Glioblastoma is the most malignant and prevalent brain tumor that still remains incurable. Recent studies reported anti-cancer effect of the broccoli-derived compound sulforaphane. We explored the mechanisms of sulforaphane-mediated apoptosis in human glioblastoma T98G and U87MG cells. Wright staining and ApopTag assay confirmed apoptosis in glioblastoma cells treated with sulforaphane. Increase in intracellular free Ca2+ was detected by fura-2 assay, suggesting activation of Ca2+-dependent pathways for apoptosis. Western blotting was used to detect changes in expression of Bax and Bcl-2 proteins resulting in increased Bax:Bcl-2 ratio that indicated a commitment of glioblastoma cells to apoptosis. Upregulation of calpain, a Ca2+-dependent cysteine protease, activated caspase-12 that in turn caused activation of caspase-9. With the increased Bax:Bcl-2 ratio, cytochrome c was released from mitochondria to cytosol for sequential activation of caspase-9 and caspase-3. Increased calpain and caspase-3 activities generated 145 kD spectrin breakdown product and 120 kD spectrin breakdown product, respectively. Activation of caspase-3 also cleaved the inhibitor-of-caspase-activated-DNase. Accumulation of apoptosis-inducing-factor in cytosol suggested caspase-independent pathway of apoptosis as well. Two of the inhibitor-of-apoptosis proteins were downregulated because of an increase in 'second mitochondrial activator of caspases/Direct inhibitor-of-apoptosis protein binding protein with low pI.' Decrease in nuclear factor kappa B and increase in inhibitor of nuclear factor kappa B alpha expression favored the process of apoptosis. Collectively, our results indicated activation of multiple molecular mechanisms for apoptosis in glioblastoma cells following treatment with sulforaphane.

DNA obtained from Chlamydia trachomatis (serovar L2) was partially digested with DNase I and inserted into the beta-galactosidase gene of bacteriophage lambda gt11. Seven recombinants were selected that produced immunoreactive fusion proteins which were detected with anti-C. trachomatis rabbit serum. One recombinant, designated lambda gt11/L2/33, reacted with various monoclonal antibodies that recognize species-, subspecies-, and type-specific determinants on the chlamydial major outer membrane protein (MOMP). Immunoblot analysis of a lambda gt11/L2/33 lysogen revealed a fusion protein that expressed a approximately 15,000-dalton carboxyl-terminal peptide of the chlamydial MOMP. This moiety of the MOMP possesses epitopes responsible for each of the unique reactivities demonstrated by anti-MOMP monoclonal antibodies. The lambda gt11/L2/33 recombinant contained a 1.1-kilobase DNA insert which hybridized to DNA isolated from each of the 15 C. trachomatis serovars.

Antibodies to DNA in the left-handed (Z) conformation bind to acid-fixed polytene chromosomes of both Chironomus thummi and Drosophila melanogaster, as shown by direct and indirect immunofluorescence. Comparison of the phase-contrast, immunofluorescence, and DNA staining patterns shows a predominant localization of the antibody to the regions of high contrast and DNA density, the bands. The immunofluorescence is completely abolished by competition with polynucleotides in the Z conformation but not by those in the B form. DNase but not RNase treatment eliminates the antibody staining. Actinomycin D inhibits binding, whereas mithramycin has no effect. The highly reproducible immunofluorescence patterns obtained with the anti-Z-DNA antibodies demonstrate variations in fluorescence intensity between particular bands, which can be quantitated by laser scanning and photon counting techniques. The telomeric regions and DNA strands associated with end-to-end chromosome linkage and ectopic pairing are exceptionally bright. At saturation, average values of 1 IgG molecule per 3,000 base pairs and 1 per 15,000 base pairs are found in the intensely and weakly staining regions, respectively. An alternative statement is that the left-handed Z-DNA conformation is present at a frequency of 0.02-0.1%. The measured differences reflect variations in the local density of Z-DNA sites and not in the affinity for the specific antibody, which appears to be relatively constant throughout the chromosomes (Kd approximately equal to 10 nM). These observations taken together with results of biophysical studies on the properties of Z-DNA in solution suggest that regions of DNA in the left-handed conformation could be involved in higher-order structural organization of chromosomes and possibly in modulation of their functional state.

To detect nuclear proteins that might be involved in induction of cellular mitogenesis, we examined the effect of various mitogens on early changes in synthesis of nuclear proteins in murine B lymphocytes. Using two-dimensional gel electrophoresis, we found that activation of B cells by mitogens (anti-immunoglobulin antibody, lipopolysaccharide, phorbol 12-myristate 13-acetate (PMA)/A23187) was associated with a rapid and prominent (5-20-fold) increase in the synthesis of a 40-kDa/pI 5.0 nuclear protein, here termed numatrin. Numatrin was found to be absent from the cytosol (soluble fraction) of resting as well as activated B cells and was markedly resistant to DNase/RNase digestion and 2 N NaCl extraction, indicating that this protein is tightly bound to the nuclear matrix. Kinetic studies showed that the increase in synthesis of numatrin was detected 60-120 min following mitogen activation, reached a peak at 16 h, and declined to almost control level by 48 h, correlating with the peak of cellular DNA synthesis. The increase in synthesis of numatrin in normal B cells was found to be associated exclusively with cellular commitment for mitogenesis because activation of B cells by stimuli such as B cell stimulating factor 1, PMA alone, and calcium ionophore A23187, which do not stimulate an increase in DNA synthesis, also failed to induce an increase in the synthesis of numatrin. Inhibition of anti-Ig-induced proliferation (by PMA pretreatment) was associated with a 63% inhibition in the synthesis of numatrin. Addition of 8-mercaptoguanosine to these PMA-treated cells was associated with restoration of the increase in synthesis of numatrin, concomitant with induction of proliferation. Elevated synthesis of numatrin was also detected in the malignant B lymphoma cells: Raji, BAL-17, and WEHI-231. Taken collectively, these results suggest that numatrin, a tightly bound nuclear matrix protein, is a growth-regulated protein which might have an important role in regulation of cellular mitogenesis in normal and malignant B lymphocytes.

We tested the hypothesis that tumor necrosis factor (TNF)-alpha induces a peroxynitrite (ONOO(-))-dependent increase in permeability of pulmonary microvessel endothelial monolayers (PMEM) that is associated with generation of nitrated beta-actin (NO(2)-beta-actin). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. beta-Actin was extracted from PMEM lysate with a DNase-Sepharose column. The extracted beta-actin was quantified in terms of its nitrotyrosine/beta-actin ratio with anti-nitrotyrosine and anti-beta-actin antibodies, sequentially, by dot-blot assays. The cellular compartmentalization of NO(2)-beta-actin was displayed by showing confocal localization of nitrotyrosine-immunofluorescence with beta-actin-immunofluorescence but not with F-actin fluorescence. Incubation of PMEM with TNF (100 ng/ml) for 0.5 and 4.0 h resulted in increases in permeability to albumin. There was an increase in the nitrotyrosine/beta-actin ratio at 0.5 h with minimal association of the NO(2)-beta-actin with F-actin polymers. The TNF-induced increase in the nitrotyrosine/beta-actin ratio and permeability were prevented by the anti-ONOO(-) agent Urate. The data indicate that TNF induces an ONOO(-)-dependent barrier dysfunction, which is associated with the generation of NO(2)-beta-actin.

To study the pathophysiological roles of overexpressed caspase-1 (CASP1), originally designated as IL-1 beta-converting enzyme, we generated transgenic mice in which human CASP1 is overexpressed in their keratinocytes. The transgenic mice spontaneously developed recalcitrant dermatitis and skin ulcers, characterized by the presence of massive keratinocyte apoptosis. The skin of the mice contained the active form of human CASP1 and expressed mRNA for caspase-activated DNase, an effector endonuclease responsible for DNA fragmentation. Their skin and sera showed elevated levels of mature IL-18 and IL-1 beta, but not of IFN-gamma. The plasma from these animals induced IFN-gamma production by IL-18-responsive NK cells. Administration of heat-killed Propionibacterium acnes, a potent in vivo type 1 cell inducer, caused IFN-gamma-mediated lethal liver injury in the transgenic mice, which was completely inhibited by treatment with neutralizing anti-IL-18 Ab. These results indicated that in vivo overexpression of CASP1 caused spontaneous apoptotic tissue injury and rendered mice highly susceptible to exogenous type 1 cell-inducing condition in collaboration with endogenously accumulated proinflammatory cytokines.

The sera from patients with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) variation of the autoimmune disease scleroderma contain autoantibodies that specifically recognize the kinetochore by immunofluorescence. Two major antigens of molecular masses 18 and 80 kD are consistently identified by Western blotting of proteins of isolated chromosomes using CREST sera. In this paper, the possible roles that these two proteins play in the interaction of metaphase chromosomes with tubulin and microtubules are examined using two different procedures. In one set of experiments. Chinese hamster ovary (CHO) chromosomes were extracted with 1-2 M NaCl before incubating with phosphocellulose-purified tubulin under in vitro microtubule assembly conditions. After this treatment, the kinetochores of the residual chromosome scaffolds can still initiate the in vitro assembly of microtubules. Immunoblots of the chromosome scaffold proteins demonstrate that the 18-kD protein has been solubilized by the 1-2 M NaCl extraction, suggesting that this protein is not essential for microtubule assembly at the kinetochore. In a second approach, tubulin was covalently cross-linked to kinetochores of CHO chromosomes using the reversible cross-linking reagent dithiobis (succinimidyl propionate). After DNase I digestion, the chromosomes were solubilized and subjected to anti-tubulin affinity chromatography. Tubulin-kinetochore protein complexes were specifically eluted and analyzed by PAGE and immunoblotting with scleroderma CREST serum. Only a small number of proteins were eluted from the antitubulin affinity column as shown by Coomassie Blue-stained gels. In addition to tubulin, an 80-kD polypeptide, bands at 110 and 24 kD, as well as a faint band at 54 kD, can be resolved. Several minor bands can also be seen in silver-stained gels. The 80-kD protein band from whole metaphase chromosomes reacted with scleroderma CREST serum by immunoblotting and therefore probably represents the major centromere antigen CENP-B. This report provides evidence for a specific protein complex on metaphase chromosomes that is contiguous with kinetochore-bound tubulin and may be involved in microtubule-kinetochore interactions during mitosis.

A nucleic acid-rich fraction extracted and purified from BCG (MY-1) augmented natural killer (NK) cell activity of mouse spleen cells in vitro, and produced factor(s) which showed anti-viral activity and rendered normal macrophages cytotoxic towards tumor cells. These cellular responses were induced by the MY-1 digested preliminarily with RNase, but not by the MY-1 digested with DNase, indicating that DNA contained in MY-1 was essential for the responses. The function of the factor to activate macrophages was destroyed by treatment with a small amount of anti-interferon (IFN)-gamma antiserum or under acidic conditions (pH 2), but not by treatment with anti-IFN-alpha/beta antiserum, while the anti-viral activity was destroyed almost completely by treatment with anti-IFN-alpha/beta antiserum. It appears that DNA from BCG stimulated mouse spleen cells in vitro, resulting in augmentation of NK activity and production of IFN-alpha/beta and -gamma.

The human alpha-like globins undergo a switch from the embryonic zeta-chain to the alpha-chain early in human development, at approximately the same time as the beta-like globins switch from the embryonic epsilon-to the fetal gamma-chains. We investigated the chromatin structure of the human alpha-globin gene cluster in fetal and adult erythroid cells. Our results indicate that DNase I-hypersensitive sites exist at the 5' ends of the alpha 1- and alpha 2-globin genes as well as at several other sites in the cluster in all erythroid cells examined. In addition, early and late fetal liver erythroid cells and adult bone marrow cells contain hypersensitive sites at the 5' end of the zeta gene, and in a purified population of 130-day-old fetal erythroid cells, the entire zeta-to alpha-globin region is sensitive to DNase I digestion. The presence of features of active chromatin in the zeta-globin region in fetal liver and adult bone marrow cells led us to investigate the transcription of zeta in these cells. By nuclear runoff transcription studies, we showed that initiated polymerases are present on the zeta-globin gene in these normal erythroid cells. Immunofluorescence with anti-zeta-globin antibodies also showed that late fetal liver cells contain zeta-globin. These findings demonstrate that expression of the embryonic zeta-globin continues at a low level in normal cells beyond the embryonic to fetal globin switch.

The vaccinia virus (VV) A10L gene codes for a major core protein, P4a. This polypeptide is synthesized at late times during viral infection and is proteolytically cleaved during virion assembly. To investigate the role of P4a in the virus life cycle and morphogenesis, we have generated an inducer-dependent conditional mutant (VVindA10L) in which expression of the A10L gene is under the control of the Escherichia coli lacI operator/repressor system. Repression of the A10L gene severely impairs virus growth, as observed by both the inability of the virus to form plaques and the 2-log reduction of viral yields. This defect can be partially overcome by addition of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). Synthesis of viral proteins other than P4a occurred, although early shutoff of host protein synthesis and expression of viral late polypeptides are clearly delayed, both in the absence and in the presence of IPTG, compared with cells infected with the parental virus. Viral DNA replication and concatemer resolution appeared to proceed normally in the absence of the A10L gene product. In cells infected with VVindA10L in the absence of the inducer virion assembly is blocked, as defined by electron microscopy. Numerous spherical immature viral particles that appear devoid of dense viroplasmic material together with highly electron-dense regular structures are abundant in VVindA10L-infected cells. These regularly spaced structures can be specifically labeled with anti-DNA antibodies as well as with a DNase-gold conjugate, indicating that they contain DNA. Some images suggest that these DNA structures enter into spherical immature viral particles. In this regard, although it has not been firmly established, it has been suggested that DNA uptake occurs after formation of spherical immature particles. Overall, our results showed that P4a and/or its cleaved products are essential for the correct assembly of the nucleoprotein complex within immature viral particles.

In mice that fail to express the phagolysosomal endonuclease DNase II and the type I IFN receptor, excessive accrual of undegraded DNA results in a STING-dependent, TLR-independent inflammatory arthritis. These double-knockout (DKO) mice develop additional indications of systemic autoimmunity, including anti-nuclear autoantibodies and splenomegaly, that are not found in Unc93b1(3d/3d) DKO mice and, therefore, are TLR dependent. The DKO autoantibodies predominantly detect RNA-associated autoantigens, which are commonly targeted in TLR7-dominated systemic erythematosus lupus-prone mice. To determine whether an inability of TLR9 to detect endogenous DNA could explain the absence of dsDNA-reactive autoantibodies in DKO mice, we used a novel class of bifunctional autoantibodies, IgM/DNA dual variable domain Ig molecules, to activate B cells through a BCR/TLR9-dependent mechanism. DKO B cells could not respond to the IgM/DNA dual variable domain Ig molecule, despite a normal response to both anti-IgM and CpG ODN 1826. Thus, DKO B cells only respond to RNA-associated ligands because DNase II-mediated degradation of self-DNA is required for TLR9 activation.

The core of the Dane particle was shown to contain a DNA polymerase and a circular double stranded DNA with a molecular weight of 1.6 X 10(6) daltons which served as the primer-template for the enzyme. The product of the DNA polymerase reaction was in a base paired form and was covalently attached to the circular DNA. Neither the circular DNA nor the attached DNA product of the enzyme reaction was attacked by the DNase or released from intact cores until the cores were disrupted with sodium dodecyl sulfate, suggesting that they are internal components of the core. The DNA polymerase is a specific marker for Dane particles and can be used to distinguish sera with high and low concentrations of Dane particles. The DNA polymerase reaction can also be used to radiolabel Dane particle cores for a specific and sensitive radioimmunoprecipitation assay for antibody against the hepatitis B core antigen (anti-HBc).

Polycyclic aromatic hydrocarbons (PAHs) are known to be activated by the cytochrome P450 (P450) 1 family. However, the precise role of individual P4501 family members in PAH bioactivation remains to be fully elucidated. We therefore investigated the formation of PAH-DNA adducts in the epidermis of Cyp1a2(-/-), Cyp1b1(-/-), and Ahr(-/-) knockout mice. A panel of different PAHs was used, ranging in carcinogenic potency. Mice were treated topically on the dorsal skin with the following tritium-labeled PAHs: dibenzo[a,l]pyre-ne (DB[a,l]P), 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (B[a]P), dibenzo[a,h]anthracene (DB[a,h]A), benzo[g]chrysene (B[g]C), and benzo[c]phenanthrene (B[c]P). At 24 h after treatment, mice (two male and two female mice per group) were sacrificed, and epidermal DNA was isolated and hydrolyzed with DNase I; subsequently, DNA adducts were quantitated by liquid scintillation counting. In the DB[a,l]P-treated mice, levels of DNA adducts were significantly lower in Cyp1a2(-/-) and Cyp1b1(-/-) mice by 57 and 46%, respectively, as compared to wild-type (WT) mice (C57BL/6 background). The levels of DB[a,l]P DNA adducts formed in Ahr(-/-) mice were 26% lower, but this was not statistically significant. The levels of DMBA-DNA adducts in Cyp1a2(-/-) mice were not different than the WT mice but were significantly lower in Cyp1b1(-/-) and Ahr(-/-) mice by 64 and 52%, respectively. DMBA-DNA adduct samples were further analyzed by HPLC following further digestion to deoxyribonucleosides. HPLC analysis of individual DMBA-DNA adducts revealed differences in the ratio of syn-DMBA-diol epoxide- to anti-DMBA-diol epoxide-derived adducts in the Ahr(-/-) and Cyp1b1(-/-) mice. The ratio of syn-/anti-derived adducts in WT mice was 0.49. This ratio was 0.23 in the Cyp1b1(-/-) mice and 0.87 in the Ahr(-/-) mice. In contrast to the results with DB[a,l]P and DMBA, the levels of B[a]P-, DB[a,h]A-, B[g]C-, and B[c]P-DNA adducts were significantly lower in Ahr(-/-) mice by 73, 75, 50, and 81%, respectively, as compared to WT mice but were not significantly lower in the Cyp1a2(-/-) or Cyp1b1(-/-) mice. Collectively, these and other results support a role for both P4501A1 and P4501BBA; P4501A2, P4501BB[a,l]P; and P4501A1 in the bioactivation of B[a]P, DB[a,h]A, B[g]C, and B[c]P in mouse epidermis. Furthermore, in the metabolic activation of DMBA in mouse epidermis, P4501BBA-diol epoxide adducts, whereas P4501A1 shows a preference for the formation of anti-DMBA-diol epoxide adducts.

The group A streptococcal C5a peptidase (SCPA) is a major surface virulence protein that facilitates the establishment of local infection by group A streptococci (GAS). We measured the human immune response to SCPA, using a standardized indirect enzyme-linked immunosorbent assay. Paired acute and convalescent serum samples from children with GAS-associated pharyngitis were assayed, and a strong immune response to SCPA was demonstrated that was independent of the infecting M type and the age of the patient. Western blot analysis of bacterial extracts revealed that all tested M types expressed SCPA. The immune response to SCPA correlated with the anti-streptolysin O and anti-DNase B responses. These data confirm the immunogenicity of SCPA in humans. Previous knowledge of SPCA's role in virulence, its highly conserved nature, and the results of mouse protection studies make SCPA an ideal vaccine candidate for the prevention of GAS disease.

Brain fatty acid-binding protein (B-FABP) is expressed in the radial glial cells of the developing central nervous system as well as in a subset of human malignant glioma cell lines. Most of the malignant glioma lines that express B-FABP also express GFAP, an intermediate filament protein found in mature astrocytes. We are studying the regulation of the B-FABP gene to determine the basis for its differential expression in malignant glioma lines. By DNase I footprinting, we have identified five DNA-binding sites located within 400 base pairs (bp) of the B-FABP transcription start site, including two nuclear factor I (NFI)-binding sites at -35 to -58 bp (footprint 1, fp1) and -237 to -260 bp (fp3), respectively. Competition experiments, supershift experiments with anti-NFI antibody, and methylation interference experiments all indicate that the factor binding to fp1 and fp3 is NFI. By site-directed mutagenesis of both NFI-binding sites, we show that the most proximal NFI site is essential for B-FABP promoter activity in transiently transfected malignant glioma cells. Different band shift patterns are observed with nuclear extracts from B-FABP(+) and B-FABP(-) malignant glioma lines, with the latter generating complexes that migrate more slowly than those obtained with B-FABP(+) extracts. All bands are converted to a faster migrating form with potato acid phosphatase treatment, indicating that NFI is differentially phosphorylated in B-FABP(+) and B-FABP(-) lines. Our results suggest that B-FABP expression in malignant glioma lines is determined by the extent of NFI phosphorylation which, in turn, is controlled by a phosphatase activity specific to B-FABP(+) lines.

Since the late 1980s several studies have described the increased incidence and severity of invasive group A streptococcal (GAS) infections. However, most studies on GAS pathogenesis have focused on information obtained during outbreaks. We analyzed isolate distribution and host susceptibility as part of a nationwide prospective surveillance study performed between January 2001 and August 2002. GAS isolates collected from 201 patients with invasive infections, 335 patients with noninvasive infections, and 17 asymptomatic carriers were characterized with respect to their emm types and superantigen genotypes. The superantigen-neutralizing capacity and levels of antibodies against streptolysin O and DNAse B were determined for isolates from the sera from 36 invasive cases and 91 noninvasive cases. emm type 1 (emm-1) isolates were significantly more common among invasive cases, whereas emm-4, emm-6, and emm-12 dominated among the noninvasive cases. The distributions of the phage-associated superantigen genes (speA, speC, speH, speI, ssa) differed among invasive and noninvasive isolates, mainly due to their linkage to certain emm types. No significant differences in serum superantigen-neutralizing capacities were observed. The levels of anti-streptolysin O and anti-DNAse B antibodies were highest in the sera from invasive cases. Our study emphasizes the importance of obtaining data during years with stable incidences, which will enable evaluation of future outbreak data.

The deposition of anti-dsDNA antibodies in the glomerulus is believed to play a critical role in the pathogenesis of nephritis in SLE. However, an absolute correlation between serum levels of anti-dsDNA antibodies and renal disease has not been found. Recently a glomerular binding assay (GBA) has been developed to detect IgG binding to isolated rat glomeruli. We have used the GBA to study sera from four groups of SLE patients: (A) + anti-dsDNA antibodies, active nephritis; (B) - anti-dsDNA antibodies, active nephritis; (C) + anti-dsDNA antibodies, no nephritis; and (D) - anti-dsDNA antibodies, no nephritis. The serum anti-dsDNA antibodies in group A and group C patients could not be distinguished on the basis of isotype, charge, or cross-reactivity with histones. Nevertheless, the mean intensity of glomerular immunofluorescence was significantly higher in group A than in the three other patient groups and distinguished between patients with serum anti-dsDNA antibodies who had nephritis and those without clinically apparent nephritis. GBA reactivity was unaffected by DNase treatment of sera, but was partially inhibited by preincubation with dsDNA. These findings are consistent with the hypothesis that some anti-dsDNA antibodies cross-react with glomerular components and that the presence of this cross-reactivity is associated with, and may be responsible for, the development of nephritis. In addition, we have identified a group of SLE patients with renal disease and typical renal histopathology and immune deposits who do not have serum anti-dsDNA antibodies or antibodies that directly bind to glomeruli in the GBA. The mechanism of renal immune deposition in these patients remains to be determined.

Earlier work by us as well as others has demonstrated that filamentous actin is mainly localized in the cortical surface of chromaffin cell. This F-actin network acts as a barrier to the chromaffin granules, impeding their contact with the plasma membrane. Chromaffin granules contain alpha-actinin, an anchorage protein that mediates F-actin association with these vesicles. Consequently, chromaffin granules crosslink and stabilize F-actin networks. Stimulation of chromaffin cell produces disassembly of F-actin and removal of the barrier. This interpretation is based on: (1) Cytochemical experiments with rhodamine-labeled phalloidin indicated that in resting chromaffin cells, the F-actin network is visualized as a strong cortical fluorescent ring; (2) Nicotinic receptor stimulation produced fragmentation of this fluorescent ring, leaving chromaffin cell cortical areas devoid of fluorescence; and (3) These changes are accompanied by a decrease in F-actin, a concomitant increase in G-actin, and a decrease in the F-actin associated with the chromaffin cell cytoskeleton (DNAse I assay). We also have demonstrated the presence in chromaffin cells of gelsolin and scinderin, two Ca(2+)-dependent actin filament-severing proteins, and suggested that chromaffin cell stimulation activates scinderin with the consequent disruption of F-actin networks. Scinderin, a protein recently isolated in our laboratory, is restricted to secretory cells and is present mainly in the cortical chromaffin cell cytoplasm. Scinderin, which is structurally different from gelsolin (different pIs, amino acid composition, peptide maps, and so on), decreases the viscosity of actin gels as a result of its F-actin-severing properties, as demonstrated by electron microscopy. Stimulation of chromaffin cells either by nicotine (10 microM) or high K+ (56 mM) produces a redistribution of subplasmalemmal scinderin and actin disassembly, which preceded exocytosis. The redistribution of scinderin and exocytosis is Ca(2+)-dependent and is not mediated by muscarinic receptors. Furthermore, our cytochemical experiments demonstrate that chromaffin cell stimulation produces a concomitant and similar redistribution of scinderin (fluorescein-labeled antibody) and F-actin (rhodamine phalloidin fluorescence), suggesting a functional interaction between these two proteins. Stimulation-induced redistribution of scinderin and F-actin disassembly would produce subplasmalemmal areas of decreased cytoplasmic viscosity and increased mobility for chromaffin granules. Exocytosis sites, evaluated by antidopamine-beta-hydroxylase (anti-D beta H) surface staining, are preferentially localized in plasma membrane areas devoid of F-actin.

When rat liver nuclei are isolated in the presence of the irreversible sulfhydryl-blocking reagent iodoacetamide, digested with DNase I and RNase A, and extracted with 1.6 M NaCl, nuclear envelope (NE) spheres depleted of intranuclear material, as analysed by thin-section electron microscopy, are obtained. Two-dimensional isoelectric focusing (IEF)/SDS-PAGE and non-equilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE reveal that the predominant polypeptides are lamins A, B and C. Nuclei isolated in the absence of sulfhydryl blocking reagents yield salt- and nuclease-resistant structures which contain sparse but demonstrable intranuclear material. A number of non-histone polypeptides are seen in addition to the lamins. Nuclei treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) yield, after exposure to nucleases and 1.6 M NaCl, nuclear matrix-like structures containing an extensive intranuclear network and components of the nucleolus in addition to the NE. Increased amounts of the non-lamin, non-histone polypeptides are recovered with these structures. Subsequent treatment of these NaTT-cross-linked structures with reducing agents in 1.0 M NaCl selectively solubilizes the intranuclear components but leaves the nuclear envelope apparently intact. The lamins remain sedimentable and are virtually absent from the soluble (intranuclear) material. Instead, the major solubilized polypeptides are (a) 68 and 63 kD polypeptides which migrate in the vicinity of lamins B and C, respectively, but are distinguishable from the lamins by immunoblotting and by uni-dimensional peptide mapping; (b) a series of basic 60-70 kD polypeptides (pI greater than 8.0) which are not recognized by anti-lamin antisera; (c) an acidic (pI 5.3) 38 kD polypeptide; and (d) a number of high molecular mass (greater than 100 kD) polypeptides. These observations not only suggest a convenient method for fractionating matrix structures from rat liver nuclei into biochemically and morphologically discrete components, but also identify a subset of major non-lamin, non-histone nuclear polypeptides (comprising approx. 20% of the total nuclear protein) whose intermolecular interactions can be reversibly stabilized apparently by intermolecular disulfide bond formation by NaTT.

To study the occurrence and subcellular distribution of actin in trypanosomatid parasites, we have cloned and overexpressed Leishmania donovani actin gene in bacteria, purified the protein, and employed the affinity purified rabbit polyclonal anti-recombinant actin antibodies as a probe to study the organisation and subcellular distribution of actin in Leishmania cells. The Leishmania actin did not cross react with antimammalian actin antibodies but was readily recognized by the anti-Leishmania actin antibodies in both the promastigote and amastigote forms of the parasite. About 10(6) copies per cell of this protein (M(r) 42.05 kDa) were present in the Leishmania promastigote. Unlike other eukaryotic actins, the oligomeric forms of Leishmania actin were not stained by phalloidin nor were dissociated by actin filament-disrupting agents, like Latrunculin B and Cytochalasin D. Analysis of the primary structure of this protein revealed that these unusual characteristics may be related to the presence of highly diverged amino acids in the DNase I-binding loop (amino acids 40-50) and the hydrophobic plug (amino acids 262-272) regions of Leishmania actin. The subcellular distribution of actin was studied in the Leishmania promastigotes by employing immunoelectron and immunofluorescence microscopies. This protein was present not only in the flagella, flagellar pocket, nucleus and the kinetoplast but it was also localized on the nuclear, vacuolar and cytoplasmic face of the plasma membranes. Further, the plasma membrane-associated actin was colocalised with subpellicular microtubules, while most of the actin present in the kinetoplast colocalised with the k-DNA network. These results clearly indicate that Leishmania contains a novel form of actin which may structurally and functionally differ from other eukaryotic actins. The functional significance of these observations is discussed.

The three-dimensional structure of bovine pancreatic deoxyribonuclease I (DNase I) has been determined at 2.5 A resolution by X-ray diffraction from single crystals. An atomic model was fitted into the electron density using a graphics display system. DNase I is an alpha, beta-protein with two 6-stranded beta-pleated sheets packed against each other forming the core of a 'sandwich'-type structure. The two predominantly anti-parallel beta-sheets are flanked by three longer alpha-helices and extensive loop regions. The carbohydrate side chain attached to Asn 18 is protruding by approximately 15 A from the otherwise compact molecule of approximate dimensions 45 A X 40 A. The binding site of CA2+-deoxythymidine-3',5'-biphosphate (Ca-pdTp) has been determined by difference Fourier techniques confirming biochemical results that the active centre is close to His 131. Ca-pdTp binds at the surface of the enzyme between the two beta-pleated sheets and seems to interact with several charged amino acid side chains. Active site geometry and folding pattern of DNase I are quite different from staphylococcal nuclease, the only other Ca2+-dependent deoxyribonuclease whose structure is known at high resolution. The electron density map indicates that two Ca2+ ions are bound to the enzyme under crystallization conditions.

To determine whether the existence of anti-dsDNA producing lymphocyte clones is limited to autoimmune strains of mice, spleen cells derived from autoimmune mice (NZB, NZB X NZW F1, MRL) and from normal strains (BALB/c, DBA/2, C57BL/6, C3H/eb) were cultured with E. coli lipopolysaccharide (LPS). DNase-treated supernatants from these cultures were assayed for anti-dsDNA antibodies by employing a sensitive solid-phase radioimmunoassay with poly (dA-dT) as the antigen. All tested spleen cells secreted a small yet significant amount of anti-dsDNA upon stimulation with LPS. There was no difference in the amount or in the heavy chain type of anti-dsDNA secreted by cells from normal and autoimmune strain cells. Evidence of clonal expansion in unstimulated cells was observed only in cultures prepared from older autoimmune animals. Removal of T cells from the spleen cell preparations had no marked effect on the spontaneous or stimulated antibody secretion. Anti-dsDNA antibodies could also be induced in vivo by i.p. injection of LPS into young normal animals. Splenocytes from all tested strains spontaneously secreted anti-ssDNA and anti-TNP antibodies in culture, and these were present at relatively high levels in the serum of unstimulated animals. Stimulation with LPS increased secretion of anti-ssDNA and anti-TNP in all strains in vitro and in five of seven strains in vivo as well. It can be concluded that a) the existence of anti-dsDNA-producing clones is not limited to autoimmune strains, and b) these clones are expanded in old but not in young autoimmune mice. They are not expanded in normal mice at any age.

Minced human tonsils were digested with DNase and collagenase, and lymphoid cell-depleted low density cells were cultured and grown in granulocyte-macrophage-CSF. Large, morphologically homogenous adherent cells with elongated extensions grew continuously in culture. These nonphagocytic cells appear to be related to follicular dendritic cell (FDC) as they do not have properties of monocytic lineage cells or dendritic cells and because, like FDC, 1) they express CD11b, CD14, CD29, CD40, CD54, CD73, CD74, and VCAM-1, and do not express CD11c, CD22, T cell markers, CD18, CD25 and CD45; and 2) they bind human B lymphocytes and B cell lines, but not T lymphocytes by an adhesion blocked in part by mAb to VLA-4 (CD49d). The cultured FDC also augmented B cell proliferation stimulated by anti-mu sera and/or CD40 mAb. Cultured FDC spontaneously produced low levels of IL-6, but did not produce IL-1 alpha or TNF-alpha; however, after treatment with either IFN-gamma or LPS, they produced more IL-6. The expression of CD54 (ICAM-1) was elevated by treating the cultured FDC with either TNF-alpha, IL-1 beta, IFN-gamma or granulocyte-macrophage-CSF; in contrast, IL-4 had no effect on CD54 but rather up-regulated expression of VCAM-1. IFN-gamma, unlike the other cytokines tested, increased expression of a set of markers on cultured FDC (CD54, VCAM-1, and CD14) and converted these class II-negative cells into class II+ cells. The fact that various T cell-derived cytokines have different effects on FDC suggests that the T cell products may influence the manner by which FDC stimulate B cell proliferation and maturation.

BACKGROUND

Anorexia nervosa (AN) is a serious illness with no definitive treatment. Clinical and research evidence led to the hypothesis that some children with AN may have a pediatric autoimmune neuropsychiatric disorder associated with streptococcus (PANDAS), similar in pathogenesis to other hypothesized PANDAS disorders.

METHODS

Four youngsters (ages, 11-15 years) with PANDAS AN were treated with an open trial of antibiotics, in addition to conventional treatment. They were evaluated for eating disorder and obsessive-compulsive symptoms, and for weight gain. Evidence of streptococcal infection came from clinical evaluation, throat cultures, and two serological tests: anti-deoxyribonuclease B (anti-DNase B) and anti-streptolysin O (ASO) titers. The "rheumatic" marker D8/17 was also measured. This B-cell alloantigen is associated, in several publications, with poststreptococcal autoimmunity: Rheumatic fever (RF), Sydenham's chorea (SC), and possibly PANDAS obsessive compulsive disorder (OCD) and tic disorders.

RESULTS

There was clinical evidence of possible antecedent streptococcal infection in all four patients, two of whom had comorbid OCD, with possible infection-triggered AN. All four had the rheumatic marker: A percentage of D8/17-positive B cells of 28-38%, with a mean of 33% (12% or more is considered positive for the marker). The patients responded to conventional treatment plus antibiotics with weight restoration and decreased eating disorder and obsessive-compulsive symptoms. Three needed to gain weight and did so.

CONCLUSIONS

There may be a link between infectious disease and some cases of AN, which raises the possibility of new treatment.