DegU was shown to be essential for flagellar synthesis and bacterial motility in L. monocytogenes and is cotranscribed with the yviA gene located downstream. We also show that DegU is required for growth at high temperatures, adherence to plastic surfaces and the formation of efficient biofilms by L. monocytogenes. DegU plays a role in virulence of L. monocytogenes as well: in a murine intravenous infection model, an 11-fold increase in LD(50) was observed for the degU mutant. Taken together, our results indicate that despite the lack of the DegS kinase, DegU is fully functional as an orphan response regulator, and plays a central role in controlling several crucial adaptive responses in L. monocytogenes.

Group A streptococci (GAS) express up to four types of secreted DNases. Although GAS infections are correlated with the production of anti-DNase B antibodies, the roles of DNases in the pathogenesis of GAS infections remain unclear. From a lambda library of serotype M49 strain CS101 GAS genome, a 2,147-bp fragment expressing DNase activity on an indicator agar was identified and sequenced. One 1,155-bp open reading frame (ORF) was identified in this fragment. This ORF was found to be 48% identical on the amino acid level to group C streptococcal DNase (Sdc). The regions of highest homology corresponded to amino acid residues that were also identified as part of the active site in staphylococcal nuclease. Transcription analysis revealed a specific 1.3-kb mRNA, which corresponded to the size predicted by the promoter and transcription termination signature sequences and indicated a monocistronic mode of transcription. Allelic replacement of the ORF rendered a M49 mutant devoid of extracellular DNase activity when cultured on indicator agar. Virulence parameters such as resistance to phagocytosis were not affected by the mutation. The sda gene was cloned and expressed in Escherichia coli as a thioredoxin fusion. By performing Ouchterlony immunodiffusion on the recombinant protein and by using protein preparations from culture supernatants of wild-type bacteria and the DNase mutant, the results of immunoreactivity with DNase type-specific polyclonal rabbit antisera classified the DNase as a type D enzyme. Fifty percent of patients with sera exhibiting high titers of antistreptolysin or anti-DNase B antibodies also had SdaD-reactive antibodies in comparison with <10% of serologically normal controls. While the value of recombinant SdaD for diagnostic purposes needs to be clarified, the isogenic DNase mutant pair of M49 should allow the significance of GAS DNase D as a bacterial virulence factor to be determined.

A major goal for the treatment of patients with systemic lupus erythematosus with cytotoxic therapies is the induction of long-term remission. There is, however, a paucity of information concerning the effects of these therapies on the reconstituting B cell repertoire. Since there is recent evidence suggesting that B cell lymphopenia might attenuate negative selection of autoreactive B cells, we elected to investigate the effects of cyclophosphamide on the selection of the re-emerging B cell repertoire in wild type mice and transgenic mice that express the H chain of an anti-DNA antibody. The reconstituting B cell repertoire in wild type mice contained an increased frequency of DNA-reactive B cells; in heavy chain transgenic mice, the reconstituting repertoire was characterized by an increased frequency of mature, high affinity DNA-reactive B cells and the mice expressed increased levels of serum anti-DNA antibodies. This coincided with a significant increase in serum levels of BAFF. Treatment of transgene-expressing mice with a BAFF blocking agent or with DNase to reduce exposure to autoantigen limited the expansion of high affinity DNA-reactive B cells during B cell reconstitution. These studies suggest that during B cell reconstitution, not only is negative selection of high affinity DNA-reactive B cells impaired by increased BAFF, but also that B cells escaping negative selection are positively selected by autoantigen. There are significant implications for therapy.

Interleukin-1 beta (IL-1beta) is one of the most important inflammatory mediators in human inflammatory and immunological diseases. The regulation of human IL-1beta gene expression has been studied for several years, and a few regulatory elements have been discovered in the promoter region. However, little is known about negative regulation of IL-1beta expression at the transcriptional level, which may play an important role in anti-inflammatory and immunosuppressive effects. We have identified a negative regulatory element located in the region between -685 and -395. Within this region, a 19-bp nuclear factor binding site (-570 to -552) was characterized by DNase I footprinting and electromobility shift assay. A consensus sequence for a negative glucocorticoid response element (nGRE) and a transcription activator protein-2 binding site were noted within this footprint. Functional studies showed a 2.5-fold increase in promoter activity when this 19-bp binding site was deleted in the reporter constructs IL-1beta/CAT and IL-1beta/SV40 promoter/CAT. Dexamethasone (10(-8) M) repressed chloramphenicol acetyltransferase (CAT) production by 75% in the wild-type fragment but not in a deletion mutant lacking the 19-bp site. A protein of about 150 kD that bound to this negative regulatory sequence was identified by UV cross-linking. This is the first description of a negative regulatory region responsive to glucocorticoids in a cytokine gene.

OBJECTIVE

To examine the efficacy of deoxyribonuclease I (DNAse) therapy in the (NZB x NZW)F1 murine model of lupus.

METHODS

Lupus-prone female (NZB x NZW)F1 mice were treated daily with 0-15 microg/g of recombinant DNAse for 1-6 months. Parameters including anti-DNA autoantibody production, activation of cytokine secreting cells, kidney function and longevity were monitored.

RESULTS

DNAse treatment selectively reduced the number of B cells secreting anti-dsDNA antibodies for approximately one month. However, neither short-term nor long-term treatment altered cytokine production, delayed the onset or reduced the severity of glomerulonephritis, or prolonged survival.

CONCLUSIONS

DNAse treatment initiated before, during, or after the onset of murine lupus did not improve clinical outcome.

To establish viral infection, SV40 must expose nuclear localization signals (NLSs) that are internal in the virion architecture in order to enter the nucleus via interaction with the host's nuclear import machinery, which includes importin alpha and importin beta. The time course for SV40 association with the importins in infected cells was examined. The viral DNA associated with importin alpha by 1.5h post infection, before associating with the importin beta nuclear import receptor, by 3h post infection. Only a small fraction of cell-internalized SV40 that contained viral DNA was bound by the two importins. This fraction, termed "nuclear entry-competent SV40," was slightly smaller than the virion but, importantly, was larger than the viral chromatin and contained both Vp1 and Vp3. Furthermore, the internalized viral DNA in either anti-importin or anti-Vp3 immune complexes was sensitive to DNase I, whereas the viral DNA in mature virions was resistant. All these results suggest that once SV40 enters the cytoplasm, it undergoes an architectural modification that exposes the virion's NLSs for nuclear entry.

The effect of aggregation and secretion on membrane proteins was studied in washed human platelets. Reversible aggregation without secretion was stimulated by ADP and secretion without aggregation was stimulated by thrombin in the presence of EDTA. No loss of platelet surface glycoproteins occurred during reversible ADP-induced platelet aggregation, as measured by quantitative polyacrylamide gel electrophoresis analysis of platelets that were labeled with (125)I-diazotized diiodosulfanilic acid (DD(125)ISA) before ADP stimulation. Also, no new proteins became exposed on the platelet surface after ADP aggregation, as determined by DD(125)ISA labeling after stimulation. Thrombin-induced platelet secretion also caused no loss of platelet surface glycoproteins. However, after platelet secretion two new proteins were labeled by DD(125)ISA: (a) actin and (b) the 149,000-mol wt glycoprotein (termed GP-G), which is contained in platelet granules and secreted in response to thrombin. The identity of DD(125)ISA-labeled actin was confirmed by four criteria: (a) comigration with actin in three different sodium dodecyl sulfate-polyacrylamide gel electrophoresis systems, (b) elution from a particulate fraction in low ionic strength buffer, (c) co-migration with actin in isoelectric focusing, and (d) binding to DNase I. The identity of actin and its appearance on the platelet surface after thrombin-induced secretion was also demonstrated by the greater binding of an anti-actin antibody to thrombin-treated platelets, measured with (125)I-staphylococcal protein A.Therefore, major platelet membrane changes occur after secretion but not after reversible aggregation. The platelet surface changes occurring with secretion may be important in the formation of irreversible platelet aggregates and in the final retraction of the blood clot.

The RAG-2 gene encodes a component of the V(D)J recombinase which is essential for the assembly of antigen receptor genes in B and T lymphocytes. Previously, we reported that the transcription factor BSAP (PAX-5) regulates the murine RAG-2 promoter in B-cell lines. A partially overlapping but distinct region of the proximal RAG-2 promoter was also identified as an important element for promoter activity in T cells; however, the responsible factor was unknown. In this report, we present data demonstrating that c-Myb binds to a Myb consensus site within the proximal promoter and is critical for its activity in T-lineage cells. We show that c-Myb can transactivate a RAG-2 promoter-reporter construct in cotransfection assays and that this transactivation depends on the proximal promoter Myb consensus site. By using a chromatin immunoprecipitation (ChIP) strategy, fractionation of chromatin with anti-c-Myb antibody specifically enriched endogenous RAG-2 promoter DNA sequences. DNase I genomic footprinting revealed that the c-Myb site is occupied in a tissue-specific fashion in vivo. Furthermore, an integrated RAG-2 promoter construct with mutations at the c-Myb site was not enriched in the ChIP assay, while a wild-type integrated promoter construct was enriched. Finally, this lack of binding of c-Myb to a chromosomally integrated mutant RAG-2 promoter construct in vivo was associated with a striking decrease in promoter activity. We conclude that c-Myb regulates the RAG-2 promoter in T cells by binding to this consensus c-Myb binding site.

The murine alpha B-crystallin/small heat shock protein gene is expressed at high levels in the lens and at lower levels in the heart, skeletal muscle, and numerous other tissues. Previously we have found a skeletal-muscle-preferred enhancer at positions -427 to -259 of the alpha B-crystallin gene containing at least four cis-acting regulatory elements (alpha BE-1, alpha BE-2, alpha BE-3, and MRF, which has an E box). Here we show that in transgenic mice, the alpha B-crystallin enhancer directs the chloramphenicol acetyltransferase reporter gene driven by the alpha B-crystallin promoter specifically to myocardiocytes of the heart. The alpha B-crystallin enhancer was active in conjugation with the herpes simplex virus thymidine kinase promoter/human growth hormone reporter gene in transfected rat myocardiocytes. DNase I footprinting and site-specific mutagenesis experiments showed that alpha BE-1, alpha BE-2, alpha BE-3, MRF, and a novel, heart-specific element called alpha BE-4 are required for alpha B-crystallin enhancer activity in transfected myocardiocytes. By contrast, alpha BE-4 is not utilized for enhancer activity in transfected lens or skeletal muscle cell lines. Alpha BE-4 contains an overlapping heat shock sequence and a reverse CArG box [5'-GG(A/T)6CC-3']. Electrophoretic mobility shift assays with an antibody to serum response factor and a CArG-box-competing sequence from the c-fos promoter indicated that a cardiac-specific protein with DNA-binding and antigenic similarities to serum response factor binds to alpha BE-4 via the reverse CArG box; electrophoretic mobility shift assays and antibody experiments with anti-USF antiserum and heart nuclear extract also raised the possibility that the MRF E box utilizes USF or an antigenically related protein. We conclude that the activity of the alpha B-crystallin enhancer in the heart utilizes a reverse CArG box and an E-box-dependent pathway.

An extracellular product of group A streptococci which induces lymphocyte blastogenesis has been purified to homogeneity by DEAE-cellulose and CM-cellulose chromatography. The protein, termed streptococcal blastogen A, has a mol wt of approximately or equal to 17,500 and is inactivated by protease treatment and by heating at 100 degrees C. The purified blastogen gave rise to multiple protein bands on nondenaturing polyacrylamide gel electrophoresis, only two of which possessed blastogenic activity. Treatment of the protein with dithiothreitol before electrophoresis resulted in the apparent conversion of the multiple forms to a single active species. Blastogen A differs in electrophoretic mobility from the streptococcal pyrogenic exotoxins and its lymphocyte stimulating activity is not inhibited by rabbit antisera to the exotoxins. An enzyme immunoassay has been developed to measure human antibodies against blastogen A. A selection of sera with varying levels of anti-DNase B contained antiblastogen A-IgG.

The nonstructural protein NS3 of hepatitis C virus (HCV) possesses two enzymatic domains which are thought to be essential for the virus life cycle: an N-terminal serine-type proteinase, responsible for the processing of nonstructural polypeptides, and a C-terminal nucleoside triphosphatase/helicase, presumably involved in the unwinding of the viral genome. The human antibody response to NS3 usually appears early in the course of HCV infection and is predominantly directed against the carboxyl-terminal portion; however, its fine specificity and clinical significance are largely unknown. We have generated a human monoclonal antibody (hMAb), designated CM3.BB-cell line obtained from the peripheral blood of a patient with chronic HCV infection, which selectively recognized the purified NS3 protein expressed in bacteria or in eukaryotic cells transfected with full-length or NS3 cDNA. Fine-specificity studies revealed that CM3.BDNase fragment library but failed to bind to 12-mer peptides synthesized from the same region, suggesting recognition of a conformational B-cell epitope. Experiments using deletion mutants of clone 8 and competitive inhibition studies using a panel of NS3 peptide-specific murine MAbs indicated that limited N-terminal and C-terminal deletions resulted in a significant reduction of hMAb binding to clone 8, thus identifying a minimal antibody binding domain within clone 8. Competition experiments showed that binding of CM3.BBanti-HCV positive sera, suggesting potentially relevant clinical applications in the diagnosis and treatment of HCV infection.

A monoclonal mouse antibody, 4-B-5, that reacted with both single-stranded DNA (ssDNA) and double-stranded DNA showed direct reactivity and cell association with mouse thymocytes, as well as peripheral blood mononuclear cells. Absorption of 4-B-5 with peripheral blood mononuclear cells or with mouse thymocytes markedly reduced reactivity with both ssDNA and double-stranded DNA. However, treatment of mouse thymocytes or human T cells with DNase completely eliminated monoclonal anti-DNA reactivity with cells. The cellular reactivity was completely restored when monoclonal anti-DNA was incubated with DNase-treated cells in the presence of normal human serum. Normal human serum was found to contain 574 +/- 639 ng/ml of ssDNA. Coincubation or cell preincubation of mouse thymocytes with ssDNA produced a marked increase in 4-B-5 cell association. These findings indicate that mouse monoclonal anti-DNA can react with and can penetrate both mouse and human mononuclear cells, and that this reactivity may depend on the presence of cell membrane DNA on both types of target cells.

Patients undergoing chronic hemodialysis, as well as dialysis staff members, are at high risk of infection with hepatitis B virus (HBV). We have analyzed by PCR the presence of HBV DNA in serum and peripheral blood mononuclear cells (PBMC) from 33 hepatitis B surface antigen (HBsAg)-negative hemodialysis patients and 24 dialysis unit staff members; eight of the 24 staff members had an acute hepatitis B resolved 13 to 21 yr before. HBV DNA was detected in serum of 19 (58%) patients (12 of 17 with and 7 of 16 without anti-HBV antibodies). HBV DNA was found in PBMC of 18 (54%) patients (13 of 17 with and 5 of 16 without anti-HBV antibodies). In the staff members, serum HBV DNA was found only in the individuals who suffered a previous acute hepatitis (P < 0.005). HBV DNA was detected in PBMC of four of six staff members (all with previous acute hepatitis). In two HBV DNA-positive PBMC samples, viral RNA was detected by reverse transcription-PCR. To ascertain whether the HBV DNA detected in serum was encapsulated, seven HBV DNA-positive serum samples were digested with DNase before PCR. After treatment, HBV DNA remained detectable in four cases. In conclusion, HBV DNA in serum and PBMC is detectable in a high proportion of HBsAg-negative hemodialysis patients and may persist several years after a resolved acute hepatitis B. The viral DNA is encapsulated and remains transcriptionally active in PBMC. In the anti-HBs-negative patients, HBV DNA is, at the present time, the only means for diagnosing a past HBV hepatitis.